CN114460288A - Preparation method of functionalized magnetic beads for broad-spectrum separation of bacteria - Google Patents

Abstract

The invention discloses a preparation method of functionalized magnetic beads for wide-spectrum separation of bacteria, and belongs to the field of biotechnology. The method of the invention includes the coupling of nano-magnetic beads and cefepime (Cefepime, Cefe), the cefepime-modified nano-magnetic bead complexes capturing the target bacteria in the sample liquid, and by the action of an external magnetic field, the captured target bacteria are combined with The sample solution is separated and resuspended. The target bacteria captured by the magnetic separation of the present invention can be directly subjected to subsequent analysis; compared with the traditional magnetic separation method of bacteria, the method of the present invention overcomes the difficulty of "no omission" enrichment and separation of bacteria in the sample, and improves the accuracy of the sample. While improving the efficiency of bacterial isolation, it also reduces costs.

Description

Preparation method of functionalized magnetic beads for broad-spectrum separation of bacteria

technical field

The invention belongs to the field of biotechnology, in particular to a method for preparing functionalized magnetic beads for broad-spectrum separation of bacteria.

Background technique

Diseases caused by pathogenic microorganisms are diseases with high morbidity and mortality worldwide, causing millions of infections every year and causing major obstacles to socio-economic development. Human infection with bacteria can cause bacteremia, meningitis and other related diseases. From 1980 to 2014, more than four million people in the United States died from various infectious diseases caused by pathogenic microorganisms. Therefore, it is necessary to develop rapid screening methods for pathogenic bacteria. In view of the need to establish an efficient, rapid, and matrix-resistant detection method, sample pretreatment techniques based on functionalized nanomagnetic beads have been rapidly developed. SUMMARY OF THE INVENTION

Aiming at the defects of the prior art that only a single target bacteria can be enriched and separated and the efficiency is low, the purpose of the present invention is to provide a method for preparing functionalized magnetic beads for broad-spectrum separation of bacteria, which can Quickly separate bacteria from complex matrices, with high capture efficiency, easy operation and short separation time.

The present invention is specifically realized through the following technical solutions:

The preparation method of functionalized magnetic beads for broad-spectrum separation of bacteria includes the following steps:

1) Wash the magnetic nanobeads and resuspend them in sterile PBS solution;

2) Dissolve EDC and NHSS in sterile PBS solution respectively, then add them to the solution obtained in step 1), activate at room temperature for 1-2 hours, and wash and resuspend the activated magnetic nanobeads again;

3) Dissolve cefepime in sterile ultrapure water, then add it to the nanomagnetic bead solution washed again after activation in step 2), and react for 3 to 6 hours, and the obtained reaction product is washed and resuspended to obtain cefepime-modified nanomagnets. bead complex;

4) Add the cefepime-modified nanomagnetic bead complex prepared in step 3) to the solution containing bacteria, mix and incubate at 180 rpm for 20-40 minutes at 37°C, and then insert a magnetic stand to separate for 4-5 minutes. The product is washed and resuspended to obtain the bacteria-cefepime-nanomagnetic bead complex.

Further, the surface of the magnetic nano-beads in step 1) is carboxylated, and the particle size thereof is 180 nm.

Further, sterile PBS solution was used for the washing and resuspension.

Further, the concentration of the sterile PBS solution is 0.01M, and the pH is 7.4.

Further, the cefepime in step 3) is the fourth generation cephalosporin, and its molecular weight is 480.56.

The cefepime used in the present invention contains an amino functional group, which can be connected with the carboxyl group on the surface of the nano-magnetic beads through the amino group, and then the cefepime is connected with the penicillin-binding protein on the surface of the bacteria through covalent interaction, so as to achieve the purpose of enrichment and separation in the present invention . The specific principle is shown in Figure 1.

Further, the mass ratio of the nano-magnetic beads in step 1) to the cefepime in step 3) is 5:7.

Further, the mass ratio of EDC and NHSS described in step 2) is 2.90:3.26.

Compared with the prior art, the beneficial effects of the present invention are:

1. This method is suitable for enrichment and separation of various bacteria from complex sample matrices, such as food, environmental or hospital samples. Food samples include all kinds of fresh or frozen processed food materials, such as fresh vegetables, meat, seafood and other products; environmental samples include sewage, soil, etc.; hospital samples include various nosocomial infection samples, such as blood samples, cerebrospinal fluid, ascites, etc. . Samples can be processed according to conventional methods. For example, a solid sample can be crushed to make a solution to be tested, and a liquid sample can be directly diluted to make a solution to be tested.

