A cyclodextrin biomimetic organic iridium magnetic catalyst and its preparation method and application

Abstract

The invention discloses a cyclodextrin bionic organic iridium magnetic catalyst and its preparation method and application, belonging to the field of catalysts and applications. The catalyst comprises three parts: magnetic nanoparticles, β-cyclodextrin and adamantane-functionalized 2-pyridine benzamide-type organic iridium catalyst; the magnetic nanoparticles are surface carboxylated magnetic nanoparticles, which are connected to the amino β-cyclodextrin through an amide bond after being activated by N-hydroxysuccinimide; the β-cyclodextrin is connected to the adamantane in the structure of the adamantane-functionalized 2-pyridine benzamide-type organic iridium catalyst through a host-guest coupling effect. It also includes the application of the catalyst combined with a rhodamine 110-type quinone reduction-regulated fluorescent probe in the detection of coenzyme NADH. The cyclodextrin bionic organic iridium magnetic catalyst MBs@CD@Ir prepared by the present invention has excellent biocompatibility, and the constructed NADH fluorescent probe has the advantages of high sensitivity, good selectivity and easy recovery of the catalyst.

Description

Technical Field

[0001] The present invention relates to an organic metal complex catalyst, and to a synthesis method and application of the catalyst, and in particular to a cyclodextrin biomimetic organic iridium magnetic catalyst, a preparation method of the catalyst and application in the field of fluorescence analysis. Background Art

[0002] Organoiridium catalysts have been widely used in biological environments, especially in life science fields such as imaging, sensing and therapy. Studies have shown that diseases such as anemia and diabetes are accompanied by changes in NADH content. In particular, NADH located in mitochondria is essential for maintaining mitochondrial antioxidant defense. Its abnormal level induces the production of reactive oxygen species (ROS) and is associated with stroke, Alzheimer's disease and Parkinson's disease. In addition, NADH in mitochondria is overexpressed in many cancers. So far, highly sensitive and non-invasive fluorescence imaging methods have been developed, such as those based on genetically encoded proteins, nanoparticles and small molecule probes, for tracking NADH, especially tracking NADH in living cells. Among these modes, small molecule fluorescent probes have great potential for application in many biological environments due to their simple preparation, ease of use, high cell permeability, low toxicity and applicability to in vivo experiments.

[0003] The property of organic iridium catalysts being deactivated when in contact with thiol molecules limits their application in biological environments. Common solutions currently include material compartmentalization strategy and biotin-streptavidin strategy. However, these methods also have some shortcomings, which mainly include high cost, low stability, and no significant improvement in anti-thiol performance. Therefore, it is very important to develop an organic iridium catalyst with good biocompatibility. β-cyclodextrin (β-CD) is a well-known cyclic oligosaccharide that can form inclusion complexes with various hydrophobic substrates (such as adamantane derivatives) in aqueous solution, and has been widely used as a supramolecular construction model in various fields. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a cyclodextrin bionic organic iridium magnetic catalyst and a preparation method and application thereof. First, the catalyst has the characteristics of easy recovery and high catalytic hydrogenation activity, and the provided catalyst can be quickly recovered by magnetic separation; second, a catalyst with the above characteristics is prepared; third, the catalyst is combined with a trimethylhydroquinone hydrogenation regulation fluorescent probe to construct a sensitive and easily recoverable coenzyme NADH fluorescent sensor.

[0005] The technical solution of the present invention is as follows:

[0006] A cyclodextrin biomimetic organic iridium magnetic catalyst comprises three parts: magnetic nanoparticles, β-cyclodextrin and adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst; the magnetic nanoparticles are surface carboxylated magnetic nanoparticles, which are connected to the amino β-cyclodextrin through an amide bond after being activated by N-hydroxysuccinimide; the β-cyclodextrin is connected to the adamantane in the structure of the adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst through a host-guest coupling effect.

[0007] Preferably, the structural formula of the cyclodextrin biomimetic organic iridium magnetic catalyst is as follows:

[0008] ;

[0009] Where MBs represents magnetic nanoparticles.

[0010] The preparation method of the cyclodextrin biomimetic organic iridium magnetic catalyst comprises the following steps:

[0011] Step (1): mixing magnetic nanoparticles, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, incubating, removing excess 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide by magnetic separation, washing the obtained magnetic nanoparticles to obtain N-hydroxysuccinimide-modified magnetic nanoparticles, denoted as MBs-NHS;

[0012] Step (2): adding amino-β-cyclodextrin to the MBs-NHS obtained in step (1), and incubating to obtain cyclodextrin-modified carboxyl magnetic beads, denoted as MBs@CD;

[0013] Step (3): After washing the MBs@CD obtained in step (2), a 2-pyridinebenzamide-type organic iridium catalyst functionalized with adamantane is added, and after incubation on a shaking table, the obtained magnetic beads are washed to obtain a cyclodextrin biomimetic organic iridium magnetic catalyst, which is recorded as MBs@CD@Ir.

