CN114246981A - Magnetic nano material for very early diagnosis of related infection of orthopedic implant and preparation method thereof - Google Patents
Magnetic nano material for very early diagnosis of related infection of orthopedic implant and preparation method thereof Download PDFInfo
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- CN114246981A CN114246981A CN202111429899.5A CN202111429899A CN114246981A CN 114246981 A CN114246981 A CN 114246981A CN 202111429899 A CN202111429899 A CN 202111429899A CN 114246981 A CN114246981 A CN 114246981A
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Abstract
The invention discloses a magnetic nano material for very early diagnosis of related infection of orthopedic implants and a preparation method thereof. The invention synthesizes Fe with the surface modified with bone targeting peptide and bacterial targeting peptide3O4@SiO2Magnetic nanomaterials which can be used for very early diagnosis of orthopaedic implant related infections. In vivo and in vitro experiments prove that the magnetic nano material has bone targeting and bacteria targeting functions, can be enriched at infected parts, has good targeting capability on bone tissues and bacteria in vitro, and can be used in orthopedicsHas good very early diagnosis effect in an animal model of the implant-related infection.
Description
Technical Field
The invention relates to a magnetic nano material for very early diagnosis of related infection of orthopedic implants and a preparation method thereof, belonging to the technical field of medical diagnosis.
Background
Orthopedic implants, once implanted in the body, are at risk of developing orthopedic implant related infections, which is also one of the major reasons for joint replacement failure. The reported incidence rate is 1-2% (initial hip and knee replacement), and the recurrence rate of infection of secondary revision surgery is different from 3-25%. Although the incidence of related infection of orthopedic implants is not high, once the infection occurs, the infection is often a catastrophic disease, the implant is often ineffective, and the infection can be cured only by the combination operation of systemic antibiotics, thereby bringing huge physical and psychological burden to doctors and patients. And periprosthetic infection and aseptic loosening are often difficult to distinguish, making diagnosis further difficult, thereby impacting clinical decision-making.
With the wide development of joint replacement in China, the related infection of the bone implant becomes a clinical problem which needs to be solved urgently, and how to quickly and accurately diagnose the related infection of the implant also becomes a hot spot of the current research at home and abroad. To date, there is no effective method for early and specific diagnosis of implant-related infection in clinic, and first, biochemical examination, synovial fluid examination and imaging examination used in clinic at present cannot be used for early diagnosis, so that treatment is delayed; secondly, the infection focus cannot be positioned, so that the treatment is blindly, because the precise positioning of the infection focus around the infection is helpful for the selection of a surgical scheme, if the infection is only limited to soft tissues around the joint, debridement may be effective, but once the infection occurs at a bone interface and even in a marrow cavity, joint replacement surgery is necessary; furthermore, the related infection of the orthopedic implant is often accompanied by bacterial biofilm formation, and once the biofilm is formed, the antibiotic resistance is generated, so that the infection of the bone tissue is not healed. Therefore, the research of a means capable of diagnosing the related infection of the orthopedic implant very early and specifically is vital to the intervention of the infection and the subsequent treatment, can avoid the further deterioration of the related infection of the orthopedic implant, provides a new strategy for the diagnosis and treatment of the related infection of the orthopedic implant, and has important clinical significance.
The magnetic particles currently applied to the biomedical field are ferroferric oxide nano particles Fe with silicon dioxide modified on the surface3O4@SiO2The magnetic response capability of ferroferric oxide is integratedAnd the characteristics of large specific surface area, controllable particle size and easy surface modification of the silicon dioxide are in single particles, so that the silicon dioxide has great potential in the application fields of imaging, diagnosis, target molecule loading, imaging agents, targeted drug delivery and the like. The magnetic iron oxide nanoparticles have excellent superparamagnetism, can shorten T2 relaxation time, reduce signals in T2WI, have the advantages of high clearance rate in vivo, good biocompatibility and stability and the like, and are widely used in T2WI, and meanwhile, the magnetic iron oxide nanoparticles are approved by the US FDA to be used as a unique MRI nano contrast agent. When the core is ferroferric oxide with superparamagnetism, magnetism can be further endowed to the material for T2WI, and the existence of the mesoporous silica outer layer can improve the stability and biocompatibility of the magnetic material.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the current conventional detection methods do not allow for early, specific diagnosis of orthopedic implant related infections.
