CN109628547B - Modified magnetic bead, preparation method and application thereof - Google Patents
Modified magnetic bead, preparation method and application thereof Download PDFInfo
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- CN109628547B CN109628547B CN201811533536.4A CN201811533536A CN109628547B CN 109628547 B CN109628547 B CN 109628547B CN 201811533536 A CN201811533536 A CN 201811533536A CN 109628547 B CN109628547 B CN 109628547B
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Abstract
The invention discloses a modified magnetic bead, a preparation method and application thereof, wherein the modified magnetic bead comprises Fe3O4Nano magnetic bead and coating Fe3O4A gemini cationic surfactant on the surface of the nano magnetic bead; the preparation method comprises the following steps: mixing Fe3O4Mixing the nanometer magnetic beads and the gemini cationic surfactant solution at 40-60 ℃, and adding a cross-linking agent in the mixing process to obtain the gemini cationic surfactant nanoparticle coated Fe3O4Nano magnetic beads, namely modified magnetic beads. The modified magnetic beads prepared by the method can be used for adsorbing gram-negative bacteria in high-protein-content food. The invention utilizes gemini cationic surfactant to modify Fe3O4The modified magnetic beads obtained by the nano magnetic beads are used for forming aggregates under the condition of low bacterial concentration in the detection process of gram-negative bacteria in food with high protein content, and the problem that the traditional surfactant needs high concentration to form the aggregates is solved.
Description
Technical Field
The invention belongs to the technical field of food microorganism detection, and particularly relates to a modified magnetic bead for enriching gram-negative bacteria in a high-protein sample, a preparation method and application thereof.
Background
Food safety issues are an important issue of widespread human attention. Among many food safety problems, bacterial contamination has become a major factor affecting food safety due to its characteristics of many kinds, wide distribution, short outbreak period, infectivity, etc. The existing national food safety standard GB 4789 in China relates to food microbiology inspection, and selective culture medium or biochemical test is mainly adopted for identification. The methods are the gold standard of food microbiological inspection in China at present, and have the advantages of high detection repeatability, strong operability and the like. However, the method has long detection time and poor targeting property, is easy to cause secondary cross contamination, is only limited to be detected in a laboratory of a professional inspection institution, and cannot be used for timely and on-site detection of microorganisms in links such as food processing, transportation and storage.
The conventional enrichment culture method, the immunomagnetic bead method and the modified magnetic bead method (such as silicon hydroxylation, carboxylation, amination, oleic acid modification and the like) are commonly used for enriching bacteria at present. The traditional enrichment culture method is a process of processing a food sample, culturing for 24-96 hours under certain conditions (such as culture medium, temperature and the like), centrifuging, filtering and washing to realize the separation of bacteria and the sample. Although the traditional method has certain advantages in bacterial enrichment, the traditional method is complex to operate, easy to pollute, time-consuming and low in concentration efficiency.
The immunomagnetic bead method is a microorganism rapid separation and enrichment technology based on the antigen-antibody specific binding reaction principle, and is widely applied to rapid detection of food microorganisms by virtue of high target specificity of the immunomagnetic bead method. The enrichment time required by conventional detection is shortened. However, when adsorbing a sample with a high protein content, there are the following problems: on the one hand, proteins contained in the sample block binding sites of antibodies, thereby reducing adsorption efficiency; on the other hand, it reacts with the antibody as an "antigen" and causes false positive results.
The modified magnetic bead method is a functionalized carrier coated with chemical active groups, is based on a microorganism enrichment technology for forming electrostatic adsorption between magnetic beads and bacteria, has good biocompatibility and is widely applied. However, due to the defects of uneven size, poor dispersibility, low functionalization degree and the like in the preparation process, the adsorption efficiency and the recovery rate of the modified magnetic beads are low when the modified magnetic beads are used for enriching trace gram-negative bacteria in food with high protein content, and the application of the modified magnetic beads in the aspect of rapid detection in the field of food safety is limited.
Disclosure of Invention
Aiming at the limitation of the conventional method in the rapid detection of microorganisms safe to food, the invention aims to provide a modified magnetic bead, a preparation method and application thereof, and solves the problem that the magnetic bead prepared by the conventional method needs high concentration of microorganisms to form an aggregate.
