A multifunctional magnetic nanosphere and its preparation and application

Abstract

The present invention relates to a multifunctional magnetic nanosphere and its preparation and application, belonging to the field of bacterial separation and enrichment. In the present invention, after first modifying the surface of amino-functionalized magnetic nanospheres (MNP ‑NH2 ) with a polydopamine (PDA) shell layer, long-chain polyethylene glycol (PEG) molecular arms are connected, and then the antibiotic polymyxin B (PMB) is modified on the molecular arms to obtain MNP PDA‑PEG‑PMB . PMB has broad-spectrum bacterial binding characteristics, but has a stronger binding ability to G ‑ . Modifying PDA on the surface of MNP- NH2 can increase the number of active groups on the MNP surface and increase the content of PMB bound thereto; the long-chain PEG helps to reduce the steric hindrance when MNP binds to bacteria, and the combination of the two ultimately improves the adsorption capacity of MNP PDA‑PEG‑PMB . The MNP PDA‑PEG‑PMB microspheres adsorbed with bacteria can be directly added to solid and liquid culture media for cultivation to rapidly detect bacterial activity. MNP PDA‑PEG‑PMB combined with the thermal cracking method can be applied to the rapid detection of a series of bacterial nucleic acids in complex samples.

Description

Technical Field

[0001] The present invention belongs to the field of bacterial isolation and enrichment, and more specifically, relates to a polymyxin B-functionalized magnetic nanospheres for efficient targeted isolation and enrichment of bacteria, and its preparation and application. Background Art

[0002] According to the statistics of the World Health Organization (WHO), pathogenic bacterial infections may cause 13.3 million deaths every year, posing a great threat to the public health safety of the world (Analyst, 2012, 137(15): 3405-3421). There are a wide variety of pathogenic bacteria, diverse infection routes, and numerous therapeutic drugs. Establishing a rapid and accurate detection method for pathogenic bacteria helps to better prevent and treat bacterial infections (Mater Chem B, 2017, 5(44): 8631-8652). The detection of pathogenic bacteria includes two aspects: viability detection and nucleic acid detection. Since the bacterial content in actual samples varies, and proteins, fats, enzyme reaction inhibitors, ions, etc. therein can all affect the sensitivity and accuracy of bacterial viability and nucleic acid detection, it is often necessary to isolate and enrich the target bacteria from the sample before performing viability and nucleic acid detection to ensure the sensitivity, accuracy, and reliability of the detection (Biosens and Bioelectron, 2011, 26(11): 4368-4374). Therefore, it is very necessary to develop simple, rapid, and convenient bacterial isolation and enrichment materials and methods that can be used for rapid detection of both bacterial viability and nucleic acids.

[0003] Common pathogen enrichment methods mainly include selective culture method, physical method and biological affinity method. Bacterial selective culture enrichment only targets specific bacteria and usually takes 2 - 4 days, which is time-consuming and cannot achieve rapid detection. Physical methods mainly include centrifugation, filtration, ultrafiltration, etc. They require basic instrument equipment and are more suitable for screening bacterial populations. If specific bacteria are to be enriched, their specificity is insufficient. The biological affinity method uses the affinity between bacteria and affinity molecules on the solid-phase carrier to achieve separation, which is a mild, efficient and simple method for bacterial separation and enrichment. Among them, magnetic nanospheres MNP (Magnetic nanoparticle, MNP) have superparamagnetism, which is convenient for separation operations using magnetic fields, overcoming the problem that it is difficult to separate other nanomaterials from the sample matrix. MNP has a high adsorption capacity, and its surface is easy to connect various functional groups, making it suitable for preparing biological affinity materials. Among the recognition molecules modifying MNP, antibodies have strong specificity, but also have many disadvantages, such as high price, long incubation time required, poor stability, etc.; biological macromolecules have many functional groups, and the denaturation of the three-dimensional structure and random molecular orientation during the modification process may reduce their biological activity; organic / inorganic molecules are usually non-specific adsorption and lack selectivity. Small molecule antibiotics have selective targeting effects on bacteria, good stability, and are suitable as affinity molecules. Antibiotic polymyxin B (Polymyxin B, PMB) has broad-spectrum bacterial adsorption properties. The amino group of the PMB side chain undergoes electrostatic binding with the phosphate group of the lipopolysaccharide lipid A in the outer membrane, and at the same time, the 6th and 7th amino acids and the fatty acyl side chain have hydrophobic interactions with the fatty acid part of lipid A. Its effect on - is stronger (see - ), so PMB has certain differences in the characteristics of Figure 1 and - and + bacteria in different samples, but shows a strong - targeting effect, is cheap, and is suitable for preparing functionalized MNP used for sample pretreatment. Previous research on PMB focused on the antibacterial field (Sci Adv, 2021, 7(32): eabj1577), which limited its application in bacterial adsorption and bacterial activity detection. In addition, the steric hindrance effect between nanoparticles will reduce the bacterial adsorption capacity. Therefore, improving the biocompatibility of PMB-functionalized nanospheres and reducing their steric hindrance effect are problems that need to be solved.