[0014] Preferably, the preparation steps of the adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst are as follows:

[0015] Step 1: Compound 1, 4-p-phenylenediamine and 1-adamantane acid chloride are mixed in a molar ratio of 2:1 and dissolved in dry dichloromethane, and stirred for reaction for 1.0 to 24 hours; after the reaction is completed, the solvent is removed by pressure distillation to obtain monoadamantane-modified p-phenylenediamine Ada-Ph-NH 2 The crude product was further separated by silica gel column chromatography to obtain a pure compound Ada-Ph-NH 2 ;

[0016] 2-pyridinecarboxylic acid and N-hydroxysuccinimide are dissolved in dry dichloromethane or dry chloroform solvent and cooled to below zero degrees Celsius; then, the reaction solution is kept below zero degrees Celsius, and a dichloromethane solution of N, N'-dicyclohexylcarbodiimide is added dropwise, and after the addition is completed, the reaction is completed at room temperature; the precipitate is removed by filtration, the solvent is removed under reduced pressure, and vacuum drying is performed to obtain a crude product of NHS-activated 2-pyridinecarboxylic acid, which is recorded as Pic-NHS;

[0017] Step 2: The compound Ada-Ph-NH obtained in step 1 2 Mixing with Pic-NHS in a molar ratio of 1:1 and dissolving in dry dichloromethane, the mixture is stirred at room temperature for reaction for 10.0 to 48.0 hours; after the reaction, removing the solvent by pressure distillation to obtain a crude product of adamantane-functionalized pyridine amide ligand PicPh-Ada; the crude product of PicPh-Ada is further separated by silica gel column chromatography to obtain a pure compound PicPh-Ada;

[0018] Step 3: The compound PicPh-Ada obtained in step 2, dichloro(pentamethylcyclopentadienyl)iridium(III) dimer and ammonium hexafluorophosphate are mixed and dissolved in ethanol, and refluxed for 5 to 24 hours under nitrogen protection; after the reaction, the mixture is cooled, filtered, the filtrate is collected, and the solvent is removed by reduced pressure distillation to obtain a crude product of adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst, and a pure adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst PicPh-Ada-Ir is obtained by recrystallization; wherein the molar ratio of PicPh-Ada, dichloro(pentamethylcyclopentadienyl)iridium(III) dimer and ammonium hexafluorophosphate is (2-3):1:(4 to 10).

[0019] The application of the cyclodextrin bionic organic iridium magnetic catalyst is to combine the cyclodextrin bionic organic iridium magnetic catalyst with a fluorescent probe regulated by rhodamine 110 type quinone hydrogenation to construct a NADH fluorescent sensor; the constructed fluorescent sensor is used to detect the coenzyme NADH.

[0020] Furthermore, the structural formula of the fluorescent probe regulated by hydrogenation of rhodamine 110-type quinone is as follows:

[0021] .

[0022] Furthermore, the conditions of the fluorescence generation system of the NADH fluorescence sensor constructed by combining the cyclodextrin biomimetic organic iridium magnetic catalyst with the rhodamine 110-type quinone hydrogenation regulated fluorescent probe are as follows: the concentration of the cyclodextrin biomimetic organic iridium magnetic catalyst is 20 μg / mL and the reaction time is 85 min.

[0023] Furthermore, the NADH fluorescence sensor constructed in the application is used to detect the coenzyme NADH in the Escherichia coli cell fermentation liquid environment.

[0024] The beneficial effects of the present invention are:

[0025] First, the present invention uses carboxylated magnetic nanoparticles (MBs-COOH) as carrier materials, and uses N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) to activate the carboxyl groups on the surface of the carrier MBs-COOH to obtain NHS-modified magnetic nanoparticles MBs-NHS, which are coupled with amino-β-cyclodextrin to obtain β-cyclodextrin-modified magnetic nanoparticles MBs@CD; finally, MBs@CD is coupled with adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst to prepare cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir; the prepared magnetic catalyst MBs@CD@Ir has better dispersibility and stability in aqueous solution, successfully solves the problem of difficulty in recovering homogeneous organic iridium catalysts, and improves the biocompatibility of organic iridium catalysts. The organic iridium catalyst of the present invention has higher catalytic hydrogenation activity and milder host-guest coupling characteristics with β-cyclodextrin. It is conducive to the wider application of metal complexes in biological environments in the future.

[0026] Second, the present invention provides a simple preparation process, and the preparation method provided has the following technical advantages: the present invention uses coenzyme NADH as a hydrogen source, and can efficiently catalyze the reduction of trimethyl-p-benzoquinone to the corresponding trimethyl-semihydroquinone. The organic iridium catalyst used in the preparation of the cyclodextrin biomimetic organic iridium magnetic catalyst is an adamantane-modified 2-pyridine benzamide-type organic iridium catalyst, which not only has extremely high catalytic hydrogenation activity, and can efficiently catalyze the reduction of trimethyl-p-benzoquinone to trimethyl-semihydroquinone in the presence of NADH, but also the organic iridium catalyst can be mildly coupled with β-cyclodextrin through host-guest interaction.

