Acupuncture-ball-shaped multi-interface type metal oxide magnetic particle and preparation method and application thereof

Abstract

The invention discloses an acanthosphere-shaped multi-interface type metal oxide magnetic particle and a preparation method and application thereof.The magnetic particle comprises a core composed of magnetic nanoparticles and a shell layer composed of titanium oxide nanosheet spines arranged in a radial mode, and the magnetic particle has superparamagnetism, high specific surface area and rich active sites; and a large number of trap spaces matched with the exosome scale range, so that the method can be used for efficiently enriching and separating the exosome from the biological fluid. Meanwhile, the magnetic core has an out-phase junction composed of amorphous phase titanium oxide and anatase phase titanium oxide, a heterojunction interface composed of titanium oxide and the magnetic core and an ordered spine microstructure, the synergistic effect of the multi-interface effect and the point effect is fully exerted under laser irradiation, the excellent ultraviolet absorption capacity and electron-hole separation efficiency are shown, and the magnetic core has the good application prospect. The method is used for laser desorption ionization mass spectrometry, efficient in-situ pyrolysis of the exosome is achieved, desorption and ionization of metabolite in a lysis solution are promoted, and a high-resolution mass spectrum of the metabolite of the exosome is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanomaterials and biology detection, and relates to a spiky-spherical multi-interface type metal oxide magnetic microparticle, a preparation method thereof, and application thereof in detecting exosome metabolites in biological fluids. BACKGROUND

[0002] Exosomes exist in various biological fluids and play a key role in intercellular communication. Exosomes of different cell sources differ significantly in metabolite composition and content. Such differences make exosomes a very promising source of disease biomarkers, showing great prospects in early diagnosis, disease state monitoring, treatment and drug delivery. The metabolites inside the exosomes (referred to as exosome metabolites) are protected by a phospholipid bilayer, which can effectively isolate external interference and maintain good specificity. In contrast, free metabolites in biological fluids are diluted by a large amount of liquid flow after being secreted from parent cells, and are more likely to react with surrounding active substances, resulting in reduced or even lost specificity, which seriously affects the detectable time of metabolite specificity. Therefore, detecting and analyzing exosome metabolites can more accurately and efficiently understand the physiological state of their parent cells. Unfortunately, the current detection method of exosome metabolites is severely limited by the extraction efficiency of exosomes and the complexity of metabolite detection. In the traditional detection process, the extraction of exosomes needs to be completed first, and the methods mainly include high-speed differential centrifugation, density gradient centrifugation and space volume exclusion. These methods are extremely time-consuming and inefficient. After obtaining the exosomes, pre-lysis and modulation of the lysis solution are required, and then co-crystallization with an auxiliary matrix is performed, and finally LDI-MS detection is performed. The detection process is complex, and pre-lysis, lysis solution modulation and recrystallization steps may introduce errors, ultimately affecting the accuracy of the detection results. In addition, according to the literature, the stability of exosome metabolites after lysis will decrease. Therefore, it is urgent to develop a more accurate and efficient detection method for exosome metabolites to promote various analyses and applications based on exosome metabolites.

[0003] Magnetic titanium oxide composite nanomaterials have great potential in exosome extraction and metabolite detection due to their separability as magnetic materials and excellent performance of titanium oxide. The Lewis acid sites on the surface of titanium oxide can reversibly and specifically bind to the phosphate groups on the exosome membrane, thereby achieving effective extraction of exosomes. Meanwhile, titanium oxide has good photoelectric and photothermal properties and is often used as an auxiliary matrix for LDI-MS metabolite detection. However, the surface layer of the reported magnetic titanium oxide composite materials is usually smooth or flat, lacking suitable trapping spaces for capturing exosomes, and their specific surface area is limited and the active sites are insufficient, resulting in low enrichment efficiency and poor selectivity when applied to exosome extraction. In addition, the reported magnetic titanium oxide composite materials have relatively weak photoelectric and photothermal properties when applied to LDI-MS, which cannot achieve effective detection of exosome metabolites. The introduction of heterojunction and hetero-phase interface is considered a reliable strategy to improve the LDI-MS efficiency of metal oxide materials. By rationally designing binary metal oxide heterojunctions with matching band gaps and constructing hetero-phase interfaces between amorphous and crystalline phases, the ultraviolet absorption capacity and electron-hole separation efficiency of titanium oxide composite materials can be effectively improved. At the same time, by constructing a sharp structure, the sharp effect can be fully utilized to achieve the local convergence of energy carriers such as electrons, photons, and magnetic fields, thereby enhancing the local electric field, thermal field, and magnetic field strength and further improving the energy conversion efficiency. Therefore, by coupling multiple interface effects and sharp effects based on the optimization of materials, it is expected to obtain multifunctional materials that exceed the intrinsic properties of the materials, and to achieve efficient extraction of exosomes and mass spectrometry detection of exosome metabolites. SUMMARY

[0004] In view of the shortcomings of existing exosome metabolite detection methods, the present application discloses a thorned-spherical multi-interface type metal oxide magnetic microparticle particularly suitable for exosome extraction and LDI-MS detection. After treating biological fluids with this material, exosomes can be effectively extracted and exosome metabolites can be detected. Specifically, the present application includes the following technical solutions:

[0005] A thorned-spherical multi-interface type metal oxide magnetic microparticle comprising a core composed of magnetic nanoparticles and a shell layer composed of radially arranged titanium oxide nanosheet thorns.

[0006] The thorned-spherical multi-interface type metal oxide magnetic microparticle described above has a core composed of one or more of Fe3O4, γ-Fe2O3, NiFe2O4, and CuFe2O4.

[0007] In a preferred embodiment, the shell layer of the magnetic microparticle described above is composed of radially arranged titanium oxide nanosheet thorns, the roots of the thorns are staggered and connected to form abundant trapping spaces, the length of the thorns is 50-200 nm, and the size of the trapping spaces is 10-200 nm.