2. The present invention uses nano-magnetic beads with a particle size of 180 nm, which is mainly used for the rapid enrichment of target bacteria in the matrix, while the prior art uses a particle size of 30 nm or 50 nm. The enrichment method of magnetic beads combined with dendrimer is used for magnetic signals. magnification.

3. The cefepime used in the present invention is a fourth-generation cephalosporin, which has a strong affinity with the penicillin-binding protein on the surface of bacteria, and has the advantages of good stability, low cost, and controllable quality compared with antibodies. Based on the above advantages, in a single separation process, the cost of the cefepime-nanomagnetic bead complex constructed by the present invention is greatly lower than that of immunomagnetic separation, and the material shelf life is longer than that of immunomagnetic beads.

4. Bacterial infection is often the coexistence of a variety of pathogenic bacteria. The cefepime used in the present invention is a molecular recognition agent for broad-spectrum recognition of bacteria, and is used to separate bacteria in a matrix. Identification by chain reaction (PCR) or traditional culture methods. In contrast, antibodies can only identify the corresponding target bacteria specifically, and other pathogenic bacteria present in the matrix cannot be identified, so they are not suitable for rapid screening of samples of a large number of unknown target bacteria.

5. The cefepime-nano magnetic bead complex synthesized by the present invention first activates the carboxyl group on the surface of the nano magnetic bead through EDC and NHSS, and then utilizes the activated carboxyl group to covalently bond with the amino group on cefepime, and the Cefepime is coupled to the surface of magnetic nanobeads. Compared with the method of bovine serum albumin or polyethylene glycol-mediated coupling, the method for synthesizing the cefepime-nanomagnetic bead complex of the present invention is simpler, takes less time and costs less.

6. In the present invention, cefepime is modified on the surface of the nano-magnetic beads. Compared with the method in which the vancomycin-modified nano-magnetic beads can only separate Gram-positive bacteria, the cefepime used in the present invention can identify more Bacteria, because the target of cefepime (penicillin-binding protein) is a membrane protein necessary for the synthesis of bacterial cell walls, so its broad spectrum is stronger, more types of bacteria can be separated, and it has higher separation efficiency.

7. The cefepime-nanomagnetic bead complex synthesized by the present invention interacts with the penicillin-binding protein on the surface of bacteria through the β-lactam ring in cefepime to form a covalent bond, compared with antibodies or polymers, etc. The identification molecule is based on the method of hydrogen bonding or electrostatic interaction. The cefepime-nanomagnetic bead complex of the present invention has stronger interaction force with bacteria and stronger ability to separate bacteria, and is especially suitable for enrichment and separation of bacteria in complex samples .

Description of drawings

FIG. 1 is a schematic diagram of a preparation process of a functionalized magnetic bead in a specific embodiment of the present invention;

FIG. 2 is a schematic diagram of another preparation process of functionalized magnetic beads in a specific embodiment of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The selected nano-magnetic beads (180nm) of the present invention were purchased from Shanghai Aorun Co., Ltd.

The cefepime (molecular weight 480.56) selected in the present invention was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

NHSS (N-hydroxythiosuccinimide sodium salt), EDC (ethyl 3-(3-dimethylamino)carbodiimide hydrochloride) and the like are conventional reagents, and will not be repeated here.

The preparation method of 0.01M PBS used in the present invention is as follows: 8.0g NaCl, 0.2g KCl, 0.24g KH ₂ PO ₄ , 1.44g Na ₂ HPO ₄ are dissolved in 800 mL of distilled water, the pH is adjusted to 7.4 with 5M NaOH, and the volume is adjusted to 1000 mL. have to.