[0027] Third, the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir provided by the present invention can be used in combination with a fluorescent probe regulated by hydrogenation of rhodamine 110-type quinone to construct a NADH fluorescent sensor. After the trimethyl-p-benzoquinone in the molecular structure of the fluorescent probe regulated by hydrogenation of rhodamine 110-type quinone is hydrogenated and reduced to trimethyl-semihydroquinone, the quinone functional group in the probe molecule will fall off due to intramolecular cyclization, thereby turning on the fluorescence of the probe molecule and generating a strong fluorescent signal. The constructed fluorescent sensor has the advantages of strong controllability, high signal-to-noise ratio and good selectivity for the detection of coenzyme NADH, has higher sensitivity for the detection of coenzyme NADH, and can realize sensitive detection of coenzyme NADH in the environment of Escherichia coli cell fermentation broth, and has potential biological application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The scanning electron microscope SEM photo (A) and particle size distribution diagram (B) of the cyclodextrin bionic organic iridium magnetic catalyst MBs@CD@Ir prepared in Example 2 of the present invention;

[0029] Figure 2 This is a schematic diagram of the X-ray photoelectron spectroscopy XPS structural characterization of the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir prepared in Example 2 of the present invention;

[0030] Figure 3 The cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir (C) and its precursor materials MBs-COOH (A) and MBs@CD (B) prepared in Example 2 of the present invention catalyze the fluorescent probe to produce a fluorescence spectrum in the presence of coenzyme NADH;

[0031] Figure 4 This is a schematic diagram of the detection mechanism of the NADH fluorescence sensor constructed by combining the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir prepared in Example 2 of the present invention with a fluorescent probe regulated by the hydrogenation of rhodamine 110-type quinone;

[0032] Figure 5 This is a fluorescence detection condition optimization diagram of the NADH fluorescence sensor constructed by combining the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir prepared in Example 2 of the present invention with the fluorescence probe regulated by rhodamine 110-type quinone hydrogenation. A) is a graph showing the fluorescence intensity change of the constructed fluorescence sensor to NADH under different catalyst MBs@CD@Ir dosage conditions; B) is a graph showing the fluorescence intensity change of the constructed fluorescence sensor to NADH under different reaction times;

[0033] Figure 6 This is a fluorescence spectrum of the NADH fluorescence sensor constructed by combining the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir prepared in Example 2 of the present invention with the fluorescent probe regulated by the hydrogenation of rhodamine 110-type quinone to detect different concentrations of NADH ( Figure 6 A in the figure), fluorescence intensity of different concentrations of NADH at 523 nm fluorescence emission wavelength ( Figure 6 B) and the standard curve of the constructed fluorescent sensor for NADH detection ( Figure 6 Figure 2. Illustration of Figure 2 (inset in B). DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] 1. The structure of the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir of the present invention.

[0036] The cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir provided by the present invention has the following structural formula:

[0037] .

[0038] Its structure mainly includes three parts: magnetic nanoparticles (MBs), organic iridium catalysts and β-cyclodextrin (β-CD). Among them, the magnetic nanoparticles are carboxylated magnetic nanoparticles (MBs-COOH) with regular spheres and a particle size of 180.53 ± 14.2 nm; the organic iridium catalyst is a 2-pyridine benzamide organic iridium catalyst functionalized with adamantane; and β-cyclodextrin (β-CD) is amino β-cyclodextrin. Among them, the carboxylated magnetic nanoparticles (MBs-COOH) are activated by N-hydroxysuccinimide NHS and connected to the amino β-cyclodextrin through an amide bond; then, the β-cyclodextrin on the surface of the magnetic nanoparticles continues to connect to the organic iridium catalyst through a host-guest interaction.

[0039] The structural formula of the organic iridium catalyst is as follows:

[0040]

[0041] The structural formula of the β-cyclodextrin (β-CD) is as follows:

[0042] .

[0043] 2. Examples of the preparation method of the present invention.

[0044] Example 1. Preparation of a 2-pyridinebenzamide-type organic iridium catalyst functionalized with adamantane used in the preparation of cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir.

[0045] Step 1: Dissolve the compound 1,4-p-phenylenediamine (1.0 g, 9.26 mmol, 2.0 equiv) in 10.0 mL of dry dichloromethane, transfer to an ice bath and cool to 0 degrees Celsius, slowly dropwise add a dichloromethane (5.0 mL) solution of 1-adamantane acid chloride (916.7 mg, 4.629 mmol, 1.0 equiv) under stirring; after the addition is complete, transfer the reaction solution to room temperature and continue stirring for 2.0 hours, and plate chromatography is used to detect the reaction until the reaction is complete; after the reaction is completed, remove the solvent by pressure distillation to obtain monoadamantane-modified p-phenylenediamine Ada-Ph-NH 2 The crude product was further separated by silica gel column chromatography to obtain a pure compound Ada-Ph-NH 2 (1.09 g, 87.6% yield).