[0008] In a preferred embodiment, the multi-interface of the magnetic microparticle comprises a heterojunction interface composed of amorphous titanium oxide and anatase titanium oxide, and a heterojunction interface composed of titanium oxide and iron oxide; the microparticle as a whole presents a spiky ball-shaped three-dimensional structure, with a particle size of 300-550 nm; a specific surface area of 100-300 m 2 / g, and a magnetism of 20-60 emu / g.

[0009] Preferably, the titanium oxide shell layer is a metal-doped titanium oxide shell layer, the metal element being selected from one, two or three of niobium, hafnium and tantalum, and the molar ratio of the metal element to titanium element in the shell layer being 0.1-10%. The introduction of metal doping can increase the carrier concentration of the shell layer, optimize the interface band structure and enhance the light-induced electron migration ability.

[0010] In a specific embodiment, niobium doping can be used to significantly enhance the electron transport ability of the spiky ball shell layer; hafnium doping can be used to reduce the recombination of photo-induced electrons by regulating the lattice and interface defects; and tantalum doping can be used to improve the conductivity of the shell layer while maintaining the stability of the spiky ball structure.

[0011] Preferably, the shell layer can be a niobium and hafnium co-doped system, which forms a synergy through electron regulation by niobium and lattice regulation by hafnium, and increases the electron concentration and interface matching degree through co-doping.

[0012] The second aspect of the present application is to provide a method for preparing the spiky ball-shaped multi-interface type metal oxide magnetic microparticle, comprising the following steps:

[0013] (1) coating an amorphous titanium oxide shell layer on the surface of the magnetic nanoparticle by sol-gel method to obtain a composite magnetic microparticle;

[0014] (2) inducing the amorphous titanium oxide shell layer to crystallize and form a special spiky structure by hydrothermal treatment and regulating the alkalinity of the system and the reaction time to obtain a spiky ball-shaped composite magnetic microparticle;

[0015] (3) performing ion exchange reaction on the spiky ball-shaped composite magnetic microparticle obtained in step (2) to remove alkali metal ions;

[0016] (4) performing programmed temperature calcination and reducing atmosphere annealing treatment on the spiky ball-shaped composite magnetic microparticle obtained in step (3) to remove organic components and regulate the crystalline state to obtain a spiky ball-shaped multi-interface type metal oxide magnetic microparticle.

[0017] Preferably, in the step (1), the magnetic nanoparticle is prepared by solvothermal method, has uniform size, a particle size of 200-400 nm, is highly dispersed in a polar solvent and has superparamagnetism.

[0018] The sol-gel method is to coat the surface of magnetic nanoparticles with titanium source under catalyst to form amorphous titanium oxide shell. The titanium source used in the sol-gel method is selected from one or more of tetrabutyl titanate, titanium isopropoxide, and titanium isopropylate; the catalyst used is an alkaline catalyst selected from one or more of concentrated ammonia, tetramethylammonium hydroxide, sodium hydroxide, and potassium hydroxide; the mass percentage of magnetic nanoparticles in the reaction system is 5-10wt%, the mass percentage of titanium source is 0.1-0.5wt%, and the mass percentage of catalyst is 0.05-0.5wt%.

[0019] Preferably, in step (1), when coating the amorphous titanium oxide shell, the titanium source and the metal halide containing niobium, hafnium or tantalum are dissolved together in anhydrous alcohol solvent to prepare a metal alkoxide precursor solution, and the amorphous shell is introduced with metal doping in situ during formation by adding the solution dropwise into the magnetic nanoparticle dispersion system in the presence of an alkaline catalyst. The metal halide is at least one of niobium pentachloride, hafnium tetrachloride, and tantalum pentachloride.

[0020] Preferably, in step (1), niobium pentachloride, hafnium tetrachloride, or tantalum pentachloride is used with the titanium source to prepare a niobium-hafnium or niobium-tantalum co-doped metal alkoxide precursor solution.

[0021] Preferably, the amount of niobium pentachloride and hafnium tetrachloride is adjusted at a mass ratio of 1:9-9:1, and the molar ratio of metal elements in the metal alkoxide precursor solution relative to the titanium source is kept within the range of 0.1-10% to ensure stable doping amount and form a uniform metal-doped titanium oxide shell.

[0022] Preferably, in the above step (2), hydrothermal treatment is performed, the mass percentage of composite magnetic particles in the reaction system is 10-30wt%, the reaction temperature is 140-220℃, and the reaction time is 12-48 hours; the alkali agent used to control the alkalinity of the system is selected from sodium hydroxide or potassium hydroxide, and the molar concentration of hydroxyl ions is 3-6mol / L.

[0023] Preferably, in the above step (3), the ion exchange reaction is performed by first oscillating in a 0.05-0.1mol / L hydrochloric acid solution on a shaker for 1 hour, rinsing with deionized water for 3 times, and then oscillating in a 0.05-0.1mol / L ammonium nitrate solution on a shaker for 1 hour.

[0024] Preferably, in the step (4), the temperature programmed calcination is performed at a rate of 2℃ / min, 200℃ for 30min, 300℃ for 60min, 400-450℃ for 4-6h, and then decreased to room temperature at a rate of 1℃ / min; and the reduction atmosphere annealing is performed at a rate of 2℃ / min in H2 / Ar (5% / 95%) atmosphere, 400-450℃ for 8-10h, and then decreased to room temperature at a rate of 1℃ / min.

[0025] In a third aspect of the present application, the use of the above-described acantharic multi-interface metal oxide magnetic microparticles in preparing a biological detection reagent is provided.

[0026] The biological detection refers to the detection of exosome metabolites in biological fluids.

[0027] In a preferred embodiment, the biological detection reagent is used for enriching and separating exosomes from biological fluids, and then used for matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) detection of exosome metabolites.