Example 1

1. Synthesis of cefepime-nanomagnetic bead complex, prepared according to the following steps:

(1) Aspirate 1 mL of magnetic nanobeads (10 mg/mL), add it to 9 mL of sterile PBS solution of pH 7.4, and then wash the magnetic nanobeads under the action of an external magnetic field, repeating 3 times. Nanomagnetic beads were resuspended in 9mL sterile PBS solution;

(2) Weigh 2.9mg EDC and dissolve in 290μL sterile PBS, weigh 3.26mg NHSS and dissolve it in 326μL sterile PBS, then add the dissolved EDC and NHSS to the washed nanomagnetic bead solution, Then add 9mL sterile PBS, activate at room temperature for 1-2h;

(3) Wash the activated nano-magnetic beads three times with sterile PBS and resuspend in 8.6 mL of sterile PBS solution;

(4) Weigh 14 mg of cefepime, dissolve it in 1400 μL of sterile ultrapure water, then add it to the activated nano-magnetic bead solution, react for 3 to 5 hours, then wash with sterile PBS for 3 times and resuspend in In 10 mL of sterile PBS solution, a cefepime-modified nano-magnetic bead complex with a final concentration of 1 mg/mL was obtained for enrichment and separation of bacteria.

2. Enrichment capture: Take 1 mL of the sample solution to be tested containing bacteria, add 100 μg of cefepime-modified nano-magnetic bead complex, place it on a constant temperature shaker shaker, and incubate at 180 rpm at 37 ° C for 20 to 40 min to form Bacteria-cefepime-nano magnetic bead complex; insert the centrifuge tube into the magnetic stand for separation for 4-5 minutes, after magnetic separation, wash with sterile PBS and resuspend to obtain the bacteria-cefepime-nano magnetic bead complex .

Example 2

Enrichment effect experiment

(1) Take 1 mL of Escherichia coli O157:H7 and Staphylococcus aureus with a concentration of 10 ⁴ CFU/mL in a 1.5 mL sterile centrifuge tube, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and resuspend with an equal volume of sterile PBS solution .

(2) The technical solution groups of the present invention are respectively set up: a nano-magnetic bead group, a vancomycin-nano-magnetic bead group, and a cefepime-nano-magnetic bead group to enrich bacteria.

(3) After the magnetic separation, the supernatant was transferred to a sterile centrifuge tube, and the separated Escherichia coli O157:H7-functionalized nano-magnetic beads or Staphylococcus aureus-functionalized nano-magnetic beads were washed with PBS for two Next, mix well, and resuspend E. coli O157:H7-functionalized magnetic nanobead complex or Staphylococcus aureus-functionalized magnetic nanobead complex with 1 mL of sterile PBS solution.

The functionalized nano-magnetic beads refer to nano-magnetic beads, vancomycin-nano magnetic beads, and cefepime nano-magnetic beads.

(4) Calculation of capture rate: After gradient dilution of the enriched target bacteria resuspension in each group, each gradient was counted on an agar plate, and the capture efficiency of target bacteria was calculated by the capture efficiency formula, and each experiment was repeated three times. The calculation formula of the capture efficiency of each group is as follows: [total number of colonies enriched and adsorbed/(total number of colonies in supernatant + total number of colonies enriched and adsorbed)]×100%.

The schemes for enriching and capturing target bacteria in each group are as follows:

a. The technical scheme group of the present invention (cefepime-nanomagnetic beads) enrichment and capture target bacteria scheme is as in Example 1, the details are as follows: 100 μg of cefepime-nanomagnetic bead complex is added to the centrifuge tube containing the target bacteria, Incubate at 37°C at 180rpm for 20-40min on a constant temperature shaker. Place the centrifuge tube on a conventional magnetic stand to separate for 4-5min.

b. The scheme of enrichment and capture of target bacteria by nano-magnetic bead group is as follows:

Add 100 μg of magnetic nanobeads to the centrifuge tube containing the target bacteria, place on a constant temperature shaker shaker, and incubate at 37° C. at a speed of 180 rpm for 20 to 40 min. Finally, place the centrifuge tube on a conventional magnetic stand for 4-5 min.

c. The plan for enrichment and capture of target bacteria by vancomycin-nanomagnetic bead group is as follows:

Add 100 μg of vancomycin-nanomagnetic beads to the centrifuge tube containing the target bacteria, place on a constant temperature shaker shaker, and incubate at 37° C. at a speed of 180 rpm for 20-40 min. Finally, place the centrifuge tube on a conventional magnetic stand for 4-5 min.