In order to achieve the purpose, the invention adopts the following technical scheme:
a modified magnetic bead comprising Fe3O4Nano magnetic bead and coating Fe3O4A gemini cationic surfactant on the surface of the nanometer magnetic bead.
Preferably, the alkyl hydrophobic chain length m of the gemini cationic surfactant is 8C-18C, and the hydrophobic flexible chain length s of the connecting group is 10C; said Fe3O4The surface of the nano magnetic bead is modified by silane or oleic acid.
The invention also discloses a preparation method of the modified magnetic bead, which comprises the following steps: mixing Fe3O4Mixing the nanometer magnetic beads and the gemini cationic surfactant solution at 40-60 ℃, and adding a cross-linking agent at least once in the mixing process to obtain the gemini cationic surfactant coated Fe3O4Nano magnetic beads, namely modified magnetic beads.
Preferably, Fe is added3O4Placing the nano magnetic beads into the dispersion liquid, adding a gemini cationic surfactant solution, performing ultrasonic dispersion for 5-15 min, stirring for 0.5-2 h at the temperature of 40-60 ℃ and the rotating speed of 300-500 r/min, and adding a cross-linking agent every 10-20 min in the stirring process;
wherein the dispersion liquid is composed of deionized water, absolute ethyl alcohol and NH3·H2O is mixed according to the volume ratio of 1 (0.1-0.75) to 0.5-1;
Fe3O4the volume ratio of the nano magnetic beads to the dispersion liquid is (0.5-1) to (1-2).
Specifically, Fe3O4The nano magnetic beads are placed in the dispersion liquid and stirred at a temperature of 25-40 ℃ and a rotation speed of 300-500 r/min for 0.5 e to eAdding NaNO dropwise every 10-20 min during stirring for 2h2As a protective solution.
In particular, the Fe3O4The mass ratio of the nano magnetic beads to the gemini cationic surfactant is (5-25) to 1.
Specifically, the mass concentration of the gemini cationic surfactant in the gemini cationic surfactant solution is 0.5-2.5%; the Gemini cationic surfactant solution is acetic acid solution, glycine-hydrochloric acid solution or citric acid-sodium citrate solution of Gemini cationic surfactant; the pH of the gemini cationic surfactant solution is 2.5-5.0.
Specifically, the cross-linking agent is glutaraldehyde solution or 1, 4-butanediol diglycidyl ether solution.
The invention also discloses application of the modified magnetic beads in adsorption of gram-negative bacteria in high-protein-content food.
Specifically, a food sample to be tested is added into a Tris-HCl solution with the pH value of 2-7 for dissolution, and then is mixed with a modified magnetic bead solution at the temperature of 30-60 ℃ for 20-50 min to obtain a magnetic bead-bacterium compound; wherein the mass concentration of the modified magnetic beads in the modified magnetic bead solution is 20-50 mg/mL;
and finally, washing the magnetic bead-bacterium compound by using a PBS (phosphate buffer solution) with the pH of 2.5-4.5, and taking supernate to determine the bacterial colony number.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention utilizes gemini cationic surfactant to modify Fe3O4The modified magnetic beads obtained by the nano magnetic beads are used for forming aggregates under the condition of low bacterial concentration in the detection process of gram-negative bacteria in food with high protein content, and the problem that the traditional surfactant needs high concentration to form the aggregates is solved.
(2) The modified magnetic bead particles prepared by the method are uniformly dispersed, have regular particle shapes, and have strong adsorption effect on trace gram-negative bacteria in food with high protein content.
(3) Compared with the conventional immunomagnetic beads, the modified magnetic beads prepared by the invention avoid the interference of protein in a high-protein sample on the immunomagnetic beads, and improve the bacteria detection efficiency and sensitivity.
(4) In the preparation of the modified magnetic beads of the present invention, Fe3O4Skillfully using NH in dispersion liquid of nano magnetic beads3·H2O and NaNO2As a protective liquid, NH3·H2O can adjust the pH value of the dispersion liquid on one hand and NaNO added later on the other hand2Reaction to NaOH and N2NaOH maintains the alkalinity of the dispersion, N2Can take away the oxygen dissolved in the water solution and prevent the magnetic beads from being oxidized.
