CN114518453A - Multi-walled carbon nanotube compound and preparation method and application thereof - Google Patents

Multi-walled carbon nanotube compound and preparation method and application thereof Download PDF

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CN114518453A
CN114518453A CN202210091643.6A CN202210091643A CN114518453A CN 114518453 A CN114518453 A CN 114518453A CN 202210091643 A CN202210091643 A CN 202210091643A CN 114518453 A CN114518453 A CN 114518453A
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walled carbon
carbon nanotube
escherichia coli
nanotube composite
aptamer
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CN114518453B (en
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庞立冬
姜毓君
满朝新
杨鑫焱
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Northeast Agricultural University
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Abstract

The invention discloses a multi-wall carbon nanotube composite and a preparation method and application thereof. The invention also discloses a fluorescent aptamer sensor prepared from the multi-walled carbon nanotube composite and a preparation method thereof. Compared with the prior art, the method for synthesizing the multi-walled carbon nanotube composite by adopting the one-step method is simple, safe and effective, the obtained multi-walled carbon nanotube composite has magnetism, can be adsorbed by aptamer marked by frame fluorescence, can be used for preparing a fluorescent aptamer sensor, and can be used for quickly detecting escherichia coli, particularly quickly detecting escherichia coli O157: H7.

Description

Multi-walled carbon nanotube compound and preparation method and application thereof
Technical Field
The invention belongs to the technical field of escherichia coli detection, and particularly relates to a multi-walled carbon nanotube compound and a preparation method and application thereof.
Background
The multi-wall carbon nano-tube can be connected with biomolecules such as aptamer with different targeting functions under the covalent or non-covalent action due to the special structure, surface functional group modification and pi bond accumulation. Gu et al use polyethylene glycol as a binder to covalently link hydroxylated multi-walled carbon nanotubes with amino-modified aptamers for diagnosis of prostate cancer, and have been validated in both cell and animal experiments. Barbosa et al, which use ultrasound as a main treatment means, non-covalently link multi-walled carbon nanotubes and aptamers for detecting human colon cancer cells, have obtained good results in cytotoxicity tests. Although single multi-walled carbon nanotubes have good connection characteristics, the single multi-walled carbon nanotubes cannot meet the increasing demand, and if the multi-walled carbon nanotubes are endowed with magnetism, the separation process of materials can be simplified, and the applicability of the nano materials can be enhanced. The solvothermal method and the chemical coprecipitation method are the two most commonly used methods, and can reduce iron ions on the surface of the multi-walled carbon nanotube to obtain the multi-walled carbon nanotube attached with magnetic particles, and are applied in many fields. Deng et al used solvothermal method to attach Fe to the surface of multi-walled carbon nanotubes3O4And the composite material is verified to have good activity of catalyzing acid orange II. Liu-Hao et al prepared magnetic multi-walled carbon nanotubes by a chemical coprecipitation method and explored the effect of removing methylene blue and copper in water. Therefore, after the multi-wall carbon nano tube is endowed with magnetism, more application functions are added, but the two traditional methods for preparing the magnetic multi-wall carbon nano tube are time-consuming and labor-consuming, high-temperature conditions or reagents with irritation and corrosiveness and the like are often needed, corresponding protective measures should be taken in the test process, otherwise, personal injury can be caused by misoperation. Obviously, the method does not adapt to the development trend of green chemistry, and generates certain limitation to non-professional workers, so that an effective composite method which is simple, safe, convenient and strong in adaptability is of great significance.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the problems in the prior art, the invention provides a multi-walled carbon nanotube composite, a preparation method and application thereof.
The technical scheme is as follows: in order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a multi-wall carbon nanotube composite is mainly prepared from multi-wall carbon nanotubes and carbonyl iron powder.
Preferably, the ratio of the multi-walled carbon nanotube to the carbonyl iron powder is 1: (1-6), more preferably 1: 3.
Preferably, the multi-walled carbon nanotubes are selected from carboxylated multi-walled carbon nanotubes.
The preparation method of the multi-wall carbon nanotube composite comprises the following steps:
(1) adding the multi-walled carbon nanotube into a buffer solution, uniformly mixing, then adding EDC and NHS, and reacting;
(2) and (2) after the reaction in the step (1) is finished, adding carbonyl iron powder, uniformly mixing, and continuing to react to obtain the iron-based catalyst.
Preferably, in step (1), the buffer is MES buffer at pH 4.0-6.0, more preferably pH 5.0.
Preferably, in the step (1), the reaction time is 110-130 min; the reaction was carried out at 25. + -. 2 ℃ with shaking at 160-.
Preferably, in the step (2), the reaction time is 20-70min, more preferably 60 min; the reaction was carried out at 25. + -. 2 ℃ with shaking at 160-.
The multi-walled carbon nanotube composite is applied to the rapid detection of escherichia coli, in particular to the rapid detection of escherichia coli O157: H7.
A fluorescent aptamer sensor is made of the multi-walled carbon nanotube composite.
The preparation method of the fluorescent aptamer sensor comprises the following steps:
(1) adding deionized water into the multi-walled carbon nanotube compound, dispersing, and mixing with the escherichia coli aptamer;
(2) taking the mixture, ultrasonically dispersing for 50-70min, and standing at 4 +/-2 ℃;
(3) and (4) after magnetic separation, washing the mixture for 3 to 5 times by using deionized water, and collecting the solution after heavy suspension to obtain the magnetic separation material.