[0028] The use of the biological detection reagent can include the following steps:

[0029] I. The biological fluid is diluted 10-30 times with physiological saline, and then filtered with a 0.22μm water-based filter membrane, and the filtrate is collected to remove cells, cell debris and other blocks in the biological fluid;

[0030] II. The filtrate is incubated with the acantharic multi-interface metal oxide magnetic microparticles for 15min, and the exosomes are enriched by oscillating the reaction at a rate of 70-150rpm on a shaking table;

[0031] III. After the incubation, magnetic separation is performed, then the physiological saline is washed, and the magnetic separation is repeated for 3 times, then the deionized water is resuspended to obtain a resuspension, and the non-specifically combined impurities are eluted;

[0032] IV. The resuspension is added dropwise to a stainless steel target plate, naturally dried, and then subjected to laser desorption ionization mass spectrometry detection and analysis to obtain the mass spectrum of the exosome metabolites in the biological fluid.

[0033] The biological fluid in the step I can be plasma, serum, urine or tears, and preferably is plasma.

[0034] The specific conditions of the laser desorption ionization mass spectrometry in step I above can be as follows: using a Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer, using a 355 nm Nd:YAG laser light source, a laser frequency of 2000 Hz, an acceleration voltage of 20 kv, a collection mode of positive ion reflector mode, and a mass-to-charge ratio range of 100-1000 Da; obtaining metabolic mass spectrum data from flexContro 13.4, and exporting the data in flexAnalysis 3.4.

[0035] The spike ball-shaped multi-interface type metal oxide magnetic microparticle developed by the application is composed of a magnetic core and a radially arranged titanium oxide nanosheet spike shell layer, has superparamagnetism, a high specific surface area, abundant active sites, and a large number of trap spaces that are adapted to the size range of exosomes, can efficiently enrich and separate exosomes, and realizes efficient extraction of exosomes. Meanwhile, the magnetic microparticle has a large number of heterojunctions composed of amorphous phase and anatase titanium oxide and heterojunction interfaces composed of titanium oxide and the magnetic core, and an ordered spike microstructure, fully plays the synergistic effect of the multi-interface effect and the tip effect under laser irradiation, exhibits excellent ultraviolet absorption capacity and electron-hole separation efficiency, can be used for laser desorption ionization mass spectrometry, realizes efficient in-situ cleavage of exosomes and promotes the analysis and ionization of metabolites in the cleavage solution, and obtains a high-resolution mass spectrum of exosome metabolites. The biological fluid exosome metabolite detection method based on the spike ball-shaped multi-interface type metal oxide magnetic microparticle eliminates the steps of exosome pre-cleavage and cleavage solution conditioning, realizes process simplification and optimization, and will exhibit great potential in various analyses and applications based on exosome metabolites. BRIEF DESCRIPTION OF DRAWINGS

[0036] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0037] Figure 1 A scanning electron microscope photograph of the spike ball-shaped multi-interface type metal oxide magnetic microparticle of Example 1 of the present application.

[0038] Figure 2 A transmission electron microscope photograph of the spike ball-shaped multi-interface type metal oxide magnetic microparticle of Example 1 of the present application.

[0039] Figure 3 A high-resolution transmission electron microscope photograph of the spike ball-shaped multi-interface type metal oxide magnetic microparticle of Example 1 of the present application.

[0040] Figure 4 A hysteresis loop of the spike ball-shaped multi-interface type metal oxide magnetic microparticle and the magnetic core of Example 1 of the present application.

[0041] Figure 5 X-ray diffraction pattern of the thorn-ball-shaped multi-interface type metal oxide magnetic microparticles of Example 1 of the present invention.

[0042] Figure 6 XPS pattern of the thorn-ball-shaped multi-interface type metal oxide magnetic microparticles of Example 1 of the present invention.

[0043] Figure 7 UV absorption spectrum of the magnetic microparticles of Example 1 and Comparative Example 1 of the present invention.

[0044] Figure 8 Photoelectric response graph of the magnetic microparticles of Example 1 and Comparative Example 1 of the present invention.

[0045] Figure 9 High-resolution electron microscope photograph of the exosomes extracted from human plasma using the thorn-ball-shaped multi-interface type metal oxide magnetic microparticles as an extraction agent of Example 2 of the present invention.

[0046] Figure 10 Particle size distribution of the exosomes extracted from human plasma using the thorn-ball-shaped multi-interface type metal oxide magnetic microparticles as an extraction agent of Example 2 of the present invention.

[0047] Figure 11 Protein blot gray scale graph of the exosomes extracted from human plasma using the thorn-ball-shaped multi-interface type metal oxide magnetic microparticles as an extraction agent of Example 2 of the present invention.

[0048] Figure 12 Metabolite spectrum graph of the exosomes obtained by performing exosome extraction and laser desorption ionization mass spectrometry using the thorn-ball-shaped multi-interface type metal oxide magnetic microparticles as an exosome extraction agent and an auxiliary matrix of Example 3 of the present invention.

[0049] Figure 13 Metabolic substance spectrum cosine similarity of the exosomes, exosome lysate, exosome lysate supernatant, and plasma of Example 4 of the present invention.

[0050] Figure 14 Scanning electron microscope photograph of the smooth fine particle single-interface type titanium dioxide magnetic microparticles of Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0051] The application prepares a spiky globular multi-interface type metal oxide magnetic microparticle, and establishes a biological fluid exosome metabolite detection method based on the material. Compared with the traditional method, the method established by the application omits the steps of exosome pre-lysis and lysis liquid conditioning, simplifies the analysis process, and realizes accurate and efficient exosome metabolite detection. The term "heterojunction" is also called nanoheterojunction, which is a nanostructure composed of different materials, and has unique electrical, optical and magnetic properties. The formation of such structure makes the electron reflect, refract and transmit at the interface, thereby obtaining a series of enhanced physical and chemical properties. For example, semiconductor heterojunction refers to the interface region formed by the contact of two different semiconductors, which can improve the photo-thermal and photoelectric performance of the interface. "Hetero-phase junction" is a new physical and chemical property obtained by combining two different crystalline states. For example, the hetero-phase junction formed by amorphous titanium oxide and anatase titanium oxide, in which the amorphous titanium oxide provides a large number of surface defects and unsaturated bonds, and the defect state can act as an electron trapping center to inhibit electron-hole recombination; and the long-range ordered anatase titanium oxide crystal structure can efficiently generate electron-hole pairs under light excitation, which is the main source of photo-generated carriers; the synergistic effect of amorphous phase and anatase phase plays a synergistic effect on the hetero-phase interface, greatly inhibits the recombination probability of electrons and holes in the anatase body, promotes the spatial separation of photo-generated charges, and then realizes the large improvement of photoelectric performance.