Drawings
FIG. 1 is a TEM photograph of the modified magnetic beads prepared in example 1.
FIG. 2 is a TEM photograph of the modified magnetic beads prepared in example 1 after adsorbing bacteria.
FIG. 3 is a colony count culture, A-control colony, B-eluent colony of the bacterial plate.
Fig. 4 shows the particle size of modified magnetic beads prepared by mixing Fe3O4 dispersion and gemini cationic surfactant solution under different mixing processes.
Fig. 5 is a topographical view of magnetic beads prepared using surfactant SDS.
FIG. 6 is a topographical view of magnetic beads prepared using surfactant Tritonx-100.
Fig. 7 is a topographical view of magnetic beads prepared using surfactant CTAB.
FIG. 8 is a graph of the adsorption rates of four different surfactants at different mass concentrations and addition volumes.
FIG. 9 shows adsorption rates of Gemini surfactants with different alkyl hydrophobic chain lengths.
FIG. 10 shows adsorption rates of Gemini surfactants with varying lengths of hydrophobic flexible linker.
FIG. 11 shows Fe obtained by the mixing process of the nano magnetic beads and the dispersion liquid in Table 23O4@SiO2The particle size of the nano magnetic beads.
Fig. 12 shows the adsorption rate of the modified magnetic beads of the present invention after mixing with a sample solution under different processes.
The invention is described in detail below with reference to the drawings and the detailed description.
Detailed Description
Fe used in the present invention3O4The nanometer magnetic bead is a novel functional material, has the characteristics of good biocompatibility, easy modification of the surface and the like, and is widely applied in the field of biomedicine. Gemini cationic surfactants (Gemini) are amphiphiles containing amphiphilic oil groups and amphiphilic water groups, and compared with traditional single-chain surfactants with single head (single hydrophilic group) and single tail (single hydrophobic group), the gemmini surfactants have the advantages of low critical micelle concentration, higher surface activity and larger temperature application range.
In the invention, Fe3O4A gemini cationic surfactant modified on the surface of nano magnetic beads is used for preparing magnetic beads for rapidly enriching gram-negative bacteria in a sample with high protein content. Solves the problems of low efficiency, complex process and the like of high protein sample bacteria enrichment in the traditional method, and has low cost and easy industrial production.
Fe in the invention3O4The nano magnetic beads can be naked magnetic beads or magnetic beads with silane-based modification, oleic acid modification, citric acid modification and carboxyl modification on the surface, and preferably silane-based modification or oleic acid modification. The gemini cationic surfactant disclosed by the invention has an m-s-m structure.
Modified magnetic beads of the present invention comprise Fe3O4Nano magnetic bead and coating Fe3O4A gemini cationic surfactant on the surface of the nanometer magnetic bead. Preferably, the alkyl hydrophobic chain length m of the gemini cationic surfactant is 8C to 18C, and the hydrophobic flexible chain length s of the linking group is 10C, wherein 8C means 8 carbon atoms, 18C means 18 carbon atoms, and 10C means 10 carbon atoms.
The modified magnetic beads of the present invention are prepared by the following method:
mixing Fe3O4Mixing the nano magnetic beads with a gemini cationic surfactant solution at 40-60 ℃, and adding a cross-linking agent in the mixing process to obtain the gemini cationic surfactant nano particlesGrain coated Fe3O4And finally, washing the nano magnetic beads alternately by using an ethanol solution and deionized water to remove the residual surfactant, thus obtaining the modified magnetic beads.
Wherein, Fe3O4The mass ratio of the nano magnetic beads to the gemini cationic surfactant is (5-25) to 1. Fe3O4The volume ratio of the nano magnetic beads to the dispersion liquid is (0.5-1) to (1-2).