[0004] Polydopamine (PDA) is prepared by the self-polymerization of dopamine and has a large number of hydrophilic hydroxyl and amino groups, showing good biocompatibility. Modifying the surface of MNP with a PDA shell can not only provide the active groups required for subsequent modification but also prevent the direct contact between MNP and the biological system, improving the biocompatibility of MNP, which helps to solve the problem of the bacterial toxicity of PMB. Polyethylene glycol (PEG) is a linear long-chain polymer with good water solubility and biocompatibility and has diverse functional groups. Longer PEG side chains will provide more active groups, which can further reduce steric hindrance and help improve the adsorption capacity of MNP microspheres. Therefore, sequentially modifying PDA and PEG on MNP helps to provide the active groups required for subsequent modification, reduce the toxicity of PMB to bacteria, and improve the bacterial adsorption capacity of functionalized MNP. However, there is currently no report on polymyxin-functionalized MNP that sequentially modifies PDA and PEG for bacterial adsorption and activity detection.

[0005] The rapid detection of bacterial microorganisms is a difficult problem that urgently needs to be solved at present. The entire detection process of conventional bacterial activity detection in food, such as bacterial culture, counting, and biochemical tests, takes at least 5 days (Food, 2021, 10(10): 2402). Antibiotic-functionalized MNP can efficiently and rapidly enrich bacteria, showing promise for the rapid detection of bacterial activity. The extraction steps of existing commercial bacterial nucleic acid extraction kits are complicated and time-consuming, about 3 - 6 hours, which affects the rapid detection of bacterial nucleic acids. Combining antibiotic-functionalized MNP with a rapid nucleic acid extraction method is expected to achieve the rapid detection of bacterial nucleic acids. The simplest and fastest nucleic acid extraction method is the direct lysis method based on high temperature, namely the thermal lysis method. This method only uses boiling water to heat for 15 min, which is simple and fast and does not require the use of other chemical reagents. PDA, PEG, and PMB have good thermal stability (Food Chem, 2022, 379: 132148 and Small, 2022, 2106533), which provides the possibility of the combined application of MNP bacterial adsorption and the thermal lysis method. When the combined application of functionalized MNP bacterial adsorption and the thermal lysis method is carried out, the bacterial enrichment and the rapid nucleic acid extraction of bacteria can be organically combined, greatly simplifying the nucleic acid extraction steps, significantly shortening the time, and removing the interference of the matrix in the sample, improving the quality of rapidly extracted bacterial nucleic acids. For example, high temperature will cause the protein in the sample to denature and wrap the nucleic acid, and will also promote the release of enzyme reaction inhibitors, etc., reducing the quality of nucleic acid extraction, which is more significant in samples with high protein content (such as milk) (Microb Pathog, 2019, 129: 266 - 270 and Vet Res Forum, 2017, 8(1): 55 - 58). After separating bacteria using functionalized MNP and then extracting the nucleic acid of the bacteria bound to MNP by the thermal lysis method, this key problem affecting the nucleic acid extraction by the thermal lysis method can be overcome.

[0006] In actual samples, multiple types of bacteria often coexist. Currently, there is a lack of methods and materials for simultaneously separating and enriching different types of bacteria in the same sample. Therefore, the present invention utilizes the broad-spectrum bacterial binding property of PMB, and the prepared MNP PDA-PEG-PMB can effectively adsorb G - and G + bacteria simultaneously in tap water and urine samples with relatively simple sample matrices, and can only adsorb G - bacteria in milk with a relatively complex matrix. To achieve the efficient enrichment of G - and G + in complex samples, we combined MNP PDA-PEG-PMB with vancomycin-functionalized MNP (MNP + PEG-Van ) that can target and bind G - + and G + bacteria in milk simultaneously. Through the above design, MNP PDA-PEG-PMB is expected to efficiently enrich a series of bacteria, and can simultaneously perform rapid detection of bacterial activity and bacterial nucleic acids. Summary of the Invention

[0007] Aiming at the main problems that need to be solved by antibiotic-functionalized MNP in the rapid detection of bacterial activity and bacterial nucleic acids, the present invention first prepares MNP PDA-PEG-PMB with high biocompatibility, low toxicity, high adsorption capacity, and high heat tolerance. The polymyxin-functionalized magnetic nanospheres are amino-functionalized magnetic nanospheres surface-modified with a polydopamine shell layer, polyethylene glycol molecular arms, and polymyxin B.

[0008] According to the first aspect of the present invention, there is provided a polymyxin-functionalized magnetic nanosphere, wherein the polymyxin-functionalized magnetic nanosphere is an amino-functionalized magnetic nanosphere surface-modified with a polydopamine shell layer, polyethylene glycol molecular arms, and polymyxin B; the polydopamine shell layer coats the amino-functionalized magnetic nanosphere; both ends of the polyethylene glycol are modified with aldehyde groups, and the aldehyde groups at both ends are respectively connected to the amino groups on the polydopamine shell layer and polymyxin B.

[0009] Preferably, the amino-functionalized magnetic nanosphere is an amino-functionalized iron oxide nanosphere or an amino-functionalized iron oxide nanosphere.

[0010] According to another aspect of the present invention, there is provided a method for preparing the polymyxin-functionalized magnetic nanosphere, wherein after modifying the polydopamine shell layer on the surface of the amino-functionalized magnetic nanosphere, the molecular arm polyethylene glycol and polymyxin B are sequentially connected; both ends of the molecular arm polyethylene glycol are modified with aldehyde groups, and the aldehyde groups at both ends are respectively connected to the amino groups on the polydopamine shell layer and polymyxin B.