[0052] The application will be described in detail below by examples, and those skilled in the art should understand that the following examples are only used to illustrate the application, and are not limited to the application.

[0053] Example 1: Preparation of spiky globular multi-interface type metal oxide magnetic microparticle

[0054] (1) Preparation of ferroferric oxide magnetic nanoparticles: 1.3 g of FeCl3·6H2O was added to 40 mL of ethylene glycol and dissolved under magnetic stirring; then 0.6 g of sodium citrate dihydrate (C6H5Na3O7·2H2O) was added and dissolved under magnetic stirring; then 2.4 g of sodium acetate was added and continuously dissolved under magnetic stirring; finally, it was transferred to a polytetrafluoroethylene reaction kettle and reacted at 200℃ for 10 hours, then washed twice with deionized water and ethanol, and then dried in a vacuum oven at 40℃ for 10 hours, to obtain ferroferric oxide magnetic nanoparticles with a particle size of about 200 nm.

[0055] (2) Coating amorphous titanium oxide shell: 50 mg of the above-mentioned Fe304nanoparticles were dispersed in 50 mL of ethanol, and 0.2 mL of 37 wt% concentrated ammonia water was added under mechanical stirring at 250 rpm. After stirring for 5 min, 0.5 mL of tetrabutyl titanate was added dropwise, and the addition was completed within 10 min. After reaction in a water bath at 45 °C for 8 h, the product was washed with ethanol for 3 times, and then dried in a vacuum oven at 40 °C for 10 h to obtain the composite magnetic microparticles with a particle size of about 400 nm.

[0056] (3) Formation of thorn-like structure: 20 mg of the above-mentioned composite magnetic microparticles were dispersed in 40 mL of 3.0 mol / L NaOH aqueous solution, and the reaction was carried out in a polytetrafluoroethylene reactor at 150 °C for 24 h. The product was washed with deionized water until the pH was neutral.

[0057] (4) Removal of sodium ions by ion exchange reaction: After magnetic separation, the product was transferred to 30 mL of 0.1 mol / L hydrochloric acid solution and reacted at a low speed of 75 rpm on a shaking table for 1 h. Then, the product was washed with deionized water for 3 times, and then transferred to 30 mL of 0.1 mol / L ammonium nitrate solution and reacted at a low speed of 75 rpm on a shaking table for 1 h. The product was then separated by magnetic separation, and then dried in a vacuum oven at 40 °C for 8 h.

[0058] (5) Heat treatment for regulating crystallization state: The product was calcined at a heating rate of 2 °C / min, 200 °C for 30 min, 300 °C for 1 h, and 450 °C for 4 h, and then cooled to room temperature at a rate of 1 °C / min. Then, the product was annealed in a reducing atmosphere: H2 / Ar (5% / 95%) at a heating rate of 2 °C / min, 450 °C for 8 h, and then cooled to room temperature at a rate of 1 °C / min. Finally, the product was directly obtained from the reaction system. Figure 1 The scanning electron microscope photograph showed that the thorn-like particles had a length of about 150 nm and a trap space size of about 100 nm.

[0059] Comparative Example 1: Preparation of smooth fine particle single-interface type titanium dioxide magnetic microparticles

[0060] (1) Preparation of Fe304magnetic nanoparticles: 1.3 g of FeCl3·6H2O was added to 40 mL of ethylene glycol and dissolved under magnetic stirring. Then, 0.6 g of sodium citrate dihydrate (C6H5Na3O7·2H2O) was added and dissolved under magnetic stirring. Then, 2.4 g of sodium acetate was added and continuously dissolved under magnetic stirring. Finally, the mixture was transferred to a polytetrafluoroethylene reactor and reacted at 200 °C for 10 h. Then, the product was washed with deionized water and ethanol for two times, respectively, and then dried in a vacuum oven at 40 °C for 10 h to obtain Fe304magnetic nanoparticles with a particle size of about 200 nm.

[0061] (2) Coating amorphous titanium oxide shell: 50 mg of the Fe304nanoparticles were dispersed in 50 mL of ethanol, and 0.2 mL of 37 wt% concentrated ammonia was added under mechanical stirring at 250 rpm. After stirring for 5 min, 0.5 mL of tetrabutyl titanate was added dropwise, and the addition was completed within 10 min. The reaction was carried out in a water bath at 45 °C for 8 h, and then the product was washed with ethanol for 3 times and dried in a vacuum oven at 40 °C for 10 h to obtain the composite magnetic microparticles with a particle size of about 400 nm.

[0062] (3) Formation of smooth fine particle single interface: 20 mg of the above-mentioned composite magnetic microparticles were dispersed in 40 mL of deionized water, and the reaction was carried out in a polytetrafluoroethylene reaction kettle at 150 °C for 24 h. The product was washed with deionized water for 3 times.

[0063] (4) Ion exchange reaction: After magnetic separation, the product was transferred to 30 mL of 0.1 mol / L hydrochloric acid solution and reacted at a low speed of 75 rpm on a shaking table for 1 h, and then washed with deionized water for 3 times. After magnetic separation, the product was transferred to 30 mL of 0.1 mol / L ammonium nitrate solution and reacted at a low speed of 75 rpm on a shaking table for 1 h. The product was then magnetically separated and dried in a vacuum oven at 40 °C for 8 h.