In order to solve the technical problems, the invention provides a magnetic nano material for very early diagnosis of related infection of orthopedic implants, wherein the magnetic nano material is Fe the surface of which is modified with bone targeting peptide and bacterial targeting peptide3O4@SiO2Magnetic nanomaterials.
Preferably, the bone targeting peptide is aspartic acid hexapeptide, and the amino acid sequence is: DDDDDD.
Preferably, the bacterial targeting peptide is UBI29-41The amino acid sequence is as follows: TGRAKRRMQYNRR are provided.
Preferably, the mass ratio of the bone targeting peptide to the bacterial targeting peptide is 1: 1.
The invention also provides a preparation method of the magnetic nano material for the very early diagnosis of the related infection of the orthopedic implant, which comprises the following steps: fe in core-shell structure3O4@SiO2Magnetic nano particles are used as raw materials, and then amino modification is carried out to obtain Fe3O4@SiO2-NH2Then respectively adding bone targeting peptide nanoparticles containing carboxyl and bacterial targeting peptide nanoparticlesAnd performing condensation reaction by adopting an EDC/NHS coupling method to form an amido bond, thereby preparing the magnetic nano material.
Preferably, said Fe3O4@SiO2The magnetic nano-particles are magnetic Fe modified by oleic acid3O4The nanocrystalline is used as a core and is obtained after a silicon dioxide layer is prepared on the surface of the nanocrystalline.
Preferably, the amino modification is amination by using 3-aminopropyltriethoxysilane to obtain Fe3O4@SiO2-NH2。
Preferably, the bone targeting peptide nanoparticle containing carboxyl is an amphiphilic polymer modified with carboxyl and bone targeting peptide; the bacterial targeting peptide nano particle containing carboxyl is an amphiphilic polymer modified with carboxyl and bacterial targeting peptide.
Preferably, the amphiphilic polymer is polyethylene glycol, and the molecular weight of the polyethylene glycol is 3000-5000 g/mol.
Preferably, the bone targeting peptide nanoparticle containing carboxyl is COOH-PEG-D6(ii) a The bacterial targeting peptide nano particle containing carboxyl is COOH-PEG-UBI.
The invention also provides an application of the magnetic nano material in preparation of a reagent and/or a kit for very early diagnosis of related infection of orthopedic implants, and the magnetic nano material is the magnetic nano material for very early diagnosis of related infection of orthopedic implants in the technical scheme.
The technical principle of the invention is as follows:
the invention relates to magnetic Fe modified by oleic acid3O4The nanocrystalline (about 20 nm) is used as an inner core, and a layer of compact silicon oxide is coated on the surface of the inner core to obtain the core-shell structured nano composite material (Fe)3O4@SiO2) Introduction of APTES to Fe by condensation reaction3O4@SiO2To obtain Fe with amino modification3O4@SiO2-NH2. Then modifying the surface with carboxyl functional groups by using an EDC/NHS coupling method and simultaneously modifying the modified bandBone-targeted, bacteria-targeted polyethylene glycol through Fe3O4@SiO2-NH2Condensation of amino groups on the surface of the material to form amide bonds, i.e. bone targeting on the surface (D)6) And bacterial targeting (UBI)29-41) Modification to confer bone targeting (bone targeting head D)6) Targeting to bacteria (bacterial targeting target UBI)29-41) Function to enable it to concentrate at the site of infection.
Compared with the prior art, the invention has the beneficial effects that:
the invention shows through relevant in vivo and in vitro experiments that the synthesized magnetic nano material has good bone targeting and bacteria targeting performances in vitro; the magnetic nano material can specifically position and realize the early diagnosis of infection in animal orthopedic implant related infected animal models, and makes up the defects of a clinical detection method.