[0011] Preferably, the method specifically includes the following steps:

[0012] (1) Modify the polydopamine shell on the aminated magnetic nanospheres: Mix the aminated magnetic nanospheres, dopamine hydrochloride, and ethylenediamine, and the generated polydopamine coats the surface of the aminated magnetic nanospheres;

[0013] (2) Modify the polyethylene glycol molecular arms: Add polyethylene glycol modified with aldehyde groups at both ends to the intermediate product obtained in step (1), and the aldehyde groups react with the amino groups on the polydopamine through aldehyde-amine condensation reaction;

[0014] (3) Modify polymyxin B: Add polymyxin B to the intermediate product obtained in step (2), mix well, and then add a redox catalyst to make the aldehyde groups on the polyethylene glycol react with the amino groups on the polymyxin B through aldehyde-amine condensation reaction to obtain polymyxin-functionalized magnetic nanospheres.

[0015] Preferably, in step (1), the aminated magnetic nanospheres are aminated magnetite nanospheres or aminated iron oxide nanospheres.

[0016] According to another aspect of the present invention, there is provided the application of the polymyxin-functionalized magnetic nanospheres as described above for adsorbing bacteria.

[0017] Preferably, adsorb Gram-negative bacteria and / or Gram-positive bacteria in different samples.

[0018] Preferably, the polymyxin-functionalized magnetic nanospheres are combined with another magnetic nanosphere for adsorbing Gram-negative bacteria and Gram-positive bacteria in various samples; the other magnetic nanosphere is a carboxylated magnetic nanosphere surface-modified with polyethylene glycol and vancomycin, and both ends of the polyethylene glycol are modified with amino groups and are respectively connected to the carboxylated magnetic nanosphere and vancomycin.

[0019] Preferably, after adsorbing bacteria, the nucleic acid of the bacteria bound to the polymyxin-functionalized magnetic nanospheres is extracted by the thermal cleavage method and then used for qPCR detection to improve the sensitivity and detection speed of nucleic acid identification, thereby realizing the rapid identification of bacterial nucleic acids in samples.

[0020] Preferably, after adsorbing bacteria, the polymyxin-functionalized magnetic nanospheres do not affect the activity of the adsorbed bacteria, and the polymyxin-functionalized magnetic nanospheres adsorbed with bacteria are directly added to a solid or liquid culture medium for cultivation to realize the rapid detection of bacterial activity.

[0021] Generally speaking, compared with the prior art by the above technical solutions conceived by the present invention, the following beneficial effects can be achieved:

[0022] (1) In the present invention, on the MNP -NH2After surface modification of the PDA shell, the active groups such as amino and carboxyl groups on the surface of MNP can be increased, and the content of PMB bound to the microspheres also increases accordingly. At the same time, PEG reduces the steric hindrance during the adsorption of MNP to bacteria, so the adsorption capacity is greatly improved. MNP PDA-PEG-PMB can efficiently adsorb G - and G + bacteria in PBS, tap water and urine with simple matrices, but can only enrich G - bacteria in milk with complex matrices.

[0023] (2) MNP PDA-PEG-PMB combined with MNP + targeting and binding to G PEG-van bacteria can simultaneously and efficiently enrich G - and G + bacteria in complex samples (milk).

[0024] (3) The highly biocompatible PDA and PEG modified on MNP PDA-PEG-PMB can effectively reduce the toxicity of PMB to bacteria. After MNP PDA-PEG-PMB adsorbs bacteria, it does not affect the bacterial activity. The MNP PDA-PEG-PMB bound to bacteria can be directly added to solid medium or liquid medium for cultivation to quickly determine the bacterial activity.

[0025] (4) The MNP PDA-PEG-PMB constructed in the present invention has the advantages of high heat tolerance and reusability, and can directly extract the nucleic acids of the bacteria it binds by thermal cracking method for qPCR with high nucleic acid quality requirements. Bacterial separation and nucleic acid extraction are completed within 45 minutes, significantly improving the nucleic acid detection speed, and are applicable to rapid bacterial nucleic acid detection.

[0026] (5) The MNP PDA-PEG-PMB magnetic nanospheres of the present invention are prepared under simple conditions and do not require complex synthesis steps, being suitable for large-scale industrial production. Brief Description of the Drawings

[0027] Figure 1 is a schematic diagram of the preparation steps of the multifunctional magnetic nanospheres MNP PDA-PEG-PMB of the present invention (A), a schematic diagram of the principle of PMB binding to G - bacteria (B) and a schematic diagram of the separation, enrichment of bacteria and rapid detection of bacterial activity and bacterial nucleic acids by MNP PDA-PEG-PMB ;

[0028] Figure 2 is MNP -NH2 (A), MNP PDA (B), MNP PDA-PEG (C), MNP PDA-PEG-PMB(D) Transmission electron microscope image of (D) and MNP PDA-PEG-PMB Particle size distribution diagram (E);

[0029] Figure 3 is MNP -NH2 (A), MNP PEG (B), MNP PEG-PMB (C) Transmission electron microscope image of (C) and MNP PEG-PMB Particle size distribution diagram (D);

[0030] Figure 4 is MNP PDA-PEG-PMB Infrared spectra (A) and thermogravimetric analysis (C) of MNP and each microsphere at different functionalization stages; MNP PEG-PMB Infrared spectra (B) and thermogravimetric analysis (D) of MNP and each microsphere at different functionalization stages;