[0064] (5) Heat treatment to regulate the crystalline state: The above-mentioned product was calcined at a heating rate of 2 °C / min, 200 °C for 30 min, 300 °C for 1 h, and 450 °C for 4 h, and then cooled to room temperature at a rate of 1 °C / min to obtain the smooth fine particle single interface type titanium dioxide magnetic microparticles. The product can be obtained from Figure 14 The scanning electron microscope photograph showed that the surface layer of the product was composed of smooth fine particles.

[0065] The absorption intensity of the spiky globular multi-interface type titanium dioxide magnetic microparticles prepared in Example 1 under 355 nm ultraviolet light irradiation was about 10% higher than that of the smooth fine particle single interface type magnetic microparticles prepared in Comparative Example 1 under the same detection conditions. The data are shown in Table 1 and Figure 7 .

[0066] Table 1

[0067] Absorbance (a.u.) Comparative Example 1 0.898 Example 1 0.993

[0068] The above ultraviolet light absorption intensity detection was carried out on a Lambda 1050+ ultraviolet / visible / near-infrared spectrophotometer (PerkinElmer, USA). The detection parameters were as follows: scanning range 200-800 nm, sampling interval 1 nm, and data mode absorbance.

[0069] The photocurrent density of the spiky, multi-interface titanium dioxide magnetic microparticles prepared in Example 1 was increased by approximately 70% compared to the smooth, fine-grained, single-interface magnetic microparticles prepared in Comparative Example 1 under the same detection conditions. Data are shown in Table 2. Figure 8 .

[0070] Table 2

[0071] Photo current density (x 10 -6 A / cm 2 )]]> Comparative Example 1 2.04 Example 1 3.41

[0072] The photocurrent density was detected using a Shanghai Chenhua CHI600F series electrochemical analyzer and a CHF-XM-500W xenon lamp. In the three-electrode system, the current response was recorded by applying periodic illumination and shading at a potential of 0.1V using the current-time curve method. The three-electrode system consisted of a working electrode made of the magnetic microparticles to be tested, a platinum mesh as the counter electrode, and an Ag / AgCl electrode as the reference electrode. The electrolyte was a 0.1M Na2SO4 solution. The alternation of 50 seconds of illumination and 50 seconds of darkness was achieved by manually shading the light source, with an interval of 0.1 seconds.

[0073] Material characterization: Scanning electron microscope images of spiky, multi-interface metal oxide magnetic microparticles, as shown below. Figure 1 Transmission electron micrographs of spiky, multi-interface metal oxide magnetic particles, such as... Figure 2 High-resolution transmission electron microscopy images of spiky, multi-interface metal oxide magnetic particles, such as... Figure 3 ; Hysteresis loops of spiky, multi-interface metal oxide magnetic particles and their magnetic cores, such as Figure 4 The X-ray diffraction pattern of the spiky, multi-interface metal oxide magnetic microparticles is shown below. Figure 5 XPS images of spiky, multi-interface metal oxide magnetic particles are shown below. Figure 6 The ultraviolet absorption spectrum of spiky, multi-interface metal oxide magnetic particles is shown below. Figure 7 The photoelectric response diagram of spiky, multi-interface metal oxide magnetic particles is shown below. Figure 8 Scanning electron microscope images of smooth, fine-grained, single-interface titanium dioxide magnetic microparticles are shown below. Figure 14 .

[0074] Example 2: Extraction of exosomes from human plasma using spiky, multi-interface metal oxide magnetic microparticles as an extractant.

[0075] (1) After thawing the plasma on ice after removing it from the -80℃ freezer, take 30 μL into a 1.5 mL centrifuge tube and dilute it 30 times with 1X phosphate-buffered saline (PBS) to 900 μL. Gently pipette the solution until homogeneous. The PBS was filtered through a 0.22 μm aqueous filter membrane before use to remove impurities.

[0076] (2) The above dilution liquid is filtered with a 0.22 μm water filter membrane, and the filtrate is collected. Cells, cell debris and other blocks in the biological fluid are removed.

[0077] (3) 500 μL of the above filtrate is added to a PBS dispersion liquid containing 500 μL of the magnetic microparticles of the spiky spherical multi-interface metal oxide with a concentration of 10 mg / mL, and incubated for 15 min on a shaker at a speed of 150 rpm.

[0078] (4) After magnetic separation of the above suspension, the suspension is washed with PBS for 3 times, then 10 wt% ammonia water is added and shaken for 5 min at 4°C, and the enriched exosomes are eluted, and then the exosome suspension is obtained by magnetic separation.

[0079] (5) The above suspension is quickly added to a 1.5 mL ultrafiltration tube (Millipore, 100 KD membrane), centrifuged at 4°C at 10000 x g for 3 min, the filtrate is discarded and resuspended with PBS, and then liquid phase replacement is repeated with PBS for 3 times, finally resuspended with PBS to obtain an exosome dispersion liquid.

[0080] The extracted exosomes are characterized in time, and the results are shown in the corresponding figures. The high-resolution electron microscope photo of the exosomes extracted from human plasma using the spiky spherical multi-interface metal oxide magnetic microparticles as the extraction agent is as shown in Figure 9 ; the particle size distribution of the exosomes extracted from human plasma using the spiky spherical multi-interface metal oxide magnetic microparticles as the extraction agent is as shown in Figure 10 ; and the protein blot gray scale diagram of the exosomes extracted from human plasma using the spiky spherical multi-interface metal oxide magnetic microparticles as the extraction agent is as shown in Figure 11 .

[0081] Example 3: Detection of plasma exosome metabolites

[0082] The spiky spherical multi-interface metal oxide magnetic microparticles are used as the exosome extraction agent and auxiliary matrix at the same time, and the plasma is subjected to exosome extraction and exosome metabolite detection.