Drawings
FIG. 1 is Fe3O4And Fe3O4@D&Transmission electron microscopy of U;
FIG. 2 is Fe3O4And Fe3O4@D&The cytotoxicity detection result of U;
FIG. 3 is Fe3O4And Fe3O4@D&The hemolytic activity detection result of U;
FIG. 4 is Fe3O4And Fe3O4@D&The in vitro bone targeting performance test result of U; wherein a is fluorescently labeled Fe3O4And Fe3O4@D&U binding capacity to Hydroxyapatite (HAP); b is for a (fluorescent-labeled Fe)3O4And Fe3O4@D&U binding capacity for Hydroxyapatite (HAP);
FIG. 5 is Fe3O4And Fe3O4@D&The result of the in vitro bacterial targeting performance test of U; wherein a is the detection of fluorescently labeled Fe by flow cytometry3O4And Fe3O4@D&U ability to target bacteria; b is a statistical analysis of a;
FIG. 6 is Fe3O4And Fe3O4@D&Results of in vitro nuclear magnetic resonance of U; wherein a is Fe of different concentrations3O4And Fe3O4@D&U T2 weighted MRI image and its pseudocolor map, b is Fe3O4And Fe3O4@D&T2 relaxation rate of U at different concentrations;
FIG. 7 is Fe3O4And Fe3O4@D&U's in vivo nuclear magnetic resonance localization diagnosis of orthopedic implant-related infection results; wherein a is Fe3O4And Fe3O4@D&T2-weighted MRI images of U-tail vein injection SD rats and their pseudo-color maps, b is the signal intensity of T2-value of ROI of left femur of infected rat, c is Fe concentration in tissues from rat including femur, heart, kidney, heart, liver, spleen, lung and kidney.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.
Bone targeting peptide nanoparticles COOH-PEG-D used in the following examples6Purchased from gill biochemical (shanghai) ltd, cat #: 766170, bacterial targeting peptide nanoparticles COOH-PEG-UBI was purchased from Gill Biochemical (Shanghai) Co., Ltd., Cat No.: 659445.
example 1
Preparing a magnetic nano material:
(1) synthesis of a precursor: fe3O4The precursor (iron-oleate complex) of (a) is prepared mainly by reacting a metal chloride with sodium oleate to prepare a metal oleate complex. 10.8g of ferric chloride (FeCl) was weighed into a three-necked flask3·6H2O) and 36.5g of sodium oleate were dissolved in a mixed solvent composed of 80mL of ethanol, 60mL of distilled water and 140mL of n-hexane. The resulting solution was heated to 70 ℃ and held at that temperature for 4 hours. After the reaction was completed, 30mL of distilled water was equally divided into 3 parts, and the upper organic layer was washed 3 times with a solution containing an iron oleate complex in a separatory funnel. After washing, the mixture is put in a vacuum ovenDried at 80 ℃ overnight to evaporate the n-hexane to give iron oleate complex as a waxy solid.
(2)Fe3O4And (3) synthesis of nanocrystals: 9 g of iron oleate complex, 2.13g of oleic acid, were dissolved in 50mL of 1-octadecene in a three-necked flask capable of withstanding temperatures above 300 ℃ at room temperature. The reaction mixture was heated constantly to 50 ℃ under vacuum in a heating mantle for 20min, then the temperature was raised to 90 ℃ and held constant at this temperature for 20min, after which it was heated again to 120 ℃ and held at this temperature for 30min, nitrogen was passed in and the vacuum pump was then switched off. When the reaction temperature reaches 320 ℃, violent reaction occurs, the initial transparent solution becomes turbid and brownish black, the temperature is kept constant for 30min, and whether the bumping phenomenon occurs or not is observed at any time. The resulting solution containing the nanocrystals was then cooled to room temperature under stirring, and n-hexane, ethanol and isopropanol were sequentially added to the solution to precipitate the nanocrystals. Finally, the nanocrystals were centrifuged at 3000 rpm.
(3)Fe3O4@SiO2Synthesis of core-Shell Structure-Collection of the synthesized Fe3O4The nanocrystals were added to 60mL of water followed by sonication for 30min with 6g CTAB and 0.18g triethanolamine with gentle stirring for 2 h. 2mL of TEOS was dispersed in cyclohexane at 10 wt% to form a homogeneous mixed solution, and added to the upper solution at 5rpm with a peristaltic pump and mechanically stirred at room temperature for 48 h. The obtained nano-particle Fe3O4@SiO2Washed 3 times with ethanol and water each.
(4)Fe3O4@SiO2Amination of nanoparticle surface 1g Fe3O4@SiO2Adding into 100mL toluene, stirring, adding 1mL APTES (3-aminopropyltriethoxysilane, C) after 5min9H23NO3Si) is refluxed for 20 hours at the temperature of 80 ℃, and then is centrifuged and washed by absolute ethyl alcohol at the rotating speed of 11000rpm, and Fe is obtained after freeze drying for 36 hours3O4@SiO2-NH2And (3) powder.
