Nanometer magnetic bead for specific capture of DNA of bacteria and fungi, preparation method of nanometer magnetic bead and rapid sterile detection method of cell therapy product

Abstract

The invention discloses a nano magnetic bead for specific capture of DNA (deoxyribonucleic acid) of bacteria and fungi, a preparation method of the nano magnetic bead and a rapid sterile detection method of a cell therapy product. The method comprises the following steps: S1, coating Fe3O4 with polydopamine to form a modified Fe3O4 magnetic bead; s2, compounding the MOF shell layer to the modified Fe3O4 magnetic beads to form Fe3O4-coated MOF magnetic beads; s3, gold nanoparticles are anchored to the Fe3O4 (at) MOF magnetic beads to form Fe3O4 (at) MOF (at) Au magnetic beads; and S4, modifying the Fe3O4 (at) MOF (at) Au magnetic bead with a specific capture probe aiming at DNA of bacteria and fungi to form a Fe3O4 (at) MOF (at) Au-Probe probe magnetic bead. The modified DNA probe disclosed by the invention can efficiently capture free bacteria and fungus nucleic acid fragments in a sample, so that template enrichment is realized.

Description

Technical Field

[0001] The present invention relates to the field of medical detection, and in particular to a nanomagnetic bead for specifically capturing bacterial and fungal DNA and a preparation method thereof, and a rapid sterility detection method for cell therapy products. Background Art

[0002] In recent years, with the continuous advancement of basic theories, technical approaches, and clinical research in stem cell and immune cell therapy, cell therapy products have provided new treatment approaches and methods for some serious and intractable diseases. To standardize cell therapy research and encourage technological innovation, my country's regulatory approval agencies have issued a series of guidelines and technical standards. These include: the National Medical Products Administration (NMPA) issued the "Good Manufacturing Practice for Pharmaceuticals - Cell Therapy Product Appendix (Draft for Comment)" on January 4, 2022; the Center for Drug Evaluation (CDE) issued the "Technical Guidelines for Clinical Trials of Immune Cell Therapy Products (Trial)" on February 9, 2021, and the "Technical Guidelines for Clinical Trials of Human Stem Cells and Their Derivatives (Draft for Comment)" on August 24, 2020; and the National Stem Cell Translational Resource Bank issued the "Stem Cell Preparation Release Inspection Specifications (Trial)" on September 22, 2021. While these documents have provided a better R&D environment and technical support for cell therapy products, progress in quality control-related inspection and testing technologies, particularly those for detecting biological contamination of control materials, has been slow. Cell therapy products typically have a shelf life of only a few hours and face urgent clinical needs, posing a significant challenge to pharmacopoeial sterility testing methods, which typically require a 14-day testing cycle. Therefore, the development of rapid sterility testing methods for process control and rapid release of cell-based preparations is essential.

[0003] PCR methods targeting bacterial and fungal DNA offer high sensitivity, good specificity, and ease of use, enabling sterility testing of samples within 3 hours. However, because this method cannot distinguish between the active state of microorganisms in a sample, interference from DNA from dead bacteria can lead to false-positive results, making it unsuitable for routine drug sterility testing. Raw materials and excipients for cell therapy products are sourced reliably and controllably. Through rigorous quality control, production materials are free of microbial contamination, eliminating interference from DNA from dead bacteria. Therefore, PCR methods based on microbial DNA detection are ideal candidates to replace pharmacopoeial sterility testing methods.

[0004] Since cell therapy products need to be produced and prepared in a Class A clean environment, even if microbial contamination occurs, the bioburden is generally low, and traditional PCR methods cannot directly detect contamination amounts below 10 CFU (low detection sensitivity). Past research data show that the sterility test method of the "Chinese Pharmacopoeia" can detect microbial contamination as low as 1 CFU. Therefore, using traditional PCR methods to directly perform sterility tests on cell therapy products cannot meet the requirement of the "Chinese Pharmacopoeia" that "the effect of the application of alternative methods should be better than that of the equivalent pharmacopoeial methods."

[0005] Therefore, it is particularly important to develop a method that can improve the low sensitivity of traditional PCR methods in detecting targets in samples. Summary of the Invention

[0006] Based on the above problems, the present invention provides a method for preparing nanomagnetic beads for the specific capture of bacterial and fungal DNA. The modified DNA probes by this method can efficiently capture free bacterial and fungal nucleic acid fragments in the sample, thereby achieving template enrichment.