[0083] (1) The plasma is taken out from the -80°C refrigerator, melted on the ice surface, and then taken into a 30 μL to 1.5 mL centrifuge tube, diluted with 1X physiological saline by 30 times to 900 μL, and uniformly blown with a gun head. The physiological saline is filtered with a 0.22 μm water filter membrane before use.

[0084] (2) The above dilution liquid is filtered with a 0.22 μm water filter membrane, and the filtrate is collected. Cells, cell debris and other blocks in the biological fluid are removed.

[0085] (3) Take 500 μL of the above filtrate and add it to a physiological saline dispersion containing 500 μL of the above-mentioned spiky-spherical multi-interface metal oxide magnetic microparticles with a concentration of 10 mg / mL. Shake and incubate for 15 min at a speed of 150 rpm on a shaker to enrich the exosomes.

[0086] (4) After magnetic separation of the above-mentioned suspension, rinse with deionized water for 3 times, then quantitatively add deionized water to 5 mg / mL to obtain a dispersion of spiky-spherical multi-interface metal oxide magnetic microparticles enriched with exosomes.

[0087] (5) Take 1 μL of the above-mentioned dispersion and drop it onto a stainless steel target plate. After natural drying, perform laser desorption ionization mass spectrometry detection. Use a Bruker UltrafleXtreme MALDI-TOF mass spectrometer, use a 355 nm Nd:YAG laser light source, the laser frequency is 2000 Hz, the acceleration voltage is 20 kv. The acquisition mode is the positive ion reflector mode, and the acquisition mass-to-charge ratio range is 100-1000 Da. Obtain the mass spectrometry data from flexControl3.4, and export the data in flexAnalysis3.4 to obtain the exosome metabolite mass spectrum.

[0088] The exosome metabolite spectrum obtained by using the spiky-spherical multi-interface metal oxide magnetic microparticles as the exosome extraction agent and auxiliary matrix for exosome extraction and laser desorption ionization mass spectrometry detection is as follows Figure 12 .

[0089] Example 4: In-situ exosome lysis and exosome metabolite detection by spiky-spherical multi-interface metal oxide magnetic microparticles in laser desorption ionization mass spectrometry detection

[0090] To verify the in-situ lysis ability, this example uses spiky-spherical multi-interface metal oxide magnetic microparticles as auxiliary matrix to detect metabolites from ① exosomes extracted from plasma, ② exosome lysis solution, ③ supernatant of exosome lysis solution, and ④ diluted plasma, respectively. Then calculate and evaluate the similarity of the metabolite mass spectra of the four different samples using cosine similarity.

[0091] (1) Take 5 mg of the spiky-spherical multi-interface metal oxide magnetic microparticles prepared in Example 1, disperse them in 1 mL of deionized water, and mix them uniformly at room temperature by ultrasonic agitation to obtain a uniformly dispersed spiky-spherical multi-interface metal oxide magnetic microparticle suspension.

[0092] (2) Add the spiky-spherical multi-interface metal oxide magnetic microparticle suspension obtained in step (1) to a stainless steel target plate, and after natural drying, drop 1 μL of exosomes (①) with an appropriate concentration onto the corresponding stainless steel target point. After natural drying of the sample point, perform laser desorption ionization mass spectrometry detection.

[0093] (3) The spiky-spherical multi-interface metal oxide magnetic microparticle suspension obtained in step (1) is dropped onto a stainless steel target plate, and after it is naturally dried, 1 μL of an exosome lysate (②) of an appropriate concentration is dropped onto the corresponding stainless steel target point, and after the sample point is naturally dried, laser desorption ionization mass spectrometry detection is performed.

[0094] (4) The spiky-spherical multi-interface metal oxide magnetic microparticle suspension obtained in step (1) is dropped onto a stainless steel target plate, and after it is naturally dried, 1 μL of supernatant of an exosome lysate (③) of an appropriate concentration is dropped onto the corresponding stainless steel target point, and after the sample point is naturally dried, laser desorption ionization mass spectrometry detection is performed.

[0095] (5) The spiky-spherical multi-interface metal oxide magnetic microparticle suspension obtained in step (1) is dropped onto a stainless steel target plate, and after it is naturally dried, 1 μL of diluted plasma (④) of an appropriate concentration is dropped onto the corresponding stainless steel target point, and after the sample point is naturally dried, laser desorption ionization mass spectrometry detection is performed.

[0096] The laser desorption ionization mass spectrometry detection in the above step (2), step (3), step (4), and step (5) is as follows: a Bruker UltrafleXtreme MALDI-TOF mass spectrometer is used, a 355 nm Nd:YAG laser light source is adopted, the laser frequency is 2000 Hz, the acceleration voltage is 20 kv. The collection mode is a positive ion reflector mode, and the mass-to-charge ratio range collected is 100-1000 Da. Mass spectrometry data is obtained from flexControl3.4, and the data is exported in flexAnalysis3.4 to obtain the metabolite mass spectrum of the corresponding sample.

[0097] The exosome (①) in the above step (2) is an exosome extracted from plasma in Example 2;

[0098] The exosome lysate (②) in the above step (3) is a lysate obtained by resuspending in deionized water after ultrasonic crushing and repeated freezing and thawing of the same amount of exosomes in step (2). The ultrasonic crushing and repeated freezing and thawing process is as follows: the same volume of methanol is added to the same amount of exosome suspension, ultrasonic crushing is performed at 4°C for 1 min, then strong freezing is performed under liquid nitrogen for 1 min, and thawing is performed on the ice surface for 2 min; then ultrasonic crushing is performed at 4°C for 1 min, strong freezing is performed under liquid nitrogen for 1 min, and thawing is performed on the ice surface for 2 min; the above ultrasonic crushing, strong freezing, and thawing steps are repeated 5 times, and the obtained product is resuspended in deionized water to the initial volume after freeze-drying to obtain the exosome lysate.

[0099] The supernatant (3) of the exosome lysate in the above step (4) is obtained by centrifuging the same amount of exosomes at 4°C, 13000xg for 10 min after the same method as step (3) is used to obtain the exosome lysate.