[0031] Figure 5 is MNP PDA-PEG-PMB Scanning electron microscope images of binding to E. coli (A) and S. aureus (B);

[0032] Figure 6 is MNP PDA-PEG-PMB Growth and survival rates (D) of captured E. coli (A) and S. aureus (B) in liquid and solid media (C);

[0033] Figure 7 is 10 2 -10 9 CFU / mL E. coli adsorbed by 500 μg / mL MNP PDA-PEG-PMB and each microsphere at different functionalization stages, colony growth (A) and adsorption rate (B) in the supernatant; Figure 7 C in is 500 μg / mL MNP PDA-PEG-PMB Adsorption rates of 10 3 CFU / mL E. coli, P. aeruginosa, S. aureus and S. epidermidis respectively;

[0034] Figure 8 is 500 μg / mL MNP PDA-PEG-PMB Adsorption kinetic curves of 500 μg / mL MNP for 10 3 CFU / mL E. coli and S. aureus;

[0035] Figure 9 is 500 μg / mL MNP PDA-PEG-PMB and each microsphere at different functionalization stages in LB liquid medium with different mass fractions for 10 3 CFU / mL bacteria adsorption rate;

[0036] Figure 10 A in is 500 μg / mL MNP PDA-PEG-PMB The microspheres at different functional stages were treated at high temperature for 10 3 Adsorption rate of CFU / mL bacteria; Figure 10 B in the sample is 500 μg / mL MNP PDA-PEG-PMB After high temperature treatment, reuse 1-3 times for 10 3 Adsorption rate of CFU / mL bacteria; Figure 10 C in the figure is the MNP stored at different times PDA-PEG-PMB 10 3 Adsorption rate of CFU / mL bacteria;

[0037] Figure 11 500 μg / mL MNP PDA-PEG-PMB 、MNP PEG-Van 、MNP PDA-PEG-PMB With MNP PEG-Van The mixed microspheres were used to detect 10 2 Adsorption rate of CFU / mL E. coli (A) and S. aureus (B). DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0039] Figure 1 A in the equation is MNP PDA-PEG-PMB Schematic diagram of the preparation steps; Figure 1 The B in the equation is PMB combined with G - Schematic diagram of bacteria; Figure 1 C in the equation is MNP PDA-PEG-PMB Schematic diagram of separation and enrichment of bacteria and rapid detection of bacterial activity and bacterial nucleic acid.

[0040] The present invention discloses a polymyxin functionalized magnetic nanoparticle (MNP) for targeted and efficient separation and enrichment of a series of pathogens. PDA-PEG-PMB ), MNP PDA-PEG-PMB It has a highly efficient adsorption effect on a series of bacteria, does not affect the activity of the adsorbed bacteria and is resistant to high temperatures. It can be used for rapid detection of bacterial activity and bacterial nucleic acid at the same time.

[0041] MNP of the present invention PDA-PEG-PMB The preparation method of aminated magnetic nanoparticles (MNP-NH2 ) After the surface is modified with a polydopamine (PDA) shell layer, a long-chain molecular arm polyethylene glycol (PEG) and the antibiotic polymyxin B (PMB) are sequentially connected; the specific preparation process is as follows:

[0042] Step 1: Modify the PDA shell layer on MNP -NH2 : Mix equal volumes of 5 mg / mL MNP -NH2 , 50 mg / mL dopamine hydrochloride, and 90 mg / mL ethylenediamine, and mechanically stir in a 30 °C water bath for 12 - 16 h. After the reaction is completed, wash twice with deionized water, and finally disperse in phosphate buffer (PB) to obtain MNP PDA ;

[0043] Step 2: Modify the PEG molecular arm on MNP PDA : Add 3 - 12 mg / mL CHO-PEG-CHO (5000 DA) to the above solution, ultrasonically disperse, and then mechanically stir in a 30 °C water bath for 12 - 24 h. Then wash 2 - 3 times with PB buffer solution to obtain MNP PDA-PEG ;

[0044] Step 3: Modify PMB on MNP PDA-PEG : Add 0.5 - 2 mg / mL PMB to the above solution, mechanically stir in a 30 °C water bath for 12 - 18 h, then add the redox catalyst sodium cyanoborohydride (NaCNBH 3 ) and continue stirring for 3 h. After the reaction is completed, wash 2 - 3 times with deionized water to obtain MNP PDA-PEG-PMB .

[0045] In some embodiments, the optimal concentration of CHO-PEG-CHO is 6 mg / mL and the connection time is 12 h; the optimal concentration of PMB is 2 mg / mL and the connection time is 18 h.

[0046] In the present invention, coating the PDA shell layer on the surface of MNP helps to increase the content of PMB bound to the surface of MNP; on the other hand, the long-chain molecular arm PEG effectively reduces the steric hindrance effect when MNP binds to bacteria, thereby further increasing the adsorption capacity. Specifically, MNP PDA-PEG-PMB can efficiently adsorb G - (Escherichia coli, E. coli) and G + (Staphylococcus aureus, S. aureus) in PBS, and the maximum adsorption capacity is 2.00×10 9 CFU / mg, and the maximum adsorption rate is 100%.