[0007] A method for preparing nanomagnetic beads for specific capture of bacterial and fungal DNA comprises the following steps:

[0008] S1, dopamine coated Fe3O4 to form modified Fe3O4 magnetic beads;

[0009] S2, MOF shell is composited to modified Fe3O4 magnetic beads to form Fe3O4@MOF magnetic beads;

[0010] S3, gold nanoparticles are anchored to Fe3O4@MOF magnetic beads to form Fe3O4@MOF@Au magnetic beads;

[0011] S4, specific capture probes for bacterial and fungal DNA were modified onto Fe3O4@MOF@Au magnetic beads to form Fe3O4@MOF@Au-Probe probe beads.

[0012] In one or more specific embodiments of the present application, S1 includes the following steps:

[0013] S11, FeCl3·6H2O, Na3CT and NH4Ac were dissolved in ethylene glycol, stirred evenly, heated to 180-220℃, reacted for 8-20h, and the product was collected with a magnet to obtain Fe3O4 magnetic beads;

[0014] S12, washing the Fe3O4 magnetic beads and then dispersing them in deionized water;

[0015] S13, dissolving dopamine in the first portion of Tris buffer and sonicating for 1 to 5 minutes to form a dopamine solution;

[0016] S14, washing the Fe3O4 magnetic beads taken out from S12 with the second portion of Tris buffer, adding them to the dopamine solution in S13 under magnetic stirring, and reacting for 3 to 8 hours to form polydopamine-modified Fe3O4 magnetic beads;

[0017] S15, washing the polydopamine-modified Fe3O4 magnetic beads and then dispersing them in DMF;

[0018] The mass ratio of FeCl3·6H2O:Na3CT:NH4Ac:polydopamine is 0.5-2:0.1-0.6:2-4:0.005-0.02, the volume of the first part of Tris buffer is 15-30 mL, and the volume of the second part of Tris buffer is 1-100 mL.

[0019] In one or more specific embodiments of the present application, S2 includes the following steps:

[0020] S21, add 1 mL of modified Fe3O4 magnetic beads and 0.2-0.4 g of zirconium chloride to DMF and sonicate for 20-40 min.

[0021] S22, completely dissolving 0.1-0.3 g of 2-aminoterephthalic acid in DMF;

[0022] S23, mixing S21 and S22, heating the mixture to 100-130°C, stirring and reacting for 3-5 hours to obtain Fe3O4@MOF magnetic beads;

[0023] S24, cleaning of Fe3O4@MOF magnetic beads;

[0024] S25, vacuum drying.

[0025] In one or more specific embodiments of the present application, S3 includes the following steps:

[0026] S31, 8–12 mg of Fe3O4@MOF magnetic beads and 30–38 mg of HAuCl4 were fully dispersed in methanol, incubated for 30–50 h, and then evaporated to dryness under reduced pressure;

[0027] S32, 1-5 mL of NaBH4 methanol solution was added dropwise to the solid formed in S31, and stirred at 1-5°C for 30-50 h to obtain Fe3O4@MOF@Au magnetic beads.

[0028] S32, washing Fe3O4@MOF@Au magnetic beads;

[0029] Wherein, the concentration of NaBH4 methanol solution is 0.2~0.3M.

[0030] In one or more specific embodiments of the present application, S4 includes the following steps:

[0031] S41, 1-3 mg of Fe3O4@MOF@Au magnetic beads, 1-3 nmol of 16SV1F probe, and 1-3 nmol of ITS4 probe were stirred and incubated in probe buffer for 10-20 h to obtain Fe3O4@MOF@Au-Probe probe magnetic beads;

[0032] S42, washing Fe3O4@MOF@Au-Probe probe magnetic beads;

[0033] The probe buffer includes 8-12 mM PBS, 0.1 M NaCl, pH 7-8.

[0034] The present invention also provides nanomagnetic beads for specifically capturing bacterial and fungal DNA.

[0035] A nanomagnetic bead for specifically capturing bacterial and fungal DNA is prepared by the above method.

[0036] In one or more specific embodiments of the present application, the diameter of the nanomagnetic beads is about 200 nm, and the saturation magnetization intensity is 40 emug. -1 , with a surface area of 592.7m 2 / g, the pore sizes in the MOF layer on the surface of the magnetic beads are approximately 0.8nm and 1.2nm, the probe mass accounts for more than 10%, the Zeta potential is -44mV, and red fluorescence can be seen under laser scanning confocal microscopy.

[0037] The present invention also provides a method for rapid sterility detection of cell therapy products.