[0100] The diluted plasma (4) in the above step (5) is an equal amount of the same plasma as in Example 1. After diluting with deionized water by 30 times, filtering with a 0.22 μm water filter membrane, the diluted plasma is obtained.

[0101] The cosine similarity calculation formula is:

[0102]

[0103] In the formula, Y represents the normalized mass spectrum peak intensity appearing in the mass spectrum of sample A or in the mass spectrum of sample B; y represents the normalized mass spectrum peak intensity appearing in the mass spectrum of sample A and in the mass spectrum of sample B; N A and N B respectively represent the total number of normalized mass spectrum peaks of sample A and sample B; N AB represents the total number of normalized mass spectrum peaks appearing in sample A and sample B.

[0104] The cosine similarity value of the metabolite mass spectrum of the exosome, the exosome lysate, the supernatant of the exosome lysate and the diluted plasma is as follows Figure 13 . Among the metabolite mass spectra of the four samples, the cosine similarity value between the metabolite mass spectrum of the exosome and the metabolite mass spectrum of the lysate is as high as 0.978, reflecting that the metabolite compositions of the two samples are highly consistent. It is verified from the side that the spinous spherical multi-interface type metal oxide magnetic microparticles prepared by the application have the ability to efficiently lyse exosomes in situ. At the same time, the similarity value between the metabolite mass spectrum of the supernatant of the lysate and the metabolite mass spectrum of the lysate is 0.930, indicating that most of the metabolites in the lysate have been successfully extracted into the supernatant and successfully detected. However, the similarity between the metabolite mass spectrum of the exosome and the metabolite mass spectrum of the supernatant of the lysate is slightly reduced (0.888), suggesting that there is a certain difference between the two. From the analysis of the detection process, it is speculated that the multi-step sample preparation from the exosome to the supernatant of the lysate introduces errors. Therefore, the material prepared by the application is used for detecting the metabolites of the exosomes in the biological fluid, and the steps of pre-lysis of the exosomes and conditioning of the lysate are omitted in the process, simplifying the analysis process, and at the same time, more accurate exosome metabolite spectrum can be obtained.

[0105] Example 5

[0106] The same as example 1, the difference is only in step (2): 0.5 mL of tetrabutyl titanate and 0.01 g of niobium pentachloride are mixed and dissolved in 5 mL of anhydrous ethanol to obtain a metal alkoxide precursor solution. 50 mg of Fe3O4 nanoparticles are dispersed in 50 mL of ethanol, 0.2 mL of 37 wt% concentrated ammonia is added under mechanical stirring at 250 rpm, and after stirring for 5 min, the above metal alkoxide precursor solution is added dropwise into the dispersion system under stirring within 10 min, and the reaction is continued in a 45°C water bath for 8 h. After the reaction is completed, it is washed with ethanol for 3 times, and then dried in a vacuum oven at 40°C for 10 h to obtain composite magnetic microparticles with a particle size of about 400 nm.

[0107] Example 6

[0108] The same as example 1, the difference is only in step (2): 0.5 mL of tetrabutyl titanate and 0.01 g of hafnium tetrachloride are mixed and dissolved in 5 mL of anhydrous ethanol to obtain a metal alkoxide precursor solution. 50 mg of Fe3O4 nanoparticles are dispersed in 50 mL of ethanol, 0.2 mL of 37 wt% concentrated ammonia is added under mechanical stirring at 250 rpm, and after stirring for 5 min, the above metal alkoxide precursor solution is added dropwise into the dispersion system under stirring within 10 min, and the reaction is continued in a 45°C water bath for 8 h. After the reaction is completed, it is washed with ethanol for 3 times, and then dried in a vacuum oven at 40°C for 10 h to obtain composite magnetic microparticles with a particle size of about 400 nm.

[0109] Example 7

[0110] The same as example 1, the difference is only in step (2): 0.5 mL of tetrabutyl titanate and 0.01 g of hafnium tetrachloride are mixed and dissolved in 5 mL of anhydrous ethanol to obtain a metal alkoxide precursor solution. 50 mg of Fe3O4 nanoparticles are dispersed in 50 mL of ethanol, 0.2 mL of 37 wt% concentrated ammonia is added under mechanical stirring at 250 rpm, and after stirring for 5 min, the above metal alkoxide precursor solution is added dropwise into the dispersion system under stirring within 10 min, and the reaction is continued in a 45°C water bath for 8 h. After the reaction is completed, it is washed with ethanol for 3 times, and then dried in a vacuum oven at 40°C for 10 h to obtain composite magnetic microparticles with a particle size of about 400 nm.

[0111] Example 8

[0112] The same as example 1, the difference is only in step (2): 0.5 mL of tetrabutyl titanate is mixed with 0.005 g of niobium pentachloride and 0.005 g of hafnium tetrachloride to be dissolved in 5 mL of anhydrous ethanol, and a metal alkoxide precursor solution is obtained by stirring. 50 mg of Fe3O4 nanoparticles are dispersed in 50 mL of ethanol, 0.2 mL of 37 wt% concentrated ammonia is added under mechanical stirring at 250 rpm, and after stirring for 5 min, the above metal alkoxide precursor solution is added dropwise into the dispersion system under stirring within 10 min, and the reaction is continued in a 45°C water bath for 8 h. After the reaction is completed, the product is washed with ethanol for 3 times, and then dried in a vacuum oven at 40°C for 10 h to obtain composite magnetic microparticles with a particle size of about 400 nm.