Preparation of the vancomycin-nano magnetic beads:

(1) Aspirate 1 mL of magnetic nanobeads (10 mg/mL), add it to 9 mL of sterile PBS solution of pH 7.4, and then wash the magnetic nanobeads under the action of an external magnetic field, repeating 3 times. Nanomagnetic beads were resuspended in 9mL sterile PBS solution;

(2) Weigh 2.9mg EDC and dissolve in 290μL sterile PBS, weigh 3.26mg NHSS and dissolve it in 326μL sterile PBS, then add the dissolved EDC and NHSS to the washed nanomagnetic bead solution, Then add 9mL sterile PBS, activate at room temperature for 1-2h;

(3) Wash the activated magnetic nanobeads three times with sterile PBS and resuspend in 9.5 mL of sterile PBS solution;

(4) Weigh 42 mg of vancomycin, dissolve it in 420 μL of sterile ultrapure water, then add it to the activated nanomagnetic bead solution, react for 3 to 5 hours, then wash with sterile PBS for 3 times, and resuspend in In 10 mL of sterile PBS solution, a vancomycin-modified nano-magnetic bead complex with a final concentration of 1 mg/mL was obtained for enrichment and separation of bacteria.

The capture efficiency of each group is shown in Table 1.

Table 1

The experimental results showed that the capture efficiency of cefepime-nanomagnetic bead group for Staphylococcus aureus and Escherichia coli O157:H7 was significantly higher than that of nano-magnetic bead group and vancomycin-nanomagnetic bead group, which indicated that compared with vancomycin The covalent bond formed between cefepime and the penicillin-binding protein on the bacterial surface has a stronger affinity, so more target bacteria can be separated and enriched in a short time. And compared with vancomycin-nanomagnetic beads, which can only recognize Gram-positive bacteria, cefepime-nanomagnetic beads can recognize more bacteria, including Gram-positive bacteria and Gram-negative bacteria. Based on the above data, it is shown that using cefepime as a bacterial recognition molecule not only has a stronger affinity with bacteria, but also can recognize more types of bacteria, which can better improve the capture efficiency of bacteria.

Example 3

(1) Take 1 mL of methicillin-resistant Staphylococcus aureus with a concentration of 10 ⁴ CFU/mL in a 1.5 mL sterile centrifuge tube, centrifuge at 12000 rpm for 5 min, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.

(2) The technical solution groups of the present invention are respectively set: with/without clavulanic acid treatment group, the target bacteria can be enriched with cefepime-nanomagnetic beads.

(3) After the magnetic separation, the supernatant was transferred to a sterile centrifuge tube, and the separated methicillin-resistant Staphylococcus aureus-cefepime-nanomagnetic bead complex was washed twice with PBS and mixed well , and resuspended the methicillin-resistant Staphylococcus aureus-cefepime-nanomagnetic bead complex with 1 mL of sterile PBS solution, and the capture rate was obtained as in Example 2.

The schemes for enriching and capturing target bacteria in each group are as follows:

A. The technical scheme group of the present invention (clavulanic acid treatment group) enriches and captures the target bacteria scheme as follows:

Add clavulanic acid with a final concentration of 0.1 mg/mL to the resuspended methicillin-resistant Staphylococcus aureus solution, place it on a constant temperature shaker shaker, and incubate at 37°C at 180 rpm for 2 to 3 hours. After incubation, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and resuspend the bacterial solution with an equal volume of sterile PBS solution. 100 μg of cefepime-nanomagnetic beads were added to the centrifuge tube containing the target bacteria, placed on a constant temperature shaker shaker, and incubated at 37° C. at a speed of 180 rpm for 20-40 min. Finally, place the centrifuge tube on a conventional magnetic stand for 4-5 min.

b. The plan for enrichment and capture of target bacteria in the clavulanic acid-free treatment group is as follows:

100 μg of cefepime-nanomagnetic beads were added to the centrifuge tube containing the target bacteria, placed on a constant temperature shaker shaker, and incubated at 37° C. at a speed of 180 rpm for 20-40 min. Finally, place the centrifuge tube on a conventional magnetic stand for 4-5 min.

The capture efficiency of each group is shown in Table 2.

Table 2

Approach Clavulanic acid-free treatment group Clavulanic acid treatment group Capture efficiency/% 32.24±1.04% 96.31±0.57%

The experimental results showed that the capture efficiency of the clavulanic acid treatment group was significantly higher than that of the no clavulanic acid treatment group, which indicated that after the inhibition of the activity of β-lactamase by clavulanic acid, methicillin-resistant Staphylococcus aureus had a negative effect on the β-lactamase activity. The hydrolysis of cefepime was significantly reduced, and the capture efficiency of cefepime-nanomagnetic beads was significantly improved, and a higher capture efficiency could be achieved in a short period of time.