[0046] The compound Ada-Ph-NH 2 To characterize the structure of 1 H NMR (400 MHz, Chloroform-d) δ 7.29 – 7.25 (m, 2H), 7.11 (s, 1H), 6.66 – 6.59 (m, 2H), 3.56 (s, 2H), 2.12 – 2.03 (m, 3H), 1.94 (d, J = 2.9 Hz, 6H), 1.79 – 1.68 (m, 6H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 171.12, 138.42, 124.80, 117.32, 110.73, 36.55,34.65, 31.82, 23.50. MS (ESI): m / z cacld for ([M-Cl - ] + ) C 17 H 22 N 2 NaO 2 293.1630; found 293.1643.

[0047] By Ada-Ph-NH 2 Analysis of the NMR data and mass spectrometry data proved that the organic iridium catalyst PicPh-Ada-Ir had been successfully synthesized.

[0048] 2-Pyridinecarboxylic acid (455 mg, 3.7 mmol, 1.0 equiv) and N-hydroxysuccinimide (433.2 mg, 3.7 mmol, 1.0 equiv) were dissolved in 15.0 mL of dry dichloromethane and cooled to below zero degrees Celsius. Then, while keeping the reaction solution below zero degrees Celsius, 10 mL of dichloromethane solution of N, N'-dicyclohexylcarbodiimide (910 mg, 4.44 mmol, 1.2 equiv) was slowly added dropwise. After the addition was complete, the mixed solution was transferred to room temperature and stirred for 2.0 hours. After the reaction was completed, the precipitate was removed by filtration, and the solvent was removed by distillation under reduced pressure from the obtained filtrate. The crude product NHS-activated 2-pyridinecarboxylic acid (Pic-NHS) was obtained and directly used in the next step without further purification.

[0049] Step 2: The compound Ada-Ph-NH obtained in step 1 2(1.0 g, 3.7 mmol, 1.0 equiv) was mixed with compound Pic-NHS (814 mg, 3.7 mmol, 1.0 equiv) and dissolved in 20.0 mL of dry dichloromethane. The mixture was stirred at room temperature for 24 hours. After the reaction, the solvent was removed by pressure distillation to obtain a crude product of adamantane-functionalized pyridineamide ligand PicPh-Ada. The crude PicPh-Ada product was further separated by silica gel column chromatography to obtain pure compound PicPh-Ada (1.05 g, yield 75.8%).

[0050] The structure of the compound PicPh-Ada was characterized by NMR and mass spectrometry. 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.50 (s, 1H), 9.06 (s, 1H), 8.69 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H), 8.11(dt, J = 7.8, 1.1 Hz, 1H), 8.03 (td, J = 7.7, 1.7 Hz, 1H), 7.81 – 7.72 (m,2H), 7.67 – 7.52 (m, 3H), 1.98 (q, J = 3.2 Hz, 3H), 1.87 (d, J = 3.2 Hz, 6H), 1.67 (t, J = 3.2 Hz, 6H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 176.31, 162.69, 150.54,148.95, 138.66, 135.96, 134.17, 122.80, 121.06, 120.82, 41.36, 38.89, 36.58,33.88, 28.23, 25.85, 24.99. MS (ESI): m / z cacld for ([M+Na] + ) C 23 H 25 N 3 NaO 2 398.1844; found 398.1887. Through the analysis of NMR data and mass spectrometry data of PicPh-Ada, the organic iridium catalyst PicPh-Ada-Ir has been successfully synthesized.

[0051] Step 3: The ligand compound PicPh-Ada (47.1 mg, 0.1254 mmol, 1.0 equiv) obtained in step 2, dichloro(pentamethylcyclopentadienyl)iridium(III) dimer (50.0 mg, 0.0627 mmol, 0.5 equiv) and ammonium hexafluorophosphate (40.9 mg, 0.251 mmol, 2 equiv) were mixed and dissolved in 10.0 mL of ethanol, and refluxed at 85°C for 24 hours under nitrogen protection; after the reaction, the reaction solution was cooled to room temperature, the filtrate was collected by filtration, and the solvent was removed by vacuum distillation to obtain a crude product of adamantane functionalized organic iridium complex; the crude product was further recrystallized using a mixed solvent of dichloromethane and ether to obtain a pure adamantane functionalized organic iridium complex PicPh-Ada-Ir (54.1 mg, 58.7%).

[0052] The molar ratio of PicPh-Ada, dichloro(pentamethylcyclopentadienyl)iridium(III) dimer and ammonium hexafluorophosphate is 2:1:4.