[0047] The MNP PDA-PEG-PMB magnetic nanospheres in the present invention, MNP PDA-PEG-PMBIt can efficiently adsorb a variety of bacteria; PMB can bind to a variety of bacteria using electrostatic and hydrophobic forces, but has a stronger binding ability to G - ; It can efficiently adsorb G + and G - bacteria simultaneously in PBS, tap water, and urine samples with a relatively simple matrix, and can only adsorb G - bacteria in milk with a relatively complex matrix.

[0048] The MNP PDA-PEG-PMB magnetic nanospheres in the present invention, when used in combination with vancomycin (Van)-functionalized MNP + that can specifically bind to G PEG-van , can efficiently enrich G - bacteria and G + bacteria in complex samples (milk) simultaneously.

[0049] The MNP PDA-PEG-PMB prepared in the present invention has good anti-interference ability, maintains adsorption stability after being treated at 95 °C, and still maintains adsorption ability after being reused three times. MNP PDA-PEG-PMB still maintains adsorption stability after being stored for 12 weeks.

[0050] The MNP PDA-PEG-PMB magnetic nanospheres in the present invention, the nucleic acids of bacteria bound by MNP PDA-PEG-PMB extracted by the thermal cracking method can be directly used for qPCR detection, improving the sensitivity and detection speed of nucleic acid identification and realizing the rapid identification of a series of bacterial nucleic acids in complex samples.

[0051] The MNP PDA-PEG-PMB prepared in the present invention binds the bacterial nucleic acids extracted by the thermal cracking method within 45 min and can be directly used for qPCR detection. The detection limits of Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) in PBS, tap water, urine, and milk are all 10 2 CFU / mL, but S.aureus in milk cannot be measured. If MNP PDA-PEG-PMB and MNP PEG-Van are used simultaneously, then 10 2 CFU / mL E.coli and S.aureus in milk can be detected, thus rapidly enriching and identifying a series of bacteria in complex samples.

[0052] The PDA and PEG with high biocompatibility on the surface of MNP PDA-PEG-PMB in the present invention reduce the toxicity of PMB to bacteria. MNP PDA-PEG-PMB does not affect the activity of the bound bacteria. The MNP PDA-PEG-PMB microspheres adsorbed with bacteria can be directly added to solid and liquid culture media for cultivation to rapidly detect the bacterial activity.

[0053] Therefore, the MNP prepared by the present invention PDA-PEG-PMB can efficiently enrich various bacteria in the sample and has good application prospects in the rapid detection of bacterial activity and bacterial nucleic acid.

[0054] Example 1 MNP PDA-PEG-PMB Preparation of magnetic nanospheres

[0055] By optimizing the connection time (5, 12, and 24 h), concentration of CHO-PEG-CHO (3, 6, and 12 mg / mL), connection time of PMB (5, 12, and 18 h), and concentration of PMB (0.5, 1, and 2 mg / mL), the optimal preparation conditions for MNP were obtained as follows: the concentration of PEG molecular arm was 6 mg / mL and the connection time was 12 h; the concentration of PMB was 2 mg / mL and the connection time was 18 h. Therefore, 5 mg / mL MNP PDA-PEG-PMB , 50 mg / mL dopamine hydrochloride, and 90 mg / mL ethylenediamine were mixed in equal volumes, mechanically stirred in a 30 °C water bath for 16 h, and after the reaction was completed, washed twice with deionized water to prepare MNP -NH2 . 6 mg / mL CHO-PEG-CHO was added to the above solution, mechanically stirred in a 30 °C water bath for 12 h, and after the reaction was completed, washed twice to prepare MNP PDA . 2 mg / mL PMB was added to the above solution, mechanically stirred in a 30 °C water bath for 18 h, then NaCNBH PDA-PEG was added, and stirring was continued for 3 h. After the reaction was completed, washed twice to finally prepare MNP 3 . At the same time, under the same conditions, PEG and PMB were directly connected to MNP PDA-PEG-PMB to obtain MNP -NH2 as a control. PEG-PMB

[0056] Example 2 Characterization of various magnetic nanospheres

[0057] From A, B, C, and D in Figure 2 and A, B, and C in Figure 3 , the transmission electron microscopy results show that the prepared MNP PDA-PEG-PMB and MNP PEG-PMB and each microsphere at different functionalization stages are all monodisperse spherical, with uniform size. The average particle sizes of MNP PDA-PEG-PMB and MNP PEG-PMB are 220 nm and 210 nm respectively. As shown by E in Figure 2 and D in Figure 3 , dynamic light scattering analysis (DLS) measured MNP PDA-PEG-PMBThe particle size distribution ranges from 61 to 550 nm, with an average particle size of 235 ± 11.7 nm, MNP PEG-PMB The particle size distribution ranges from 100 to 595 nm, with an average particle size of 225.16 ± 12.3 nm. The particle size distribution is relatively uniform, which is consistent with the TEM results.