Example 4

Pulverize the sterile vegetables, prepare the sample solution to be tested according to the national standard method, add Listeria monocytogenes to adjust the colony concentration to 10 ⁴ CFU/mL for later use; dilute the pool water and cerebrospinal fluid 10 times with PBS buffer to prepare the sample solution, add Staphylococcus aureus was used to adjust the colony concentration to 10 ⁴ CFU/mL.

The prepared cefepime-nanomagnetic beads (100 μg) were respectively added to the sample solution, placed on a constant temperature shaker shaker, and incubated at 37° C. at a speed of 180 rpm for 20 to 40 min. Placed on a conventional magnetic stand for magnetic separation for 4 to 5 minutes. The supernatant was poured into a sterile centrifuge tube, and the L. monocytogenes-cefepime-nanomagnetic bead complex or the Staphylococcus aureus-cefepime-nanomagnetic bead complex was isolated and captured with no The bacteria were washed twice with PBS, resuspended with 1 mL of sterile PBS and mixed well. The capture rate was obtained by the method in Example 2, and the rest were the same as those in Example 1.

The capture efficiency of each group is shown in Table 3.

table 3

The results show that cefepime-nanomagnetic beads have ideal capture efficiency for Listeria monocytogenes in vegetables, Staphylococcus aureus in pool water and cerebrospinal fluid, indicating that this scheme can efficiently enrich and separate food, environmental and hospital samples bacteria in.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application rather than limit them; although the present application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand: Modifications to the specific embodiments of the application or equivalent replacement of some technical features shall all be included in the scope of the technical solutions claimed in this application.

Claims (8)

1. the functionalized magnetic bead preparation method of broad-spectrum separation bacteria, is characterized in that, comprises the following steps: 1) Wash the magnetic nano-beads, and resuspend them in sterile PBS solution after washing to obtain a magnetic nano-bead solution; 2) Dissolve EDC and NHSS in sterile PBS solution respectively to obtain EDC solution and NHSS solution, then add them to the nano-magnetic bead solution obtained in step 1), activate at room temperature for 1-2 hours, and reactivate the activated nano-magnetic beads again. Wash and resuspend to obtain an activated nanomagnetic bead solution; 3) Dissolving cefepime in sterile ultrapure water to obtain a cefepime solution, adding the cefepime solution to the activated nano-magnetic bead solution obtained in step 2) and washing again, and reacting for 3 to 6 hours to obtain a reaction product After washing and resuspension, the cefepime-modified nanomagnetic bead complex was obtained; 4) Add the cefepime-modified nanomagnetic bead complex prepared in step 3) to the sample solution containing bacteria, at 37°C, at 180 rpm, mix and incubate for 20-40 minutes, and then insert into a magnetic stand to separate 4 After ~5min, the isolated product was washed and resuspended to obtain a bacterial-cefepime-nanomagnetic bead complex. 2 . The method for preparing functionalized magnetic beads for broad-spectrum separation of bacteria according to claim 1 , wherein the surface of the nano-magnetic beads in step 1) is carboxylated, and the particle size is 180 nm. 3 . 3. The method for preparing functionalized magnetic beads for broad-spectrum separation of bacteria according to claim 1, characterized in that, sterile PBS solution is used in the washing and resuspending. 4. The method for preparing functionalized magnetic beads for broad-spectrum separation of bacteria according to claim 1 or 3, wherein the sterile PBS solution has a concentration of 0.01M and a pH of 7.4. 5 . The method for preparing functionalized magnetic beads for broad-spectrum separation of bacteria according to claim 1 , wherein the cefepime in step 3) is a fourth-generation cephalosporin, and its molecular weight is 480.56. 6 . 6. The method for preparing functionalized magnetic beads for broad-spectrum separation of bacteria according to claim 1, wherein the mass ratio of the nano-magnetic beads in step 1) to the cephalosporin in step 3) is 5:7. 7. The method for preparing functionalized magnetic beads for broad-spectrum separation of bacteria according to claim 1, wherein the mass ratio of EDC and NHSS in step 2) is 2.90:3.26. 8 . The method for preparing functionalized magnetic beads for broad-spectrum separation of bacteria according to claim 1 , wherein the sample solution in step 4) includes food, environmental and hospital samples. 9 .

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