For increased dispersibility, Fe3O4Placing the NaNO magnetic beads into the dispersion liquid, stirring for 0.5-2 h at the temperature of 25-40 ℃ and the rotating speed of 350-500 r/min, and dropwise adding NaNO every 10-20 min in the stirring process2As a protective solution, magnetic beads are prevented from being oxidized; and adding a gemini cationic surfactant solution, performing ultrasonic dispersion for 5-15 min, stirring for 0.5-2 h at 40-60 ℃ and at the rotating speed of 300-500 r/min, and adding a cross-linking agent every 10-20 min in the stirring process. Wherein the diameter of the stirring paddle is preferably 50mm to 100mm for a 500ml beaker to achieve sufficient stirring.
Wherein the dispersion liquid is composed of deionized water, absolute ethyl alcohol and NH3·H2The O is composed according to the volume ratio of 1 (0.1-0.75) to 0.5-1. The dispersion of the invention can disperse Fe3O4Nano magnetic beads to increase the mixing uniformity of the nano magnetic beads and the gemini cationic surfactant, wherein NH3·H2O can adjust the pH value of the dispersion liquid on one hand and NaNO added later on the other hand2Reaction to NaOH and N2NaOH maintains the alkalinity of the dispersion, N2Can take away the oxygen dissolved in the water solution and prevent the magnetic beads from being oxidized.
In order to increase the dispersibility and uniformity of the gemini cationic surfactant, the gemini cationic surfactant is prepared into a solution, and the mass concentration of the gemini cationic surfactant in the solution is 0.5-2.5%; wherein the gemini cationic surfactant solution is acetic acid solution, glycine-hydrochloric acid solution or citric acid-sodium citrate solution of gemini cationic surfactant; the pH value of the gemini cationic surfactant solution is 2.5-5.0.
The cross-linking agent in the invention is glutaraldehyde solution or 1, 4-butanediol diglycidyl ether solution.
The modified magnetic beads prepared by the invention can be used for adsorbing gram-negative bacteria in high-protein-content food, and the specific bacteria adsorption process comprises the following steps:
the modified magnetic beads prepared by the method are prepared into a solution, and the pH value of the solution is controlled to enable the modified magnetic beads and bacteria to form an electrostatic adsorption effect, so that gram-negative bacteria in samples with high protein content, such as liquid milk, yogurt, milk powder, condensed milk, cheese and other dairy products, are adsorbed, enriched and primarily separated.
Firstly, the adsorption enrichment process is as follows: adding a sample to be adsorbed into a Tris-HCl solution with the pH value of 2-7 to dissolve the sample, adding a modified magnetic bead solution into the sample solution, and adsorbing at the rotating speed of 20-50 r/min and the temperature of 30-60 ℃ for 20-50 min to obtain a magnetic bead-bacterium compound. Wherein the volume ratio of the modified magnetic bead solution to the sample solution is 1 (500-1000); the mass concentration of the modified magnetic beads in the modified magnetic bead solution is 20-50 mg/mL. Preferably, the Tris-HCl solution has a pH of 5.8, the modified magnetic bead solution has a modified magnetic bead concentration of 50mg/mL, an addition amount of 30 μ L, an adsorption temperature of 50 ℃, and a time of 20 min.
Then, primary separation: washing the magnetic bead-bacterium compound by using a PBS (phosphate buffer solution) with the pH of 2.5-4.5, separating the magnetic beads from the adsorbed bacteria, and generally washing for about 5 times; the washed supernatant was then plated and tested to determine bacterial colony counts. 0.05M PBS buffer at pH 3.5 is preferred.
Fe used in the present invention3O4The nano magnetic beads and the gemini cationic surfactant are all sold in the market.
The following embodiments of the present invention are provided, and it should be noted that the present invention is not limited to the following embodiments, and all equivalent changes based on the technical solutions of the present invention are within the protection scope of the present invention.