[0058] MNP PDA-PEG-PMB The Fourier transform-infrared absorption spectrum analysis of MNP Figure 4 is shown as A in PDA In the spectrum of MNP -1 the peaks at 3521 cm -1 and 3446 cm -1 disappear and become a large broad peak near 3435 cm -1 , which is the result of the association of amino groups and hydroxyl groups in PDA. The characteristic absorption peaks of the benzene ring at 1830 cm -1 -544 cm -1 are caused by the deformation and stretching of the benzene ring. The combination of amino and hydroxyl groups and the confirmation of the benzene ring confirm that PDA is successfully modified on the surface of MNP -NH2 . In the spectrum of MNP PDA-PEG the infrared peak shape change between 3435 cm -1 and 1830 cm -1 is basically the same as that of MNP PDA . The amide peaks at 1620 cm -1 and 1560 cm -1 prove that PEG is successfully connected to MNP PDA . In the spectrum of MNP PDA-PEG-PMB the stretching vibration peaks of methyl groups at 2926 cm -1 -2852 cm -1 indicate that PMB is successfully introduced into MNP PDA-PEG . Similarly, in the spectra of MNP PEG and MNP PEG-PMB the introduction of amide bonds (1620 cm -1 and 1560 cm -1 ) proves that PEG is successfully connected to MNP NH2 . The methyl stretching vibration peaks (2929 cm -1 -2852 cm -1 ) prove that PMB is successfully introduced into MNP PEG ( Figure 4 B in -NH2 . Therefore, in the present invention, PDA, PEG molecular arms and PMB are modified on the surface of MNP PDA-PEG-PMB and MNP PEG-PMB are successfully prepared.

[0059] Thermogravimetric analysis shows that MNP PDAshowed a weight loss of 11.06% in the range of 200 - 1000 °C, compared with MNP -NH2 and an additional weight loss of 5.61% was due to the decomposition of PDA. Compared with MNP PDA samples, MNP PDA-PEG samples showed a weight loss of 42.68% in the temperature range of 200 - 1000 °C, and the additional weight loss of 31.62% was caused by the high-temperature decomposition of PEG in a nitrogen atmosphere. MNP PDA-PEG-PMB samples showed a weight loss of 58.01% in the temperature range of 200 - 1000 °C. Compared with MNP PDA-PEG and an additional weight loss of 15.33% was due to the decomposition of PMB ( Figure 4 C). Based on this calculation, the PMB content on the surface of MNP PDA-PEG-PMB was 119 μmol / g. MNP PEG showed a weight loss of 7.41% in the range of 200 - 1000 °C. Compared with MNP -NH2 and an additional weight loss of 1.96% could be attributed to the high-temperature decomposition of PEG. MNP PEG-PMB showed a weight loss of 12.87% in the range of 200 - 1000 °C. Compared with MNP PEG and an additional weight loss of 5.46% was due to the high-temperature decomposition of PMB. Based on this calculation, the PMB content on the surface of MNP PEG-PMB was 42.54 μmol / g ( Figure 4 D), and the PMB content on the surface of MNP PDA-PEG-PMB was significantly higher than that of MNP PEG-PMB , which proved that the modification of the PDA shell increased the PMB content on the surface of the microspheres.

[0060] Example 3 Binding characteristics of MNP PDA-PEG-PMB magnetic nanospheres to bacteria

[0061] Scanning electron microscopy (SEM) showed that after adding bacteria to the functionalized MNP, MNP PDA-PEG-PMB aggregated on the surface of E. coli ( Figure 5 A) and S. aureus ( Figure 5 B) and showed good targeting effects. In addition, MNP PDA-PEG-PMB showed a monodisperse spherical shape, uniform and stable, which was consistent with the TEM characterization results.

[0062] Example 4 Effect of MNP PDA-PEG-PMB magnetic nanospheres on bacterial growth

[0063] The MNP PDA-PEG-PMB combined with E. coli or S. aureusCultured by directly adding into the liquid medium, the growth lag phase was 0 - 0.5 h, and the exponential growth phase started after 0.5 h. MNP PDA-PEG-PMB The captured E. coli( Figure 6 in A) or S. aureus( Figure 6 in B) had similar growth characteristics to those cultured in the pure bacterial solution with the same concentration and grew well. This indicated that MNP PDA-PEG-PMB had no inhibitory effect on the growth of bacteria and did not affect the bacterial activity. Culturing the E. coli or S. aureus combined with MNP PDA-PEG-PMB in the solid medium, both the captured E. coli and S. aureus grew well( PDA-PEG-PMB in C), and the number of colonies had no significant difference from that of the positive control( Figure 6 in D). This indicated that the binding of MNP Figure 6 to bacteria had no effect on the bacterial activity and could be directly cultured in solid or liquid media to rapidly detect the bacterial activity. PDA-PEG-PMB

[0064] Example 5 Performance evaluation of the adsorption of MNP PDA-PEG-PMB magnetic nanospheres on bacteria

[0065] As shown in Table 1, the surface of MNP -NH2 was first modified with a PDA shell layer, which could increase its surface functionalized binding sites. Therefore, the adsorption capacity of MNP PDA was greater than that of MNP -NH2 . After modifying the surface of MNP PDA with a PEG shell layer, it helped to reduce the steric hindrance effect of the binding between bacteria and MNP. Therefore, the adsorption capacity of MNP PDA-PEG was greater than that of MNP PDA . The PDA shell layer could increase the functionalized binding sites, thereby increasing the binding amount of PMB, increasing the binding sites between MNP and bacteria, and increasing its adsorption capacity. At the same time, after the long-chain PEG was connected, MNP helped to improve the higher freedom of the surface PMB, and the steric hindrance effect was relatively weakened. The two together improved the bacterial adsorption capacity of MNP PDA-PEG-PMB microspheres. Therefore, the adsorption capacity of MNP PDA-PEG-PMB was higher than that of MNP PDA-PEG .