(5) And (3) carboxyl activation: 9.6mg of EDC (N-hydroxysuccinimide, C) were weighed out4H5NO3) 5.8 mg of NHS (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, C8H18ClN3) And 0.5mg of COOH-PEG-D6(D6Aspartic acid hexapeptide, DDDDDD), 0.5mg COOH-PEG-UBI (UBI is bacterial targeting peptide UBI)29-41TGRAKRRMQYNRR) was added with PBS (0.2M, pH 6.1) and stirred at ordinary temperature for 0.5 hours to activate the carboxyl group.
(6) Amidation reaction: taking 100mg of Fe3O4@SiO2-NH2The powder was mixed with 40mL of PBS (0.2M, pH 7.4) and the above solution was added, the pH was raised to 7 or more, and the mixture was stirred at room temperature for 12 hours. Repeatedly centrifuging and washing with distilled water for many times at the rotating speed of 11000rpm to obtain the bone-targeted and bacteria-targeted magnetic nano material Fe3O4@SiO2-PEG-D6&UBI, abbreviated as Fe3O4@D&U。
All the above materials were freeze-dried to give powders which were stored at 4 ℃.
The magnetic nano material Fe prepared by the method3O4@D&The transmission electron micrograph of U is shown in FIG. 1, and it can be seen from FIG. 1 that the magnetic nanomaterial Fe3O4@D&The U has uniform size and good dispersion, and the particle size range is 50 +/-2 nm.
The magnetic nano material Fe prepared by the method3O4@D&U is used for carrying out cytotoxicity detection, hemolytic activity detection, in vitro bone targeting performance detection, in vitro bacteria targeting performance detection, in vitro nuclear magnetic resonance detection and in vivo nuclear magnetic resonance positioning diagnosis on related infection of the orthopedic implant respectively, as shown in examples 2-7.
Example 2
Fe3O4And Fe3O4@D&Cytotoxicity assay of U:
bone marrow mesenchymal stem cells (rBMSC) collected from Rat tibia and femur were suspended in a minimal essential medium containing 10% fetal bovine serum and cultured in 5% CO2And cultured in a humidified incubator with 95% air at 37 ℃. Every thirdGrowth media was changed day by day and third generation rbmscs were used for experiments.
CCK-8 is used for detecting the toxicity of the nanoparticles to rBMSCs, 100 mu L of the rBMSCs for more than 3 passages are added into each well of a 96-well plate, and the final cell amount is 5 multiplied by 103One/well, putting 96-well plate into constant temperature incubator at 37 deg.C and 5% CO2After incubation for 24h under conditions, the supernatant was carefully aspirated, 100. mu.L of nanoparticles (1000, 500, 250, 100, 50,10, 5. mu.g/mL) at various concentrations were added to each well, and fresh culture broth was used as a negative control. At preset time points (1d, 3d, 5d), the wells were aspirated, 100. mu.L of CCK-8 working solution was added to each well, the 96-well plate was returned to the incubator and incubated for 2h, and the absorbance (OD) at 450nm was measured. As shown in FIG. 2, it can be seen from FIG. 2 that Fe was present on the fifth day in comparison with the control group containing no nanoparticles3O4@D&U concentrations as high as 500. mu.g/mL did not show any significant growth inhibition. The magnetic nanomaterial Fe prepared in example 1 was demonstrated3O4@D&U has good biocompatibility in vitro.
Example 3
Fe3O4And Fe3O4@D&Detection of hemolytic activity of U:
freshly collected human blood was centrifuged with heparin to remove the buffy coat and the red blood cells obtained were washed three times with Phosphate Buffered Saline (PBS), centrifuged at 1,000g for 10 minutes and resuspended to 4% (v/v) in PBS. Fresh human red blood cells (hRBC) were washed three times with PBS (35mM phosphate buffer, 0.15M NaCl, pH 7.4) and centrifuged at 1000g for 5 minutes. Two-fold serial dilutions of magnetic nanomaterial in PBS (concentration tested in the range of 10,20,50,100,200,500, 1000. mu.g/mL) were added to each well of a 96-well plate (total volume: 100. mu.l). An equal volume (100 μ L) of 4% w/v hRBC in PBS suspension was added to each well to reach a final volume of 200 μ L. The plates were incubated at 37 ℃ for 1 hour, and then the cells were pelleted by centrifugation at 1000g for 5 minutes. The supernatant (100. mu.L) was transferred to a clear 96-well plate. Hemoglobin was detected by measuring the absorbance at 414nm (Molecular Devices, Sunnyvale, Calif., USA). Determination of the value of zero hemolysis (negative vs.) Using PBS (APBS)Control), 0.1% (v/v) Triton was usedX-100(ATriton) 100% hemolysis (positive control) was determined. Percent hemolysis was calculated using the following formula:
hemolysis%sample-APBS)/(ATriton-APBS)×100；
The results of the hemolytic activity assay of the magnetic nanomaterial are shown in FIG. 3, which shows that Fe is present even at concentrations as high as 500. mu.g/mL3O4@D&U does not show any obvious hemolytic activity, which indicates that the magnetic nanomaterial prepared in example 1 has no hemolytic activity.