[0113] Example 9

[0114] The same as example 1, the difference is only in step (2): 0.5 mL of tetrabutyl titanate is mixed with 0.005 g of niobium pentachloride and 0.005 g of hafnium tetrachloride to be dissolved in 5 mL of anhydrous ethanol, and a metal alkoxide precursor solution is obtained by stirring. 50 mg of Fe3O4 nanoparticles are dispersed in 50 mL of ethanol, 0.2 mL of 37 wt% concentrated ammonia is added under mechanical stirring at 250 rpm, and after stirring for 5 min, the above metal alkoxide precursor solution is added dropwise into the dispersion system under stirring within 10 min, and the reaction is continued in a 45°C water bath for 8 h. After the reaction is completed, the product is washed with ethanol for 3 times, and then dried in a vacuum oven at 40°C for 10 h to obtain composite magnetic microparticles with a particle size of about 400 nm.

[0115] The test data of examples 5-9 are shown in table 3.

[0116] Table 3

[0117] Absorbance (a.u.) Photo current density (x 10 -6 A / cm 2 )]]> Example 5 0.998 3.95 Example 6 0.997 3.83 Example 7 0.995 3.62 Example 8 0.999 4.24 Example 9 0.996 3.73

[0118] In example 5, niobium doping is introduced in the shell formation stage, which significantly improves the conductivity and electron concentration of the titanium dioxide shell, and the electron migration along the multi-interface of the thorn ball to the magnetic core is more smooth. The overall performance is that the light absorption capacity is slightly enhanced, and the photocurrent output is relatively obviously improved compared with example 1.

[0119] In example 6, hafnium doping is adopted, which optimizes the shell lattice and interface defects to a certain extent, which is beneficial to reduce the formation of deep recombination centers. As a result, the photocurrent density is steadily improved under the premise of maintaining good light absorption capacity, and the performance is slightly lower than that of niobium doping but still significantly better than that of the undoped system.

[0120] The introduction of tantalum doping in the titanium dioxide shell layer in Example 7 can improve the carrier concentration, but the adjustment effect on the lattice structure and interface state is relatively limited. Compared with Example 1, the light absorption and photocurrent are improved, but the overall improvement is not as good as the niobium doping and hafnium doping cases.

[0121] Example 8 realizes the combined effect of conductivity enhancement and lattice and interface optimization in the spiky ball multi-interface shell layer by simultaneously introducing niobium and hafnium co-doping, making the electron migration path more coherent and the recombination probability further reduced.

[0122] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A spiky, multi-interface metal oxide magnetic microparticle comprising a core composed of magnetic nanoparticles and a shell composed of radially arranged titanium oxide nanosheet spikes.

2. The spiky, multi-interface metal oxide magnetic microparticles as described in claim 1, characterized in that, The core of the magnetic particles is one or more of Fe3O4, γ-Fe2O3, NiFe2O4, and CuFe2O4.

3. The spiky, multi-interface metal oxide magnetic microparticles as described in claim 1, characterized in that, The shell of the magnetic microparticles is composed of radially arranged titanium dioxide nanofibers, with the roots of the fibers interlocking to form abundant trap spaces. The length of the fibers is 50-200 nm, and the size of the trap spaces is 10-200 nm.

4. The spiky, multi-interface metal oxide magnetic microparticles as described in claim 1, characterized in that, The multi-interface includes a heterojunction interface composed of amorphous titanium oxide and anatase titanium oxide, and a heterojunction interface composed of titanium oxide and a magnetic core; the magnetic particles exhibit a spiky three-dimensional structure with a particle size of 300-550 nm and a specific surface area of ​​100-300 m². 2 / g, with a magnetic strength of 20-60 emu / g.

5. A method for preparing spiky, multi-interface metal oxide magnetic microparticles as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Using magnetic nanoparticles as seeds, an amorphous titanium dioxide shell of a specific thickness is coated on the surface of the magnetic nanoparticles by the sol-gel method to obtain composite magnetic microparticles; (2) By hydrothermal treatment and by controlling the alkalinity and reaction time of the system, the amorphous titanium oxide shell is induced to crystallize and form a special spiky structure, resulting in spiky spherical composite magnetic microparticles; (3) The spiky composite magnetic microparticles obtained in step (2) are subjected to ion exchange reaction to remove alkali metal ions; (4) The spiky composite magnetic microparticles obtained in step (3) are subjected to programmed heating calcination and reducing atmosphere annealing to remove organic components and regulate the crystallization state, thereby obtaining spiky multi-interface metal oxide magnetic microparticles.

6. The method for preparing spiky, multi-interface metal oxide magnetic microparticles as described in claim 5, characterized in that, In step (1), the magnetic nanoparticles are prepared by a solvothermal method, and have uniform size with a particle size of 200-400 nm.

7. The method for preparing spiky, multi-interface metal oxide magnetic microparticles as described in claim 5, characterized in that, In step (1), the titanium source used in the sol-gel method is selected from one or more of tetrabutyl titanate, titanium isopropoxide, and tetraisopropyl titanate; the catalyst used is an alkaline catalyst selected from one or more of concentrated ammonia, tetramethylammonium hydroxide, sodium hydroxide, and potassium hydroxide; the mass percentage of magnetic nanoparticles in the reaction system is 5-10 wt%, the mass percentage of titanium source is 0.1-0.5 wt%, and the mass percentage of catalyst is 0.05-0.5 wt%.

8. The method for preparing spiky, multi-interface metal oxide magnetic microparticles as described in claim 5, characterized in that, In step (2), hydrothermal treatment is performed. The mass percentage of composite magnetic particles in the reaction system is 10-30 wt%, the reaction temperature is 140-220℃, and the reaction time is 12-48 hours. The alkaline agent used to regulate the alkalinity of the system is selected from sodium hydroxide or potassium hydroxide, and the molar concentration is 3-6 mol / L based on hydroxide ions.

9. The use of the spiky, multi-interface metal oxide magnetic microparticles as described in any one of claims 1-4 in the preparation of biological diagnostic reagents.

10. The use of the spiky, multi-interface metal oxide magnetic microparticles as described in claim 9 in the preparation of biological detection reagents, characterized in that, The biological detection reagent is used to enrich and separate exosomes from biological fluids, and then to detect exosome metabolites by laser desorption / ionization mass spectrometry.

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