[0038] A rapid sterility detection method for cell therapy products comprises the following steps: Q1, extraction and enrichment of DNA templates: preparing bacterial solutions of approximately 100 cfu / ml for each of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Clostridium sporogenes, Candida albicans, and Aspergillus niger according to the provisions of General Chapter 1105 of Part IV of the Chinese Pharmacopoeia;

[0039] Take 1 ml of each prepared test bacteria, centrifuge at 12000g for 5 minutes to collect the precipitate, resuspend the bacteria in 50 μl of Lysis Buffer for Microorganism to Direct PCR lysis buffer, incubate at 80°C for 20 minutes, add 1 ml of physiological saline to dilute the lysed sample, and add 20 μl of the above-mentioned nanomagnetic beads to it. Mix thoroughly, and after 15 minutes, use a magnetic rack to absorb the nanomaterials to the tube wall, and discard the supernatant liquid;

[0040] Q2, Reaction system preparation: Prepare the PCR reaction system in a clean bench and transfer the sample to a PCR tube, including 10μM Primer 1, 1μl; 10μM Primer 2, 1μl; 2X Taq PCR Master Mix, 25μld HO, 23μl;

[0041] Q3, PCR amplification: Place the prepared reaction tube into the PCR instrument and perform PCR amplification. The amplification program is as follows: initial denaturation at 94°C for 5 min; 32 cycles: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 16 s (rRNA: 60 s; ITS: 40 s), final extension at 72°C for 10 min; store at 4°C forever.

[0042] Q4, gel electrophoresis

[0043] Q5, judge the result.

[0044] The beneficial effects of this application are:

[0045] This method coats Fe3O4 magnetic beads with a novel metal-organic framework (MOF) material to create nanomagnetic beads. Capture probes are then modified onto the surface of the beads and anchored to gold nanoparticles, forming a nanomaterial that can specifically capture bacterial and fungal DNA. Leveraging the extremely high surface area of MOFs, the method efficiently captures bacterial and fungal DNA from enriched samples. Magnetic separation allows for rapid enrichment of target microbial DNA, laying the foundation for the development of rapid sterile testing methods for cell therapy products.

[0046] The present invention uses Fe3O4 as the magnetic core and coats the surface with MOF material formed by DMF and zirconium chloride to form Fe3O4@MOF nanomaterial, which has a specific surface area of 592.7m 2 / g, and the pore sizes in the MOF layer are approximately 0.8nm and 1.2nm. Specific capture probes for bacterial and fungal DNA are modified onto the material. Taking advantage of the large specific surface area of porous MOFs materials, the modified DNA probes can efficiently capture free bacterial and fungal nucleic acid fragments in the sample, thereby achieving template enrichment. At the same time, gold nanoparticles are anchored to the surface of magnetic MOFs nanomaterials. The gold nanoparticles can effectively improve the efficiency of subsequent PCR amplification. After enrichment with the nanomaterial, the DNA template concentration in the PCR amplification system can be significantly increased, thereby improving detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the SEM characterization image of Fe3O4@MOF;

[0048] Figure 2This is the SEM characterization image of Fe3O4@MOF@Au;

[0049] Figure 3 TEM characterization image of Fe3O4@MOF;

[0050] Figure 4 TEM characterization image of Fe3O4@MOF@Au;

[0051] Figure 5 is the VSM characterization diagram of each sample;

[0052] Figure 6 XRD characterization diagram of each sample;

[0053] Figure 7 BET characterization of Fe3O4@MOF Figure 1 ;

[0054] Figure 8 BET characterization of Fe3O4@MOF Figure 2 ;

[0055] Figure 9 Characterization diagram of Fe3O4@MOF@Au-Probe;

[0056] Figure 10 FL characterization of Fe3O4@MOF@Au-Probe

[0057] Figure 11 DLS characterization diagram of each sample;

[0058] Figure 12 CLSM characterization image of Fe3O4@MOF@Au;

[0059] Figure 13 CLSM characterization image of Fe3O4@MOF@Au-Probe;

[0060] Figure 14 The electrophoresis diagram of 16S nucleic acid sequences of five bacteria in Example 5 is shown;

[0061] Figure 15 This is the electrophoresis diagram of the ITS nucleic acid sequences of two fungi in Example 5;

[0062] Figure 16 The electrophoresis diagram of 16S nucleic acid sequences of five bacteria in Comparative Example 1 is shown;

[0063] Figure 17 The electrophoresis diagram of the ITS nucleic acid sequences of the two fungi in Comparative Example 1 is shown. DETAILED DESCRIPTION

[0064] The present invention will be further described below.

[0065] The embodiments provided in the present invention are only for further explanation of the present invention and should not be construed as any limitation to the present invention.

[0066] It should be clear to those skilled in the art that, unless otherwise specified, the materials and operating methods used in the present invention are well known in the art.