Example 1
1mL of 35mg/mL Fe3O4@SiO2The (silane group modified) nano magnetic beads are placed in dispersion liquid, and the dispersion liquid is prepared from deionized water, absolute ethyl alcohol and NH3·H2Mixing O solution at a volume ratio of 1:0.1:0.5, stirring at 30 deg.C and 500r/min for 1 hr, and adding 5M NaNO dropwise every 20min21mL is used as protective solution;
preparing a gemini cationic surfactant acetic acid solution with the mass concentration of 1%, wherein the alkyl hydrophobic chain length m of the gemini cationic surfactant (Gemini) is 8-18C, and the hydrophobic flexible chain length s of a connecting group is 10C; fe subjected to the above treatment3O4@SiO2Adding the magnetic bead dispersion into acetic acid solution of surfactant, wherein the Fe3O4@SiO2The mass ratio of the nano magnetic beads to the gemini cationic surfactant is 25: 1. Dispersing uniformly by ultrasonic for 5min, continuously stirring for 0.5-2 h at 40 ℃, 500r/min and a stirring paddle diameter of 100mm, and adding 1mL of 5% glutaraldehyde crosslinking agent every 20min to modify Fe3O4@SiO2And (4) nano magnetic beads. And (3) alternately washing the nano magnetic beads for 5 times by using a 20% ethanol solution and deionized water, and removing the residual surfactant to obtain the modified magnetic beads. As shown in fig. 1, it can be seen that the obtained modified magnetic beads have uniform particle size, and the particle size of the modified magnetic beads is 75 ± 5 nm; the surface of the modified magnetic bead is coated with a gemini cationic surfactant.
The modified magnetic beads prepared in the embodiment are used for gram-negative bacteria adsorption treatment experiments in high-protein samples:
an adsorption process: weighing 1g of liquid milk, placing the liquid milk in a 50mL centrifuge tube, adding 25mL of Tris-HCl solution with pH value of 5.8 to dissolve a sample, and uniformly mixing the solution by vortex to obtain a sample solution. And adding 30 mu L of modified magnetic beads with the mass concentration of 50mg/mL into the sample solution, and processing under the conditions of the rotating speed of 50rpm, the adsorption temperature of 50 ℃ and the adsorption time of 20min to obtain the magnetic bead-bacterium compound, wherein the volume ratio of the modified magnetic bead solution to the sample solution is 1: 25. Swirling once every 5min, finally washing the magnetic bead-bacteria complex solution for 5 times by using 70% ethanol and deionized water alternately, washing to be neutral, performing magnetic separation to obtain supernatant, clarifying, discarding the supernatant, and obtaining eluent, wherein the magnetic bead-bacteria complex in the eluent is shown in figure 2.
And (3) a separation process: washing the magnetic bead-bacteria complex in the eluate with a PBS solution with pH of 3.5, separating the magnetic beads from the adsorbed bacteria, and washing for 5 times; then, the washed supernatant is taken and subjected to a plating test to determine the bacterial colony number, as shown in fig. 3(a), the bacterial colony number is very close to that of the control group in fig. 3(B), the more bacteria in the sample eluent is, the more thorough the elution is indicated, and the higher the adsorption efficiency of the magnetic beads is, it can be seen that the adsorption rate of the modified magnetic beads prepared in the embodiment almost reaches 99% ± 1%.
Example 2
This example differs from example 1 in that: fe3O4@SiO2The mixing temperature of the magnetic bead dispersion liquid and the surfactant acetic acid solution is 50 ℃.
The particle size of the modified magnetic bead prepared by the embodiment is 300 +/-20 nm, the magnetic bead is used for gram-negative bacteria adsorption treatment experiments in high-protein samples, the finally measured bacterial colony number is 600CFU, and the adsorption efficiency is 60% +/-5%.
Example 3
This example differs from example 1 in that: fe3O4@SiO2The mixing temperature of the magnetic bead dispersion liquid and the surfactant acetic acid solution is 60 ℃.
The particle size of the modified magnetic bead prepared by the embodiment is 350 +/-10 nm, the magnetic bead is used for gram-negative bacteria adsorption treatment experiments in high-protein samples, the finally measured bacterial colony number is 600CFU, and the adsorption efficiency is 60% +/-5%.
The following Table 1 shows the composition containing Fe3O4@SiO2Mixing parameter experiments of the dispersion liquid and the gemini cationic surfactant solution are different.