[0053] The structure of adamantane functionalized organic iridium complex PicPh-Ada-Ir was characterized by NMR and mass spectrometry. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.03 (s, 1H), 8.80 – 8.71 (m, 1H), 8.10 (td, J =7.7, 1.4 Hz, 1H), 7.90 (ddd, J = 7.8, 1.6, 0.7 Hz, 1H), 7.70 (ddd, J = 7.4,5.6, 1.6 Hz, 1H), 7.65 – 7.51 (m, 2H), 7.49 – 7.33 (m, 2H), 7.22 – 6.93 (m,1H), 2.02 (s, 3H), 1.92 (d, J = 3.0 Hz, 6H), 1.79 (s, 1H), 1.71 (t, J = 3.1Hz, 6H), 1.62 (s, 1H), 1.44 (s, 1H), 1.35 (s, 11H). 13 C NMR (101 MHz, DMSO-d 6)δ 176.33, 176.17, 162.69, 154.90, 151.67, 148.96, 144.36, 139.69, 138.67,135.95, 134.16, 127.05, 125.68, 122.80, 121.06, 120.81, 120.29, 120.15,95.85, 92.63, 86.79, 38.89, 36.58, 28.23, 8.77, 8.59, 8.29, 8.09. MS (ESI):m / z cacld for ([M-Cl - ] + ) C 33 H 3 Ir 3 O 2 702.2672; found 702.2761. Through the analysis of NMR data and mass spectrometry data of PicPh-Ada-Ir, the organic iridium catalyst PicPh-Ada-Ir has been successfully synthesized.

[0054] Example 2: Preparation example of cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir.

[0055] The preparation steps are as follows:

[0056] Step 1. After ultrasonically mixing commercially available carboxyl magnetic nanoparticles (MBs-COOH), 400 μL (5 mg / ml) was taken and placed in a 5.0 mL centrifuge tube. After magnetic separation to remove the dispersion, the tube was washed with water 3 times (1.0 mL × 3 times), 1.0 mL of EDCI (10.0 mg / mL) and 1.0 mL of NHS (10.0 mg / mL) solution were added, and the tube was incubated on a shaker at room temperature for 1.0 h. After the incubation, the excess EDCI and NHS were removed by magnetic separation, and the obtained magnetic nanoparticles were washed with deionized water twice (2.0 mL × 2 times) to obtain NHS-modified magnetic nanoparticles MBs-NHS.

[0057] Step 2. Add 1.0 mL of amino β-cyclodextrin solution (1.2 mg / mL) to the NHS-modified magnetic nanoparticles MBs-NHS obtained in step 1, shake on a shaker at room temperature for 3.0 h, and then place in a refrigerator at 4.0 °C overnight to obtain a cyclodextrin-modified carboxyl magnetic beads MBs@CD aqueous solution;

[0058] Step 3. Magnetic separation and water washing 3 times (1.0 mL × 3 times), add 2.0 mL of adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst solution (0.737 mg / mL), react on a shaker at room temperature overnight, magnetic separation and water washing 3 times (1.0 mL × 3 times), and redisperse in 1.0 mL of deionized water to obtain a cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir solution, which was stored in a 4 ℃ refrigerator for later use.

[0059] 3. Structural characterization of the cyclodextrin bionic organic iridium magnetic catalyst MBs@CD@Ir of the present invention.

[0060] The present invention uses scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) to characterize the prepared catalyst MBs@CD@Ir. Figure 1 As shown in Figure 2A, the SEM spectrum of the catalyst MBs@CD@Ir shows that the catalyst MBs@CD@Ir maintains the spherical morphology of the carboxyl magnetic nanoparticles with a diameter of approximately 180.53 ± 14.2 nm (see Figure 1 The results show that MBs@CD@Ir retains the carrier structure of carboxyl magnetic nanoparticles, thus ensuring a higher specific surface area of ​​MBs@CD@Ir. Figure 2 The XPS spectrum of the prepared catalyst MBs@CD@Ir is shown in Figure 2 As shown in Figure 1, the peak at 399.71 eV in the XPS spectrum of the catalyst MBs@CD@Ir corresponds to the metal-bound nitrogen species of Ir; the peak at 400.0 eV is attributed to the amide nitrogen atom formed by the combination of the carboxyl groups on the surface of the magnetic nanoparticles and the amino-β-cyclodextrin ... 7 / 2 and Ir 4f 5 / 2 The characteristic peaks of Figure 2 The peaks observed at 198.4 eV and 199.69 eV can be attributed to the Cl 2p 3 / 2 Peak and Cl 2p 1 / 2 peak, which may be derived from Cl species combined with metallic Ir (Ir-Cl) ( Figure 2 The above results indicate that both the organic iridium catalyst and the amino-β-cyclodextrin have been successfully modified on the surface of magnetic nanoparticles.