Example 4
Fe3O4And Fe3O4@D&Testing the in vitro bone targeting performance of U:
hydroxyapatite (HAP) is similar to bone tissue components, and thus HAP can be used in vitro to mimic bone tissue for bone tissue affinity detection. 50 μ g/mL FITC-labeled nanoparticles in 20mL Deionized (DI) water containing hydroxyapatite (0.75mg/mL) were stirred continuously at 37 ℃ for 4h at 550 rpm. At a predetermined time point, a 200 μ L aliquot was diluted with 1.0mL of deionized water and then centrifuged at 1,000g for 5 minutes to remove hydroxyapatite and bound nanoparticles. The concentration of nanoparticles in the supernatant was quantified by measuring the FITC fluorescence intensity and calculated by a standard curve. Bound nanoparticles were calculated by subtracting unbound nanoparticles from the total nanoparticles.
The results of the in vitro bone targeting performance test are shown in fig. 4. As can be seen from FIG. 4, it is similar to Fe alone3O4In contrast, Fe3O4@D&The bone targeting ability of U is obviously improved. The results demonstrate that the magnetic nanomaterial particles Fe prepared in example 13O4@D&U has good bone targeting properties in vitro.
Example 5
Fe3O4And Fe3O4@D&And (3) detecting the in vitro bacterial targeting performance of U:
detection of the bacterial binding efficiency of magnetic nanomaterials by flow cytometry. Briefly, methicillin-resistant staphylococcus aureus (MRSA, ATCC 43300) was cultured to mid-logarithmic growth (OD600 ═ 0.5) in Trypticase Soy Broth (TSB), and the mciroturbidimetry adjusted bacterial concentration to 1 × 106CFU/mL. The mid-log suspension of MRSA was incubated with FITC-labeled nanoparticles (10 μ g/mL) for 30 minutes at 37 ℃. Finally, the localization of the material on the bacterial cells was quantified by flow cytometry.
The results of the in vitro bacterial targeting performance test are shown in figure 5. As can be seen from FIG. 5, this is comparable to pure Fe3O4In contrast, Fe3O4@D&The bacterial targeting ability of U is obviously improved. The results demonstrate that the magnetic nanomaterial Fe prepared in example 13O4@D&U has good bacterial targeting performance in vitro.
Example 6
Fe3O4And Fe3O4@D&In vitro nuclear magnetic resonance detection of U:
the magnetic nanomaterials were placed in 1.5mL EP tubes in a concentration gradient (0, 10,20,50, 100. mu.g/mL). And the T2-weighted MR images were measured with a 7.0T MR spectrometer (brueck corporation, Billerica, massachusetts, usa).
The in vitro bone nmr test results of the magnetic nanomaterials are shown in fig. 6. As can be seen from fig. 6, the larger the concentration of the magnetic nanomaterial particles, the shorter the T2 relaxation time under nuclear magnetic resonance, the more significant the signal attenuation, and the linear relationship between the T2 relaxation time and the change in the nanoparticle concentration (correlation R)2>0.99), which shows that the magnetic nanomaterial prepared in example 1 has good magnetic properties in vitro and can reduce the relaxation time of the T2 phase of nuclear magnetic resonance.