Table 1 process parameters of examples 1 to 3
| Factors of the fact | Temperature (. degree.C.) | Rotating speed (r/min) | Mixing time (h) |
| Experiment 1 | 40 | 300 | 0.5 |
| Experiment 2 | 40 | 400 | 1 |
| Experiment 3 | 40 | 500 | 2 |
| Experiment 4 | 50 | 300 | 2 |
| Experiment 5 | 50 | 400 | 0.5 |
| Experiment 6 | 50 | 500 | 1.5 |
| |
60 | 300 | 1.5 |
| |
60 | 400 | 2 |
| |
60 | 500 | 0.5 |
FIG. 4 is Fe3O4@SiO2And the particle size of the modified magnetic beads is obtained by the dispersion liquid and the gemini cationic surfactant solution under different mixing processes. In general, the smaller the particle diameter, the better the dispersibility and uniformity of the particles in the dispersion, and the higher the adsorption efficiency. As can be seen from FIG. 4, Fe3O4@SiO2Mechanically stirring the nano magnetic beads and the gemini cationic surfactant solution for 2 hours at 40 ℃ at 500r/min to obtain Fe in the dispersion liquid3O4@SiO2The average particle size of the nano magnetic beads is about the smallest and is 75 +/-5 nm.
Example 4
This example differs from example 1 in that: fe3O4@SiO2The mass ratio of the nano magnetic beads to the gemini cationic surfactant is 5: 1.
the modified magnetic beads prepared in the embodiment are used for gram-negative bacteria adsorption treatment experiments in high-protein samples, and the finally measured bacterial colony number is 500 CFU.
Example 5
This example differs from example 1 in that: fe3O4@SiO2The mass ratio of the nano magnetic beads to the gemini cationic surfactant is 25: 1.
the modified magnetic bead prepared in the embodiment is used for gram-negative bacteria adsorption treatment experiments in high-protein samples, and the finally measured bacterial colony number is 970 CFU.
Example 6
This example differs from example 1 in that: in this example, a glycine-hydrochloric acid solution of a gemini cationic surfactant was selected.
The modified magnetic beads prepared in the embodiment are used for gram-negative bacteria adsorption treatment experiments in high-protein samples, and the finally measured bacterial colony number is the same as that in the embodiment 1.
Example 7
This example differs from example 1 in that: in this example, a citric acid-sodium citrate solution of gemini cationic surfactants was selected.
The modified magnetic beads prepared in the embodiment are used for gram-negative bacteria adsorption treatment experiments in high-protein samples, and the finally measured bacterial colony number is the same as that in the embodiment 1.
Example 8
This example differs from example 1 in that: the crosslinking agent in this example was 1, 4-butanediol diglycidyl ether solution.
The modified magnetic beads prepared in the embodiment are used for gram-negative bacteria adsorption treatment experiments in high-protein samples, and the finally measured bacterial colony number is the same as that in the embodiment 1.
Example 9
This example differs from example 1 in that: fe used in the present example3O4The surfaces of the magnetic beads are modified by oleic acid.
The modified magnetic beads prepared in the embodiment are used for gram-negative bacteria adsorption treatment experiments in high-protein samples, and the finally measured bacterial colony number is the same as that in the embodiment 1.
Example 10
This example differs from example 1 in that: fe used in the present example3O4The surface of the magnetic beads is not modified.
The modified magnetic beads prepared in the embodiment are used for gram-negative bacteria adsorption treatment experiments in high-protein samples, and the finally measured bacterial colony number is the same as that in the embodiment 1. But the storage time of the gemini cationic surfactant coated by the naked magnetic beads without modification is shorter.
Comparative example 1
This comparative example differs from example 1 in that: the dispersion does not contain NH3·H2O and NaNO2。
The modified magnetic beads prepared in the comparative example and the example 1 are placed for 15 days at normal temperature and used for gram-negative bacteria adsorption treatment experiments in high-protein samples, and finally the measured bacteria adsorption rate is 20 +/-5%.
Comparative example 2
This comparative example differs from example 1 in that: the surfactant used in this comparative example was a hydrophilic group surfactant SDS.
The morphology of the modified magnetic beads prepared in the comparative example is shown in fig. 5, and it can be seen that the magnetic beads are seriously agglomerated.
According to the adsorption treatment experiment of gram-negative bacteria in the modified magnetic bead high-protein sample prepared in the comparative example, the finally measured bacterial colony number is 50CFU, and the adsorption efficiency is 5% +/-1%.