[0067] Example 1

[0068] A method for preparing Fe3O4@MOF@Au-Probe probe magnetic beads comprises the following steps:

[0069] S1, preparation of modified Fe3O4 magnetic beads, comprising the following steps:

[0070] In step S11, 1.16 g of FeCl₃·6H₂O, 0.40 g of Na₃CT, and 3.30 g of NH₄Ac were dissolved in 60 mL of ethylene glycol and then placed in a 100 mL stainless steel autoclave with magnetic stirring for 1 hour. After the stirrer was removed, the autoclave was heated to 200°C and allowed to react for 16 hours. After the reaction, the product was collected using a magnet to obtain Fe₃O₄ magnetic beads.

[0071] S12, after repeatedly washing the Fe3O4 magnetic beads with ethanol and water three times, the Fe3O4 magnetic beads were redispersed in 15 mL of deionized water.

[0072] S13, dissolving 10 mg of dopamine in 20 mL of Tris buffer (10 mM, pH 8.2), and sonicating for 3 min to form a dopamine solution.

[0073] S14, the Fe3O4 magnetic beads were taken out, washed with 5 mL of Tris buffer, and added to the dopamine solution under magnetic stirring for 6 h to form polydopamine (PDA)-modified Fe3O4 magnetic beads.

[0074] S15, the Fe3O4 magnetic beads modified with polydopamine (PDA) were washed three times with water and DMF respectively, and then dispersed in 2 mL DMF for later use.

[0075] S2, preparation of Fe3O4@MOF magnetic beads, including the following steps:

[0076] S21, 1 mL of modified Fe3O4 magnetic beads and 0.232 g of zirconium chloride (ZrCl4) were added to 30 mL of N,N-dimethylformamide (DMF) and sonicated for 30 min.

[0077] S22, 0.165 g of 2-aminoterephthalic acid was completely dissolved in 10 mL of DMF.

[0078] S23, mixing S21 and S22, heating the mixture to 120°C, and reacting for 4 h under stirring to obtain Fe3O4@MOF magnetic beads.

[0079] S24, the Fe3O4@MOF magnetic beads were washed three times with DMF and acetone respectively.

[0080] S25, vacuum dry and set aside for use.

[0081] S3, preparation of Fe3O4@MOF@Au magnetic beads, including the following steps:

[0082] S31, 10 mg of Fe3O4@MOF magnetic beads and HAuCl4 (0.1 mmol) were fully dispersed in 6 mL of methanol, incubated for 48 h, and then evaporated to dryness under reduced pressure.

[0083] S32, NaBH4 methanol solution (4 mL, 0.25 M) was added dropwise to the solid formed in S31, and stirred at 4°C for 48 h to obtain Fe3O4@MOF@Au magnetic beads.

[0084] S32, wash the Fe3O4@MOF@Au magnetic beads with deionized water and collect them for later use.

[0085] S4, preparation of Fe3O4@MOF@Au-Probe probe magnetic beads, including the following steps:

[0086] S41, Fe3O4@MOF@Au magnetic beads (2 mg), 16SV1F probe (2 nmol) and ITS4 probe (2 nmol) were stirred and incubated in probe buffer (10 mM PBS, 0.1 M NaCl, pH 7.4) for 16 h to obtain Fe3O4@MOF@Au-Probe probe magnetic beads.

[0087] S42, after washing the Fe3O4@MOF@Au-Probe probe magnetic beads 5 times with the probe buffer, the Fe3O4@MOF@Au-Probe probe magnetic beads were stored in the probe buffer for subsequent use.

[0088] Example 2

[0089] The Fe3O4@MOF or Fe3O4@MOF@Au prepared in Example 1 was prepared with deionized water to an appropriate concentration, and 10 μL was dropped onto a clean single crystal silicon wafer. After natural drying at room temperature, the surface morphology of the sample was observed using a transmission electron microscope (TEM). Figure 1-Figure 2 ,in, Figure 1 This is the TEM characterization picture of Fe3O4@MOF. Figure 2 TEM characterization image of Fe3O4@MOF@Au.

[0090] The Fe3O4@MOF or Fe3O4@MOF@Au prepared in Example 1 was prepared with ethanol to an appropriate concentration and then dropped onto the carbon film copper grid. After natural drying at room temperature, the transmission morphology of the sample was observed using a scanning electron microscope (SEM). Figure 3-Figure 4 ,in, Figure 3 This is the SEM characterization picture of Fe3O4@MOF. Figure 4 This is the SEM characterization image of Fe3O4@MOF@Au.

[0091] from Figure 1-Figure 4 The Fe3O4@MOF nanoparticles are spherical in shape. After coating with PDA and MOF materials, a shell structure distinct from iron oxide crystal clusters can be seen around the Fe3O4@MOF nanoparticles. The shell thickness is uniform, indicating that the MOF shell has been successfully integrated onto the Fe3O4 nanoparticle surface. Treatment with HAuCl4 has no significant effect on the Fe3O4@MOF nanoparticle morphology, and the gold nanoparticles are successfully anchored to the surface.