[0066] Table 1 Adsorption capacity of MNP PDA-PEG-PMB and microspheres at different functionalized stages

[0067]

[0068] MNP PDA-PEG-PMB had an adsorption rate of up to 100% for E. coli at various concentrations, and its bacterial adsorption effect was better than that of MNP PDA-PEG ​Control groups such as ( Figure 7 A and B in). In addition, MNP PDA-PEG-PMB also has an adsorption rate of over 90% for two other bacteria (G - , Pseudomonas aeruginosa, P. aeruginosa and G + , Staphylococcus epidermidis, S. epidermidis), showing a high adsorption effect ( Figure 7 C in). This proves that MNP PDA-PEG-PMB has a high separation and enrichment effect on various G - and G + bacteria in PBS. MNP PDA-PEG-PMB reaches the adsorption equilibrium and the maximum adsorption rate (100%) for bacteria (E. coli and S. aureus) in only 30 minutes, and the adsorption capacity is as high as 2000 CFU / mg at this time ( Figure 8 ). This shows that MNP PDA-PEG-PMB has the kinetic characteristics of high adsorption capacity and rapid achievement of adsorption equilibrium.

[0069] Example 6 Evaluation of the anti-interference performance of MNP PDA-PEG-PMB magnetic nanospheres

[0070] Even in 100% LB liquid medium, the adsorption rate of MNP PDA-PEG-PMB for E. coli is still as high as 100%. The adsorption rates of various other unmodified PMB microspheres for E. coli in LB buffer are significantly reduced because they cannot bind to the lipopolysaccharide on the surface of E. coli and cannot resist the interference of the LB matrix ( Figure 9 ). This shows that PMB-functionalized MNP can resist the interference of complex matrices and maintain the bacterial adsorption efficiency, and can be used for the separation and enrichment of bacteria in actual samples.

[0071] Example 7 Stability analysis of MNP PDA-PEG-PMB magnetic nanospheres

[0072] After high-temperature treatment, the adsorption rate of MNP PDA-PEG-PMB for E. coli is still 100%, and the adsorption effect is better than that of the control groups such as MNP PDA-PEG ( Figure 10 A in). The bacterial adsorption rates of MNP PDA-PEG-PMB reused 1 - 3 times after high-temperature heating are all maintained above 80%, which shows that MNP PDA-PEG-PMB has excellent reusability ( Figure 10 B in). Within 0 - 12 weeks, as the storage time prolongs, the adsorption rate of MNP PDA-PEG-PMB for bacteria is always as high as 100% ( Figure 10 C in). These results prove that MNP PDA-PEG-PMBHigh temperature tolerance, reusability, and long-term storage.

[0073] Example 8 Evaluation of the isolation and enrichment of bacteria by various functionalized magnetic nanospheres in actual samples MNP PDA-PEG-PMB Can efficiently enrich E. coli in PBS, tap water, and urine ( Figure 11 A in) and S. aureus ( Figure 11 B in), but can only efficiently enrich E. coli in diluted milk ( Figure 11 A in). This shows that the complex sample matrix significantly reduces the ability of PMB to bind to G + . To adsorb S. aureus in milk with a complex matrix, according to the literature strategy (ACS Nano, 2008, 2(9): 1777-88), the present invention uses an amide condensation reaction to connect a PEG molecular arm (NH 2 -PEG-NH 2 ) to carboxylated MNP to obtain MNP -PEG ; then, Van is connected to MNP -PEG by an amide condensation reaction to obtain MNP + that targets and binds to G PEG-Van bacteria. MNP PEG-Van can efficiently enrich S. aureus ( Figure 11 B in) in various samples (PBS, tap water, urine, and diluted milk), and cannot enrich E. coli ( Figure 11 A in). The combined use of MNP PDA-PEG-PMB and MNP PEG-Van can simultaneously and efficiently enrich E. coli and S. aureus in various samples ( Figure 11 ).

[0074] Example 9 Isolation and enrichment of a series of bacteria in actual samples and nucleic acid detection by using functionalized magnetic nanospheres alone or in combination

[0075] Using E. coli and S. aureus as model bacteria, a standard curve was established by qPCR. The nucleic acid of bacteria extracted by the kit (positive control) was used as a quality control, and its Ct value was 38.08 ± 0.13. Substituting it into the standard curve, the ratio of the recovered concentration to the spiked concentration, that is, the recovery rate, was 105.65%, which was not significantly different from the detection results of the MNP PDA-PEG-PMB group. After MNP PDA-PEG-PMB combined with the thermal cracking method to extract nucleic acid, the lowest detection limit of E. coli in PBS by qPCR was 10 2 CFU / mL (Table 2).

[0076] Table 2 Ct values and recovery rates of E. coli detected by MNP PDA-PEG-PMB combined with qPCR

[0077]

[0078] qPCR could not identify the samples in which nucleic acids were directly extracted by thermal cracking method without MNPs enrichment (Table 3), indicating that functionalized MNP pretreatment was necessary. When the enrichment multiple of MNPs was 100, the detection limits of qPCR for E. coli and S. aureus in various samples such as PBS, tap water, urine and 25% milk were 10 2 CFU / mL. The MNP PDA-PEG-PMB group could effectively detect E. coli in various samples, but could only detect S. aureus in PBS, tap water and urine, while the mixed microsphere group could effectively detect E. coli and S. aureus in various samples. When the enrichment multiple of MNPs was 200, the detection limits of qPCR for E. coli and S. aureus in PBS and pure milk were reduced to 50 CFU / mL. The MNP PDA-PEG-PMB group could effectively identify E. coli in PBS and 100% milk, and the mixed microsphere group could effectively identify E. coli and S. aureus in PBS and pure milk simultaneously (Table 3).