Example 7
Fe3O4And Fe3O4@D&In vivo nuclear magnetic resonance localization of U diagnoses orthopedic implant related infections:
a sample of a Polyetheretherketone (PEEK) implant (kaffir material ltd, south-bound, Jiangsu province) was machined into a rod with a diameter of 1.5 mm and a height of 20 mm. Removing surface stains by ultrasonic cleaning.12 female Sprague-Dawley rats (Shanghai, Jersey) of 12 weeks of age were assigned pathogen free (SPF). Rats were divided into 2 groups: fe3O4Group Fe3O4@D&And (4) U groups. Samples of PEEK rods were contaminated with methicillin-resistant Staphylococcus aureus bacteria (concentration: 10)8CFU/mL, methicillin-resistant Staphylococcus aureus, ATCC 43300). Four groups (N ═ 6) of PEEK rods were then passed through the left femur of the implanted rat. Briefly, the knee of the rat is opened to expose the femoral condyle and the bone cavity is enlarged by electroporation until it is large enough to accommodate the PEEK rod. After the PEEK rod was implanted, the opening of the femur was sealed with bone wax, and the surgical site was closed layer by layer. Rats were housed in individual cages and fed ad libitum. After 24 hours of operation, the left femur of each experimental group of rats was exposed to an external magnetic field, and thereafter 200. mu.L of Fe was intravenously injected through the rats according to the group3O4Or Fe3O4@D&The left femur is again exposed to an external magnetic field after U. At a given time, a T2-weighted MR image was recorded with a 7.0T MR spectrometer (brueck corporation, Billerica, massachusetts, usa).
The results of the experiment are shown in FIG. 7, from which it can be seen that Fe was injected3O4The change of the T2 signals before and after the nano particles is very little, and the magnetic nano material Fe is injected in the opposite direction3O4@D&After U, the signal attenuation of the infected tissue was clearly observed.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.
Claims (10)
1. A magnetic nano material for very early diagnosis of related infection of orthopedic implants is characterized in that the magnetic nano material is Fe the surface of which is modified with bone targeting peptide and bacterial targeting peptide3O4@SiO2Magnetic nanomaterials.
2. The magnetic nanomaterial for the very early diagnosis of orthopedic implant related infections of claim 1, wherein the bone targeting peptide is aspartic acid hexapeptide having the amino acid sequence: DDDDDD.
3. The magnetic nanomaterial for the very early diagnosis of orthopaedic implant-related infections according to claim 1, wherein the bacterial targeting peptide is UBI29-41The amino acid sequence is as follows: TGRAKRRMQYNRR are provided.
4. The magnetic nanomaterial for the very early diagnosis of orthopaedic implant-related infections according to claim 1, wherein the mass ratio of the bone targeting peptide to the bacterial targeting peptide is 1: 1.
5. The method for preparing the magnetic nanomaterial for the very early diagnosis of infection associated with orthopedic implants according to any of claims 1 to 4, comprising: fe in core-shell structure3O4@SiO2Magnetic nano particles are used as raw materials, and then amino modification is carried out to obtain Fe3O4@SiO2-NH2Then adding the bone targeting peptide nanoparticles containing carboxyl and the bacteria targeting peptide nanoparticles respectively, and carrying out condensation reaction by adopting an EDC/NHS coupling method to form an amido bond, thereby preparing the magnetic nano material.
6. The method of preparing a magnetic nanomaterial for the very early diagnosis of orthopaedic implant related infections according to claim 5, wherein the Fe3O4@SiO2The magnetic nano-particles are magnetic Fe modified by oleic acid3O4The nanocrystalline is used as a core and is obtained after a silicon dioxide layer is prepared on the surface of the nanocrystalline.
7. The method of preparing a magnetic nanomaterial for the very early diagnosis of orthopaedic implant-related infections according to claim 5, characterized in thatThe amino modification is to adopt 3-aminopropyl triethoxysilane for amination so as to obtain Fe3O4@SiO2-NH2。
8. The method for preparing a magnetic nanomaterial for the very early diagnosis of infection associated with orthopedic implants according to claim 5, wherein the bone targeting peptide nanoparticle containing carboxyl is an amphiphilic polymer modified with carboxyl and bone targeting peptide; the bacterial targeting peptide nano particle containing carboxyl is an amphiphilic polymer modified with carboxyl and bacterial targeting peptide.
9. The method for preparing a magnetic nanomaterial for use in the very early diagnosis of orthopedic implant-related infections according to claim 5, wherein the amphiphilic polymer is polyethylene glycol, and the molecular weight of the polyethylene glycol is 3000-5000 g/mol.
10. The application of a magnetic nano material in preparation of a reagent and/or a kit for very early diagnosis of related infection of orthopedic implants is characterized in that the magnetic nano material is the magnetic nano material for very early diagnosis of related infection of orthopedic implants according to any one of claims 1 to 4.
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