[0092] Example 3

[0093] Vibrating sample magnetometer characterization (VSM): 5-10 mg of sample (Fe3O4, Fe3O4@MOF, or Fe3O4@MOF-Au from Example 1) was placed in a 1 cm plastic tube sealed at one end, and the other end of the tube was blocked with cotton. The plastic tube was placed in a magnetometer at room temperature and tested in the magnetic intensity range of 0-15000 Oe to obtain the sample's magnetization curve and saturation magnetization (unit: emug). -1 ). The result is as follows Figure 5 , Figure 5 VSM characterization diagram of each sample, where a represents Fe3O4, b represents Fe3O4@MOF, and c represents Fe3O4@MOF-Au. Figure 5 The results show that all samples exhibit superparamagnetism. The superparamagnetic property comes from Fe3O4 clusters, and the saturation magnetization intensity of Fe3O4 nanoparticles is 60emug. -1 After modification of the PDA layer, the saturation magnetization of Fe3O4@MOF nanospheres is about 45emug -1 After anchoring gold particles, the saturation magnetization intensity of Fe3O4@MOF-Au is about 40emug -1 The high saturation magnetization gives the Fe3O4@MOF-Au nanospheres excellent magnetic response. When a magnetic field (approximately 200 mT) is applied, the Fe3O4@MOF-Au nanospheres can be separated from the solution within 10 seconds. After the magnetic field is removed, the nanospheres can be redispersed in water with slight shaking. The excellent magnetic properties of Fe3O4, Fe3O4@MOF, and Fe3O4@MOF-Au nanospheres are beneficial for magnetic separation applications.

[0094] Powder X-ray diffraction (XRD): About 20 mg of the sample (Fe3O4 or Fe3O4@MOF of Example 1) was ground uniformly and the crystal structure of the sample was detected under the conditions of 40Kv, 25mA, Cu / Kα (γ=0.154). The results are as follows Figure 6 , Figure 6 The XRD characterization diagrams of each sample, where a represents Fe3O4 and b represents Fe3O4@MOF. Fe3O4 and Fe3O4@MOF show typical diffraction peaks of Fe3O4 at 30.3°(220), 35.2°(311), 43.5°(400), 53.4°(422), 57.1°(511) and 62.8°(440), which are consistent with the standard XRD data card of Fe3O4 (JCPDS No. 19-06290). The results show that the modification of the MOF layer has no effect on the crystal structure of Fe3O4. In addition, four new diffraction peaks appear at 10.8°, 10.8°, 24.3° and 26.5°, which can be attributed to the crystal structure of MOFs formed by zinc nitrate and 2-aminoterephthalic acid. The XRD characterization results show that the MOF shell is successfully formed. In addition, the color of the product also changes from black to brownish yellow. Zn 2+ The MOF formed with 2-aminoterephthalic acid is brown-yellow, which also indicates that the MOF shell is successfully formed.

[0095] Specific surface area characterization (BET): About 100 mg of the sample (Fe3O4@MOF of Example 1) was taken and ground evenly, and the specific surface area and porous structure of the sample were tested under N2 protection. The results are as follows Figure 7-Figure 8 . Figure 7-Figure 8 In the nitrogen adsorption-desorption characterization, Fe3O4@MOF nanospheres have a typical IV isotherm curve. Based on the Langmuir and Barrett-Joyner-Halenda (BJH) methods, the surface area of the nanospheres is 592.7 m 2 According to density functional theory calculations, the pore sizes of the MOF layer on the surface of the Fe3O4@MOF nanospheres are approximately 0.8 nm and 1.2 nm. The high specific surface area and microporous structure prove that the MOF shell is successfully composited onto the surface of the Fe3O4 nanospheres.

[0096] Example 4 Probe Modification Characterization

[0097] Thermal Gravimetric Analysis (TGA): About 20 mg of sample (Fe3O4@MOF@Au or Fe3O4@MOF@Au-Probe of Example 1) was placed in an alumina crucible and placed in the instrument. Under dynamic nitrogen protection, the temperature was raised from room temperature to 600°C at a rate of 15°C / min to obtain the weight loss change of the sample during the heating process. The results are shown in Figure 2. Figure 9 The results show that after 600°C, the heat loss of Fe3O4@MOF@Au magnetic beads is about 33%, while the heat loss of Fe3O4@MOF@Au-Probe magnetic beads is about 47%, indicating that the mass proportion of the probe in the Fe3O4@MOF@Au-Probe nanospheres is about 14%. The higher mass proportion proves that the modification efficiency of the probe on the magnetic bead surface is higher.