[0079] Table 3 Recovery rates and detection limits of bacteria in samples detected by functionalized MNPs combined with qPCR

[0080]

[0081]

[0082] Note: a and b are 10 2 CFU / mL E. coli and S. aureus respectively. c is 10 2 CFU / mL E. coli and S. aureus (1:1) mixed bacterial solution

[0083] In summary, functionalized MNPs and a simple thermal cracking method can extract nucleic acids within 45 minutes. Compared with commercial bacterial nucleic acid extraction kits, the operation is simple and the time used is shorter. The extracted nucleic acids can be directly used for qPCR detection. For samples with simple sample matrices such as PBS, tap water and urine, MNP PDA-PEG-PMB combined with qPCR can identify G - and G + bacteria simultaneously. However, for samples with complex sample matrices such as milk, it is necessary to mix and use MNP PDA-PEG-PMB and MNP PEG-Van two kinds of MNPs to enrich and identify G - and G + bacteria simultaneously. By increasing the sample volume to increase the enrichment multiple, the sensitivity of qPCR for identifying bacteria can be improved.

[0084] Those skilled in the art can easily understand that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A polymyxin-functionalized magnetic nanosphere, characterized in that, the polymyxin-functionalized magnetic nanosphere is an amino-functionalized magnetic nanosphere with a polydopamine shell, a polyethylene glycol molecular arm, and polymyxin B modified on its surface; the polydopamine shell coats the amino-functionalized magnetic nanosphere; both ends of the polyethylene glycol are modified with aldehyde groups, and the aldehyde groups at both ends are respectively connected to the amino groups on the polydopamine shell and polymyxin B.

2. The polymyxin-functionalized magnetic nanosphere according to claim 1, characterized in that, the amino-functionalized magnetic nanosphere is an amino-functionalized iron oxide nanosphere or an amino-functionalized iron oxide nanosphere.

3. The preparation method of the polymyxin-functionalized magnetic nanosphere according to claim 1 or 2, characterized in that, after modifying the polydopamine shell on the surface of the amino-functionalized magnetic nanosphere, the molecular arm polyethylene glycol and polymyxin B are sequentially connected; both ends of the molecular arm polyethylene glycol are modified with aldehyde groups, and the aldehyde groups at both ends are respectively connected to the amino groups on the polydopamine shell and polymyxin B.

4. The preparation method of the polymyxin-functionalized magnetic nanosphere according to claim 3, characterized in that, specifically includes the following steps: (1) Modifying the polydopamine shell on the amino-functionalized magnetic nanosphere: mixing the amino-functionalized magnetic nanosphere, dopamine hydrochloride, and ethylenediamine, and the generated polydopamine coats the surface of the amino-functionalized magnetic nanosphere; (2) Modifying the polyethylene glycol molecular arm: adding polyethylene glycol with aldehyde groups modified at both ends to the intermediate product obtained in step (1), and the aldehyde group reacts with the amino group on the polydopamine through aldehyde-amine condensation reaction; (3) Modifying polymyxin B: adding polymyxin B to the intermediate product obtained in step (2), mixing well, and then adding a redox catalyst to make the aldehyde group on the polyethylene glycol react with the amino group on polymyxin B through aldehyde-amine condensation reaction to obtain the polymyxin-functionalized magnetic nanosphere.

5. The preparation method of the polymyxin-functionalized magnetic nanosphere according to claim 4, characterized in that, in step (1), the amino-functionalized magnetic nanosphere is an amino-functionalized iron oxide nanosphere or an amino-functionalized iron oxide nanosphere.

6. The application of the polymyxin-functionalized magnetic nanosphere according to claim 1 or 2, characterized in that, it is used for adsorbing bacteria.

7. The application according to claim 6, characterized in that, it adsorbs Gram-negative bacteria and / or Gram-positive bacteria in different samples.

8. The application according to claim 6, characterized in that, the polymyxin-functionalized magnetic nanosphere is combined with another magnetic nanosphere for adsorbing Gram-negative bacteria and Gram-positive bacteria in various samples; the other magnetic nanosphere is a carboxyl-functionalized magnetic nanosphere with polyethylene glycol and vancomycin modified on its surface, and both ends of the polyethylene glycol are modified with amino groups and are respectively connected to the carboxyl-functionalized magnetic nanosphere and vancomycin.

9. The application according to claim 6, characterized in that, after adsorbing bacteria, the nucleic acid of the bacteria bound to the polymyxin-functionalized magnetic nanosphere is extracted by the thermal cracking method, and then used for qPCR detection.

10. The application according to claim 6, Characterized in that, After adsorbing bacteria, the polymyxin-functionalized magnetic nanospheres do not affect the activity of the adsorbed bacteria, and the polymyxin-functionalized magnetic nanospheres adsorbed with bacteria are directly added to a solid or liquid culture medium for cultivation.

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