Comparative example 3
This comparative example differs from example 1 in that: the surfactant used in this comparative example was a nonionic surfactant, Tritonx-100.
The morphology of the modified magnetic beads prepared in the comparative example is shown in fig. 6, and it can be seen that the magnetic beads are seriously agglomerated.
According to the adsorption treatment experiment of gram-negative bacteria in the modified magnetic bead high-protein sample prepared in the comparative example, the finally measured bacterial colony number is 400CFU, and the adsorption efficiency is 40% +/-1%.
Comparative example 4
This comparative example differs from example 1 in that: the surfactant used in this comparative example was the anionic surfactant CTAB.
The morphology of the modified magnetic beads prepared in the comparative example is shown in fig. 7, and it can be seen that the magnetic beads are seriously agglomerated and have irregular shapes.
According to the adsorption treatment experiment of gram-negative bacteria in the modified magnetic bead high-protein sample prepared in the comparative example, the finally measured bacterial colony number is 680CFU, and the adsorption efficiency is 68% +/-1%.
Table 2 below shows the experimental parameters for the different surfactants used in the present invention:
table 2: experimental parameters for different surfactants
FIG. 8 is a graph of the adsorption efficiency of four different surfactants of Table 1 at different mass concentrations and addition volumes. As can be seen from FIG. 8, the Gemini cationic surfactant with a mass concentration of 1% and an added volume of 1mL had the highest adsorption efficiency.
According to the procedure of example 1 above, the invention also provides a hydrophobic chain length for the alkyl group of m<8C、m>18C, m ═ 8C to 18C, and the linking group hydrophobic flexible link lengths are each s<10C、s＝10C、s>The modified magnetic beads prepared by Gemini surfactant at 10 ℃ are subjected to adsorption efficiency test, and the adsorption efficiency is 103CFU/mL bacterial colony number is a standard experiment, and the adsorption rate of the alkyl hydrophobic chain m and the connecting group s with different carbon atoms is obtained, and the result is shown in figure 9 and figure 10.
As can be seen from FIGS. 9 and 10, when the alkyl hydrophobic chain length m is 8 to 18 carbon atoms, the adsorption rate is stable as the number of carbon atoms increases, and when the number of carbon atoms is greater than 18, the adsorption rate is decreased as the number of carbon atoms increases. When the length s of the hydrophobic flexible chain of the linking group is 10 carbon atoms, the adsorption rate is the highest, and when the number of carbon atoms is more than 10, the adsorption rate decreases as the number of carbon atoms increases.
The invention also applies to dispersions of different combined concentrations and to dispersions with Fe according to the procedure of example 1 above3O4@SiO2The experiment was carried out with a mixing process of nano magnetic beads as shown in table 3:
TABLE 3 Fe3O4@SiO2Mixing experiment of nano magnetic beads and dispersion liquid
| Factors of the fact | Temperature (. degree.C.) | Rotating speed (r/min) | Composition ratio of dispersion | Mixing time (h) |
| Experiment 26 | 25 | 350 | 1:0.1:0.5 | 0.5 |
| Experiment 27 | 25 | 400 | 1:0.2:0.5 | 1 |
| Experiment 28 | 25 | 500 | 1:0.5:1 | 2 |
| Experiment 29 | 30 | 350 | 1:0.2:0.5 | 2 |
| Experiment 30 | 30 | 400 | 1:0.5:1 | 0.5 |
| Experiment 31 | 30 | 500 | 1:0.1:0.5 | 1 |
| Experiment 32 | 40 | 350 | 1:0.5:1 | 1 |
| Experiment 33 | 40 | 400 | 1:0.1:0.5 | 2 |
| Experiment 34 | 40 | 500 | 1:0.2:0.5 | 0.5 |
FIG. 11 shows Fe obtained by different mixing processes of the nano-magnetic beads and the dispersion liquid in Table 33O4@SiO2The particle size of the nano magnetic beads. In general, the smaller the particle diameter, the better the dispersibility and uniformity of the particles in the dispersion. As can be seen from FIG. 7, the dispersion was made from deionized waterAbsolute ethanol and NH3·H2O is mixed according to the volume ratio of 1:0.1:0.5, and Fe3O4@SiO2Mechanically stirring the nano magnetic beads and the dispersion liquid for 1 hour at the temperature of 30 ℃ and at the speed of 500r/min to obtain Fe in the dispersion liquid3O4@SiO2The average particle diameter of the nano magnetic beads is about 30 +/-5 nm, and Fe3O4@SiO2The particles are most dispersible in the dispersion.