[0098] Fluorescence detection characterization (FL): Cy5 was used to label the fluorescent probe, and the fluorescence intensity of Cy5 in the supernatant solution was tested before and after the probe modification to evaluate the modification efficiency of the probe on the magnetic beads. Figure 10 The supernatant before DNA probe modification exhibited a maximum absorption peak at 615 nm, with a fluorescence signal intensity of approximately 1,300,000 AU. After DNA probe modification, the fluorescence signal intensity of the supernatant decreased to 700,000 AU, demonstrating that a large amount of fluorescent probe was modified onto the magnetic bead surface. FL characterization results indicate that the DNA probe was successfully modified onto the nanomaterial surface.

[0099] Laser particle size analyzer characterization (DLS / Zeta): a) At room temperature, a small amount of sample (modified Fe3O4, Fe3O4@MOF@Au or Fe3O4@MOF@Au-Probe of Example 1) was dispersed in 1.5 mL of deionized water to examine the hydrodynamic diameter and particle size distribution of the sample. Each sample was tested three times. b) A small amount of sample was dispersed in 1 mL of deionized water to examine the surface potential of the sample. Each sample was tested three times. The results are shown in Figure 2. Figure 11 , where a refers to modified Fe3O4, b refers to Fe3O4@MOF@Au, and c refers to Fe3O4@MOF@Au-Probe. The Zeta potential of the modified Fe3O4 in water is approximately 14mV. After being wrapped with the MOF layer and anchored with negatively charged gold nanoparticles, the Zeta potential of Fe3O4@MOF@Au becomes -25mV. After being modified with a DNA probe with a large number of negatively charged phosphate groups, the Zeta potential of Fe3O4@MOF@Au-Probe becomes -44mV. DLS characterization results show that the gold nanoparticles are successfully anchored to the MOF layer and the DNA probe is successfully modified to the surface of the nanomaterial, and the nanocapture material is successfully prepared.

[0100] Laser scanning confocal microscopy (CLSM): Fe3O4@MOF@Au-Probe magnetic beads and Fe3O4@MOF@Au magnetic beads of Example 1 were added to a glass slide at a certain concentration, and the Cy5 fluorescence signal was observed under a laser scanning confocal microscope to evaluate the modification efficiency of the Cy5 fluorescent probe. Figure 12-13 In both bright field and dark field conditions, the red fluorescence of Cy5 could not be observed on the Fe3O4@MOF@Au surface. However, after the Fe3O4@MOF@Au-Probe magnetic beads were placed, the red fluorescence of Cy5 could be clearly observed on the surface of the nanospheres, demonstrating the successful preparation of Fe3O4@MOF@Au-Probe to capture nanomaterials.

[0101] Example 5

[0102] A rapid sterility test method for a cell therapy product comprises the following steps:

[0103] Q1, DNA template extraction and enrichment

[0104] Prepare bacterial suspensions containing approximately 100 cfu / ml of Staphylococcus aureus [CMCC(B)26003], Pseudomonas aeruginosa [CMCC(B)10104], Bacillus subtilis [CMCC(B)63501], Escherichia coli [CMCC(B)44102], Clostridium sporogenes [CMCC(B)64941], Candida albicans [CMCC(F)98001], and Aspergillus niger [CMCC(F)98003] according to the requirements of Section 1105 of the General Chapter of Part IV of the Chinese Pharmacopoeia. Take 1 ml of each prepared test strain and centrifuge at 12000 g for 5 minutes to collect the precipitate. Resuspend the cells in 50 μl of Lysis Buffer for Microorganisms to Direct PCR and incubate at 80°C for 20 minutes. 1 ml of physiological saline was added to dilute the lysed sample, and 20 μl of the Fe3O4@MOF@Au-Probe capture nanomaterial of Example 1 was added thereto. The mixture was thoroughly mixed and allowed to stand for 15 minutes. The nanomaterial was then adsorbed to the tube wall using a magnetic rack, and the upper liquid was discarded.

[0105] Q2, reaction system preparation

[0106] In a clean bench, prepare the PCR reaction system according to Table 1 and transfer the samples to PCR tubes.

[0107] Table 1 16s rDNA / ITS gene PCR amplification system

[0108] PCR reaction system Volume (μl) Primer1 (10 μM) 1 Primer2 (10 μM) 1 2X Taq PCR Master Mix 25 <![CDATA[dd H2O]]> 23 total 50

[0109] Q3, PCR amplification

[0110] Place the prepared reaction tube into the PCR instrument and perform PCR amplification according to the program in Table 2.

[0111] Table 2 16S rRNA / ITS gene amplification procedure

[0112]

[0113] Q4, gel electrophoresis

[0114] After amplification, use 1% agarose gel electrophoresis to detect the PCR product.