[0061] In order to further characterize the modification of iridium complex and amino β-cyclodextrin on the surface of catalyst MBs@CD@Ir, 10.0 μL of MBs-COOH or MBs@CD or MBs@CD@Ir with a concentration of 5.0 μg / ml was added as catalyst to the Tris-HCl (10 mM, pH = 7.2) solution containing the fluorescent probe (20.0 μM) shown in the following formula and coenzyme NADH (50.0 μM), and the fluorescence response of the three materials was investigated after oscillation at 37 °C for 2.0 h.

[0062] .

[0063] like Figure 3 As shown in the figure, when MBs-COOH and MBs@CD were used as catalysts, almost no fluorescence signal was observed at 523 nm, indicating that MBs-COOH ( Figure 3 A) and MBs@CD ( Figure 3 B) has no catalytic activity against P-Rho 110. Figure 3 When organic iridium catalyst (C) was used as catalyst, obvious fluorescence intensity was observed at 523 nm, indicating that the organic iridium catalyst had been successfully modified on the surface of magnetic nanoparticles.

[0064] 4. Investigation of the catalytic hydrogenation of trimethyl-p-benzoquinone by the cyclodextrin bionic organic iridium magnetic catalyst MBs@CD@Ir of the present invention.

[0065]

[0066] Example 3: Experimental example of investigating the catalytic hydrogenation activity of the catalyst MBs@CD@Ir of the present invention on trimethyl-p-benzoquinone.

[0067] The experimental conditions are as follows: trimethyl-p-benzoquinone (3.0 mg, 0.02 mmol) and coenzyme NADH (15.0 mg, 0.02 mmol) were added to 1.0 mL phosphate buffer solution (0.1 mM, pH = 7.0) in sequence. After the mixture was shaken in a shaker for 10 minutes, an aqueous solution of the catalyst MBs@CD@Ir (20.0 μL, 1.0 mg / mL) was added. The resulting reaction solution continued to shake at 37 °C for 2.0 h, and then the reaction was tested by high performance liquid chromatography. The results showed that the product was mainly trimethyl-p-benzoquinone, with a yield of 84.6%, indicating that the prepared catalyst has high catalytic performance for catalyzing the hydrogenation of trimethyl-p-benzoquinone.

[0068] 5. The cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir of the present invention is combined with a fluorescent probe regulated by rhodamine 110-type quinone hydrogenation to construct a NADH fluorescent sensor and its response mechanism.

[0069] The cyclodextrin bionic organic iridium magnetic catalyst MBs@CD@Ir of the present invention can efficiently catalyze the reduction of trimethyl-p-benzoquinone to trimethyl-semihydroquinone in the presence of NADH; at the same time, when the trimethyl-p-benzoquinone functional group in the molecular structure of the fluorescent probe regulated by the hydrogenation of rhodamine 110 type quinone used in the present invention is hydrogenated and reduced to trimethyl-semihydroquinone, the quinone functional group in the probe molecule will fall off due to the intramolecular cyclization, thereby turning on the fluorescence of the probe molecule and generating a strong fluorescent signal; based on this, the catalyst MBs@CD@Ir provided by the present invention is combined with the fluorescent probe regulated by the hydrogenation of rhodamine 110 type quinone to construct a sensitive coenzyme NADH fluorescent sensor.

[0070] The response mechanism of the NADH fluorescence sensor constructed based on the cyclodextrin bionic organic iridium magnetic catalyst MBs@CD@Ir provided by the present invention and the fluorescence probe regulated by rhodamine 110-type quinone hydrogenation is as follows: Figure 4 As shown: In the absence of NADH, the response group (trimethyl para-benzoquinone) in the probe is in an oxidized state, and the electron transfer of the fluorophore itself is blocked, resulting in the probe itself having no fluorescence; however, in the presence of NADH, trimethyl para-benzoquinone will be rapidly reduced to the corresponding semihydroquinone by the catalyst MBs@CD@Ir, and at the same time, it will induce the spontaneous cyclization of the response group in the probe molecule and then fall off the probe molecule, causing the amide bond in the probe molecule to break, generating a new benzocyclopentanolactone, and causing the fluorophore in the probe molecule to be re-dissociated, thereby generating a strong fluorescence signal.

[0071] 6. The conditions of the fluorescence generation system of the NADH fluorescence sensor constructed by combining the cyclodextrin bionic organic iridium magnetic catalyst MBs@CD@Ir with the fluorescent probe regulated by the hydrogenation of rhodamine 110-type quinone are optimized.

[0072] Example 4: An example of optimizing the fluorescence generation conditions of the NADH fluorescence sensor constructed by combining the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir of the present invention with a fluorescence probe regulated by rhodamine 110-type quinone hydrogenation.