According to the above example process, the present invention also performed experiments on Tris-HCl solutions having pH values of 3.5, 4.5, 6.8 and PBS solutions having pH values of 2.5, 4.5, respectively, as shown in Table 4 below.
TABLE 4 mixing parameters of modified magnetic beads and sample solution under different processes
Fig. 12 shows the adsorption efficiency of the modified magnetic beads and the sample solution in table 4 after mixing under different processes. As can be seen from FIG. 12, by controlling the pH of the buffer, electrostatic adsorption is formed between the modified magnetic beads of the present invention and the bacteria. Therefore, the optimal conditions for sample preparation and bacterial adsorption are Tris-HCl buffer (acetic acid to adjust pH to 5.8), modified magnetic bead mass concentration of 50mg/mL, addition of 30 μ L, adsorption time of 20min, and adsorption temperature of 50 ℃.
In conclusion, the modified magnetic bead particles prepared by the method are uniformly dispersed, have regular particle shapes, and have strong adsorption effect on gram-negative bacteria in food with high protein content.
Claims (6)
1. A modified magnetic bead, comprising Fe3O4Nano magnetic bead and coating Fe3O4A gemini cationic surfactant on the surface of the nano magnetic bead;
the modified magnetic bead is prepared by the following method:
mixing Fe3O4Nano magnetic beads are put in dispersion liquid, and the Fe3O4The surface of the nano magnetic bead is modified by silane or oleic acid; stirring for 0.5-2 h at the temperature of 25-40 ℃ and the rotating speed of 300-500 r/min, and dropwise adding NaNO at intervals of 10-20 min in the stirring process2Treated Fe was obtained as a protective solution3O4Nano magnetic bead dispersion liquid;
fe after treatment3O4Adding a gemini cationic surfactant solution into the nano magnetic bead dispersion liquid, wherein the alkyl hydrophobic chain length m of the gemini cationic surfactant is 8C-18C, and the connecting group hydrophobic flexible chain length s is 10C; ultrasonically dispersing for 5-15 min, stirring for 0.5-2 h at 40-60 ℃ and at the rotating speed of 300-500 r/min, and adding a cross-linking agent every 10-20 min in the stirring process;
the dispersion liquid is composed of deionized water, absolute ethyl alcohol and NH3•H2O is mixed according to the volume ratio of 1 (0.1-0.75) to 0.5-1; fe3O4The volume ratio of the nano magnetic beads to the dispersion liquid is (0.5-1) to (1-2).
2. The modified magnetic bead of claim 1, wherein the Fe is3O4The mass ratio of the nano magnetic beads to the gemini cationic surfactant is (5-25) to 1.
3. The modified magnetic bead of claim 1, wherein the gemini cationic surfactant solution has a gemini cationic surfactant mass concentration of 0.5% to 2.5%; the Gemini cationic surfactant solution is acetic acid solution, glycine-hydrochloric acid solution or citric acid-sodium citrate solution of Gemini cationic surfactant; the pH of the gemini cationic surfactant solution is 2.5-5.0.
4. The modified magnetic bead of claim 1, wherein the cross-linking agent is a glutaraldehyde solution or a 1, 4-butanediol diglycidyl ether solution.
5. Use of the modified magnetic beads of any one of claims 1 to 4 as an adsorbent for gram negative bacteria in high protein content food products.
6. The use of claim 5, wherein the food sample to be tested is dissolved in Tris-HCl solution with pH 2-7, and then mixed with the modified magnetic bead solution at 30-60 ℃ for 20-50 min to obtain magnetic bead-bacteria complex; wherein the mass concentration of the modified magnetic beads in the modified magnetic bead solution is 20-50 mg/mL;
and finally, washing the magnetic bead-bacterium compound by using a PBS (phosphate buffer solution) with the pH of 2.5-4.5, and taking supernate to determine the bacterial colony number.
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