[0115] Agarose gel electrophoresis results showed that the 16s nucleic acid sequences of the five bacteria ( Figure 14 ) and the ITS nucleic acid sequences of two fungi ( Figure 15 ) were able to be effectively amplified, with distinct amplification bands around 1500 bp and 500 bp, respectively. This indicates that the Fe3O4@MOF@Au-Probe capture nanomaterial can successfully capture the target sequence in the sample and amplify it through PCR reaction.

[0116] Figure 14 Figure 1 shows the amplification results of 16S nucleic acid sequences of five bacteria, M: marker; 1: Escherichia coli; 2: Pseudomonas aeruginosa; 3: Staphylococcus aureus; 4: Bacillus subtilis; 5: Salmonella.

[0117] Figure 15 Middle, M: marker; 1: Candida albicans; 2: Aspergillus niger.

[0118] Comparative Example 1

[0119] Q1, DNA template extraction

[0120] Staphylococcus aureus [CMCC(B)26003], Pseudomonas aeruginosa [CMCC(B)10104], Bacillus subtilis [CMCC(B)63501], Escherichia coli [CMCC(B)44102], Clostridium sporogenes [CMCC(B)64941], Candida albicans [CMCC(F)98001], and Aspergillus niger [CMCC(F)98003] were prepared to a concentration of approximately 100 cfu / ml according to the provisions of Section 1105 of the General Chapter of Volume 4 of the Chinese Pharmacopoeia. Genomic DNA was extracted from each of the five bacterial and two fungal species using the Bacterial Genomic DNA Rapid Extraction Kit and the Fungal Genomic DNA Rapid Extraction Kit (Shanghai) Co., Ltd., respectively, according to the manufacturer's instructions. The DNA was then eluted and dissolved in 23 μl of water.

[0121] Q2, reaction system preparation

[0122] In a clean bench, prepare the PCR reaction system according to the requirements in Table 3 and transfer the samples to PCR tubes.

[0123] Table 3 16s rDNA / ITS gene PCR amplification system

[0124] PCR reaction system Volume (μl) Primer1 (10 μM) 1 Primer2 (10 μM) 1 2X Taq PCR Master Mix 25 DNA 23 total 50

[0125] Q3, PCR amplification

[0126] Place the prepared reaction tube into the PCR instrument and perform PCR amplification according to the program in Table 4.

[0127] Table 4 16S rRNA / ITS gene amplification procedure

[0128]

[0129] Q4, gel electrophoresis

[0130] After amplification, PCR products were detected by electrophoresis using 1% agarose gel.

[0131] Q5, judge the result.

[0132] Agarose gel electrophoresis results showed that only Pseudomonas aeruginosa and Escherichia coli could be effectively amplified among the five bacterial 16s nucleic acid sequences. The ITS nucleic acid sequences of the two fungi ( Figure 17 ) can be amplified, but the amplified band is weak. Compared with the amplification results after nanomaterial enrichment, the amplification effect of non-enriched DNA is significantly poorer, indicating that the nanomaterial prepared by the present invention has a significant effect on DNA enrichment.

[0133] Figure 16 In the figure, M: marker; 1: Staphylococcus aureus; 2: Pseudomonas aeruginosa; 3: Escherichia coli; 4: Bacillus subtilis; 5: Salmonella.

[0134] Figure 17 Middle, M: marker; 1: Candida albicans; 2: Aspergillus niger.

[0135] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing nanomagnetic beads for specific capture of bacterial and fungal DNA, characterized in that: The following steps are involved: S1, dopamine coated Fe3O4 to form modified Fe3O4 magnetic beads; S2, MOF shell is composited to modified Fe3O4 magnetic beads to form Fe3O4@MOF magnetic beads; S3, gold nanoparticles are anchored to Fe3O4@MOF magnetic beads to form Fe3O4@MOF@Au magnetic beads; S4, specific capture probes for bacterial and fungal DNA were modified onto Fe3O4@MOF@Au magnetic beads to form Fe3O4@MOF@Au-Probe probe beads.

2. The method for preparing nanomagnetic beads for specific capture of bacterial and fungal DNA according to claim 1, characterized in that: S1 includes the following steps: S11, FeCl3·6H2O, Na3CT and NH4Ac are dissolved in ethylene glycol, stirred evenly, heated to 180-220℃, reacted for 8-20h, and the product was collected with a magnet to obtain Fe3O4 magnetic beads; S12, washing the Fe3O4 magnetic beads and then dispersing them in deionized water; S13, dissolving dopamine in the first portion of Tris buffer and sonicating for 1 to 5 minutes to form a dopamine solution; S14, washing the Fe3O4 magnetic beads taken out from S12 with the second portion of Tris buffer, adding them to the dopamine solution in S13 under magnetic stirring, and reacting for 3 to 8 hours to form polydopamine-modified Fe3O4 magnetic beads; S15, washing the polydopamine-modified Fe3O4 magnetic beads and then dispersing them in DMF; The mass ratio of FeCl3·6H2O:Na3CT:NH4Ac:polydopamine is 0.5-2:0.1-0.6:2-4:0.005-0.02, the volume of the first part of Tris buffer is 15-30 mL, and the volume of the second part of Tris buffer is 1-100 mL.