[0073] First, the effect of the amount of catalyst MBs@CD@Ir in the sensor fluorescence generation system on the fluorescence intensity produced by the catalytic probe hydrogenation was investigated. Different concentrations of catalyst MBs@CD@Ir (0, 10, 20, 30 μg / ml) were added to the Tris-HCl (10 mM, pH=7.2) buffer solution containing NADH (50 μM) and the fluorescent probe (20 μM), and the fluorescence test was performed after shaking at 37 °C for 2.0 h. Figure 5 As shown in Figure A, the fluorescence intensity of the system increases with the increase of the catalyst dosage, and the fluorescence intensity of the system reaches equilibrium when the catalyst concentration is 20.0 μg / ml. Therefore, the optimal MBs@CD@Ir1 dosage is 20.0 μg / ml. On this basis, the effect of reaction time on the fluorescence response of the fluorescence sensor was further investigated. Figure 5 As shown in B, the fluorescence intensity of the sensor system increases continuously with the extension of the reaction time and reaches the optimal value at 85 min. Therefore, the optimal reaction time of the sensor is selected as 85 min.

[0074] According to the above research results, the optimal conditions for the fluorescence generation system of the NADH fluorescence sensor constructed by combining the cyclodextrin bionic organic iridium magnetic catalyst MBs@CD@Ir provided by the present invention with the fluorescent probe regulated by rhodamine 110-type quinone hydrogenation are MBs@CD@Ir concentration of 20 μg / mL and reaction time of 85 min.

[0075] 7. Detection of NADH by using a NADH fluorescent sensor constructed by combining the cyclodextrin bionic organic iridium magnetic catalyst MBs@CD@Ir of the present invention with a fluorescent probe regulated by rhodamine 110-type quinone hydrogenation.

[0076] Example 5: An application example of NADH detection by constructing a NADH fluorescent sensor using the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir of the present invention in combination with a fluorescent probe regulated by rhodamine 110-type quinone hydrogenation.

[0077] In order to fully understand the detection performance of NADH by combining cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir with rhodamine 110-type quinone hydrogenation regulated fluorescent probe to construct NADH fluorescent sensor, the present invention further evaluated the detection performance of the constructed fluorescent sensor for detecting NADH standard samples under optimal conditions. Figure 6 As shown in the figure, in the range of NADH concentration from 0 to 100 μM, as the NADH concentration increases, the fluorescence intensity of the sensor gradually increases. In the range of NADH concentration from 0.39 to 12.5 μM, the fluorescence intensity and NADH concentration show a good linear relationship (y=163.2x+55.2, R 2= 0.988), with a detection limit of 7.4 nM. This indicates that the constructed fluorescent sensor has high sensitivity for the detection of NADH.

[0078] 8. The NADH fluorescence sensor constructed by combining the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir of the present invention with a fluorescent probe regulated by rhodamine 110-type quinone hydrogenation is used to detect NADH in actual samples of Escherichia coli culture fluid.

[0079] Example 6: NADH detection example in an actual sample of Escherichia coli culture fluid using a NADH fluorescent sensor constructed by combining the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir of the present invention with a fluorescent probe regulated by rhodamine 110-type quinone hydrogenation.

[0080] In order to understand the biocompatibility of the cyclodextrin biomimetic organic iridium magnetic catalyst MBs@CD@Ir provided by the present invention in biological organisms, the present invention further applied the constructed sensor to the detection of coenzyme NADH in the environment of Escherichia coli fermentation broth. Through the spike recovery experiment, different contents of NADH were added to the Escherichia coli culture broth to obtain Escherichia coli culture broths with different concentrations of NADH (0.016, 0.16, 1.6, 4, 8 mM), and 6.25 μL of Escherichia coli culture broths with different concentrations of NADH were taken and added to the fluorescence response system to obtain actual samples of Escherichia coli culture broths with different standard concentrations of NADH (0.1, 1, 10, 25, 50 μM), each group containing three groups of parallel samples, and a standard recovery test was performed. The results are shown in Table 1. The recovery rate of NADH is 96.61%~115.92%, and the relative standard deviation is 0.14%~2.67%, indicating that the fluorescent sensor has certain practicality and application prospects.

[0081] Table 1

[0082]

[0083] In summary, the catalyst provided by the present invention has the advantages of high catalytic activity and easy recovery, and can efficiently catalyze the oxidation state of the coenzyme NAD in the presence of NADH. + Reduced to reduced coenzyme NADH. In addition, the provided catalyst MBs@CD@Ir can be combined with a fluorescent probe regulated by rhodamine 110-type quinone hydrogenation to construct a sensitive NADH fluorescent sensor, which can realize the detection of NADH spiked recovery in coenzyme NADH standards and actual samples of Escherichia coli culture fluid. The constructed sensor has the advantages of controllable reaction, high sensitivity and good selectivity.