3. The method for preparing nanomagnetic beads for specific capture of bacterial and fungal DNA according to claim 1, characterized in that: S2 includes the following steps: S21, add 1 mL of modified Fe3O4 magnetic beads and 0.2-0.4 g of zirconium chloride to DMF and sonicate for 20-40 min. S22, completely dissolving 0.1-0.3 g of 2-aminoterephthalic acid in DMF; S23, mixing S21 and S22, heating the mixture to 100-130°C, stirring and reacting for 3-5 hours to obtain Fe3O4@MOF magnetic beads; S24, cleaning of Fe3O4@MOF magnetic beads; S25, vacuum drying.

4. The method for preparing nanomagnetic beads for specific capture of bacterial and fungal DNA according to claim 1, characterized in that: In S3, the following steps are included: S31, 8–12 mg of Fe3O4@MOF magnetic beads and 30–38 mg of HAuCl4 were fully dispersed in methanol, incubated for 30–50 h, and then evaporated to dryness under reduced pressure; S32, 1-5 mL of NaBH4 methanol solution was added dropwise to the solid formed in S31, and stirred at 1-5°C for 30-50 h to obtain Fe3O4@MOF@Au magnetic beads. S32, washing Fe3O4@MOF@Au magnetic beads; Wherein, the concentration of NaBH4 methanol solution is 0.2~0.3M.

5. The method for preparing nanomagnetic beads for specific capture of bacterial and fungal DNA according to claim 1, characterized in that: S4 includes the following steps: S41, 1-3 mg of Fe3O4@MOF@Au magnetic beads, 1-3 nmol of 16SV1F probe, and 1-3 nmol of ITS4 probe were stirred and incubated in probe buffer for 10-20 h to obtain Fe3O4@MOF@Au-Probe probe magnetic beads; S42, washing Fe3O4@MOF@Au-Probe probe magnetic beads; The probe buffer includes 8-12 mM PBS, 0.1 M NaCl, pH 7-8.

6. A nanomagnetic bead for specific capture of bacterial and fungal DNA, characterized in that: The nanomagnetic beads are prepared by the method according to any one of claims 1 to 5.

7. The nanomagnetic beads for specific capture of bacterial and fungal DNA according to claim 6, characterized in that The diameter of the nanomagnetic beads is about 200nm, and the saturation magnetization intensity is 40emug -1 , with a surface area of 592.7m 2 / g, the pore sizes in the MOF layer on the surface of the magnetic beads are approximately 0.8nm and 1.2nm, the probe mass accounts for more than 10%, the Zeta potential is -44mV, and red fluorescence can be seen under laser scanning confocal microscopy.

8. A rapid sterility detection method for cell therapy products, characterized in that: The method comprises the following steps: Q1, extraction and enrichment of DNA template: preparing bacterial suspensions of about 100 cfu / ml of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Clostridium sporogenes, Candida albicans, and Aspergillus niger according to the provisions of General Rules 1105 of Part IV of the Chinese Pharmacopoeia; Take 1 ml of each prepared test bacteria, centrifuge at 12000g for 5 minutes to collect the precipitate, resuspend the bacteria in 50 μl of Lysis Buffer for Microorganism to Direct PCR lysis buffer, incubate at 80°C for 20 minutes, add 1 ml of physiological saline to dilute the lysed sample, and add 20 μl of the nanomagnetic beads described in any one of claims 6-7 to it, mix thoroughly, and after 15 minutes, use a magnetic stand to adsorb the nanomaterial to the tube wall, and discard the supernatant liquid; Q2, Reaction system preparation: Prepare the PCR reaction system in a clean bench and transfer the sample to a PCR tube, including 10μM Primer 1, 1μl; 10μM Primer 2, 1μl; 2X Taq PCR Master Mix, 25μld HO, 23μl; Q3, PCR amplification: Place the prepared reaction tube into the PCR instrument and perform PCR amplification. The amplification program is as follows: initial denaturation at 94°C for 5 min; 32 cycles: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 16 s (rRNA: 60 s; ITS: 40 s), final extension at 72°C for 10 min; store at 4°C forever. Q4, gel electrophoresis Q5, judge the results.

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