Claims

1. A cyclodextrin biomimetic organic iridium magnetic catalyst, characterized in that: The catalyst comprises three parts: magnetic nanoparticles, β-cyclodextrin and adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst; the magnetic nanoparticles are surface carboxylated magnetic nanoparticles, which are activated by N-hydroxysuccinimide and connected to the amino-modified β-cyclodextrin through an amide bond; the β-cyclodextrin is connected to the adamantane in the structure of the adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst through a host-guest coupling effect; The steps for preparing the adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst are as follows: Step 1: Compound 1, 4-p-phenylenediamine and 1-adamantane acid chloride are mixed in a molar ratio of 2:1 and dissolved in dry dichloromethane, and stirred for reaction for 1.0 to 24 hours; after the reaction is completed, the solvent is removed by pressure distillation to obtain a crude product of monoadamantane-modified p-phenylenediamine Ada-Ph-NH2 compound; the obtained crude product is further separated by silica gel column chromatography to obtain a pure compound Ada-Ph-NH2; 2-pyridinecarboxylic acid and N-hydroxysuccinimide are dissolved in dry dichloromethane or dry chloroform solvent and cooled to below zero degrees Celsius; then, the reaction solution is kept below zero degrees Celsius, and a dichloromethane solution of N, N'-dicyclohexylcarbodiimide is added dropwise, and after the addition is completed, the reaction is completed at room temperature; the precipitate is removed by filtration, the solvent is removed under reduced pressure, and vacuum drying is performed to obtain a crude product of NHS-activated 2-pyridinecarboxylic acid, which is recorded as Pic-NHS; Step 2: The compound Ada-Ph-NH2 obtained in step 1 is mixed with Pic-NHS in a molar ratio of 1:1 and dissolved in dry dichloromethane, and the mixed solution is stirred at room temperature for reaction for 10.0 to 48.0 hours; after the reaction is completed, the solvent is removed by pressure distillation to obtain a crude product of adamantane-functionalized pyridine amide ligand PicPh-Ada; the crude PicPh-Ada product is further separated by silica gel column chromatography to obtain a pure compound PicPh-Ada; Step 3: The compound PicPh-Ada obtained in step 2, dichloro(pentamethylcyclopentadienyl)iridium dimer and ammonium hexafluorophosphate are mixed and dissolved in ethanol, and refluxed for reaction for 5 to 24 hours under nitrogen protection; after the reaction is completed, the mixture is cooled, filtered, the filtrate is collected, and the solvent is removed by reduced pressure distillation to obtain a crude product of adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst, and a pure adamantane-functionalized 2-pyridinebenzamide-type organic iridium catalyst PicPh-Ada-Ir is obtained by recrystallization; wherein the molar ratio of PicPh-Ada, dichloro(pentamethylcyclopentadienyl)iridium dimer and ammonium hexafluorophosphate is (2-3):1:(4-10), and the iridium in the dichloro(pentamethylcyclopentadienyl)iridium dimer is trivalent.

2. The cyclodextrin biomimetic organic iridium magnetic catalyst according to claim 1, characterized in that: The structural formula of the cyclodextrin biomimetic organic iridium magnetic catalyst is as follows: ； Where MBs represents magnetic nanoparticles.

3. The method for preparing the cyclodextrin biomimetic organic iridium magnetic catalyst according to claim 1 or 2, characterized in that The preparation steps are as follows: Step (1): mixing magnetic nanoparticles, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, incubating, removing excess 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide by magnetic separation, washing the obtained magnetic nanoparticles to obtain N-hydroxysuccinimide-modified magnetic nanoparticles, denoted as MBs-NHS; Step (2): adding amino-β-cyclodextrin to the MBs-NHS obtained in step (1), and incubating to obtain cyclodextrin-modified carboxyl magnetic beads, denoted as MBs@CD; Step (3): After washing the MBs@CD obtained in step (2), a 2-pyridinebenzamide-type organic iridium catalyst functionalized with adamantane is added, and after incubation on a shaking table, the obtained magnetic beads are washed to obtain a cyclodextrin biomimetic organic iridium magnetic catalyst, which is recorded as MBs@CD@Ir.

4. The use of the cyclodextrin biomimetic organic iridium magnetic catalyst according to claim 1 or 2, characterized in that The cyclodextrin bionic organic iridium magnetic catalyst is combined with a fluorescent probe regulated by rhodamine 110-type quinone hydrogenation to construct an NADH fluorescent sensor; the constructed fluorescent sensor is used to detect the coenzyme NADH.

5. The use according to claim 4, characterized in that The structural formula of the fluorescent probe regulated by hydrogenation of rhodamine 110 quinone is as follows: 。 6. The use according to claim 4, characterized in that: The conditions of the fluorescence generation system of the NADH fluorescence sensor constructed by combining the cyclodextrin bionic organic iridium magnetic catalyst with the rhodamine 110 type quinone hydrogenation regulated fluorescent probe are as follows: the concentration of the cyclodextrin bionic organic iridium magnetic catalyst is 20 μg / mL and the reaction time is 85 min.

7. The use according to claim 4, characterized in that: The constructed NADH fluorescence sensor was applied to the detection of coenzyme NADH in the fermentation broth of Escherichia coli cells.

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