Preferably, the escherichia coli aptamer is selected from escherichia coli O157: H7 aptamer, the 5' end of the escherichia coli aptamer is modified by a 6-FAM group, and the nucleotide sequence of the escherichia coli aptamer is shown in SEQ ID NO. 1: 6-FAM-CGGACGCTTATGCCTTGCCATCTACAGA GCAGGTGTGACGG; the ratio of the multi-walled carbon nanotube compound to the escherichia coli aptamer is 1: 1.
The preparation of the multi-wall carbon nanotube composite material is completed by adopting a one-step method, and the forming process of the composite material mainly comprises hydrolysis reaction of the carboxylated multi-wall carbon nanotube, displacement reaction of carbonyl iron powder, chemical combination reaction of the carbonyl iron powder and the multi-wall carbon nanotube and oxidation reaction of the carbonyl iron powder. EDC and NHS were used to activate and stabilize the carboxyl groups on the surface of the multi-walled carbon nanotubes, while acting as cross-linkers. With the addition of carbonyl iron powder, the carbonyl iron will react with H in the solution+A displacement reaction occurs to generate Fe with positive charge2+And the carboxylated multi-wall carbon nano-tube is attached to the surface of the carbonyl iron particle, and under the electrostatic attraction effect of opposite charges, the carboxylated multi-wall carbon nano-tube with negative charges is combined with the carbonyl iron particle with positive charges, and the two are tightly adsorbed to form the carbon nano-tube compound. The chemical reaction process and examples that take place in the preparation of the multi-walled carbon nanotube composite of the present invention are shown in figure 1.
The optical property of the multi-walled carbon nanotube composite is applied to the detection process of the fluorescent aptamer sensor, and the detection principle is shown in figure 2. Adding an Escherichia coli O157: H7 aptamer labeled with a fluorescent group into a solution in which a multi-wall carbon nano tube compound is uniformly dispersed, and non-covalently adsorbing the aptamer on the surface of the compound under the action of pi bonds on the surface of the multi-wall carbon nano tube compound, wherein the adsorption of the aptamer and the compound can generate fluorescence resonance energy transfer, so that the fluorescent group labeled by the aptamer has a quenching effect, and the novel fluorescent aptamer sensor is obtained. When the target bacteria exist in the sample to be detected, the aptamer for marking the fluorescent group can be separated from the surface of the multi-wall carbon nanotube compound to be combined with the target bacteria, and the fluorescent group in the solution can recover the fluorescent signal after the compound is separated under the action of an external magnetic field. On the contrary, if the target bacteria are not in the sample to be detected, the aptamer is still attached to the surface of the multi-wall carbon nanotube complex under the non-covalent adsorption effect, the signal of the fluorescent group is still quenched, and under the action of an external magnetic field, the multi-wall carbon nanotube complex is separated out of the solution, so that the fluorescent signal cannot be detected in the solution. Therefore, the method can indicate Escherichia coli O157: H7 with different concentrations in a sample to be detected according to the intensity of the fluorescence signal of the solution after magnetic separation.
The invention mainly utilizes redox reaction to synthesize the compound of the multi-wall carbon nano-tube and carbonyl iron powder by a one-step method, and because the adsorption action of the multi-wall carbon nano-tube per se is easy to agglomerate and the condition that the multi-wall carbon nano-tube can not be dispersed in a solvent usually occurs, the uniform dispersion in the solvent is difficult to realize by simply utilizing the natural property of the multi-wall carbon nano-tube. EDC and NHS are added into the acidic MES buffer solution of the carboxylated multi-wall carbon nano tube, so that the effects of activating carboxyl on the surface of the multi-wall carbon nano tube and improving the stability of the nano material can be achieved, and the dispersibility of the multi-wall carbon nano tube in an aqueous solution is further enhanced. In addition, the combined use of EDC and NHS also acts as a certain cross-linking agent, and the acidic MES buffer solution not only promotes the exertion of the cross-linking agent, but also provides a proper condition for the replacement reaction of the iron particles. The research gets rid of the traditional means for preparing the magnetic composite carbon nano material, obtains a simple, safe and effective composite method, adopts the multi-wall carbon nano tube with small diameter (less than 8nm), and further proves that the composite method can effectively combine the magnetic particles on the surface of the multi-wall carbon nano tube. Therefore, the invention provides possibility for synthesizing magnetic carbon composite nano material, and the composite method can be popularized to multi-wall carbon nano tubes with larger diameter or other nano materials, thereby fully exerting the unique application of various composite nano materials.
The multi-walled carbon nano-tube adopted by the invention has larger specific surface area, and has magnetism after being compounded with the carbonyl iron powder. Under the action of non-covalent adsorption on the surface of the multi-walled carbon nano tube, the fluorescence-labeled aptamer can be adsorbed, a fluorescent group is quenched under the action of fluorescence resonance energy transfer, and a fluorescence signal can be recovered to different degrees when target objects with different concentrations exist, so that the fluorescence-type aptamer sensor constructed based on the multi-walled carbon nano tube compound is obtained and is applied to actual milk samples.
Has the advantages that: compared with the prior art, the method for synthesizing the multi-walled carbon nanotube composite by adopting the one-step method is simple, safe and effective, the obtained multi-walled carbon nanotube composite has magnetism, can be adsorbed by aptamer marked by frame fluorescence, can be used for preparing a fluorescent aptamer sensor, and can be used for quickly detecting escherichia coli, particularly quickly detecting escherichia coli O157: H7.
Drawings
FIG. 1 is a schematic diagram of the chemical reaction process and principle of the present invention for preparing multi-walled carbon nanotube composites.
FIG. 2 is a schematic diagram of the detection principle of the multi-walled carbon nanotube composite applied to a fluorescent aptamer sensor.
FIG. 3 shows the zeta potential analysis results of the multi-walled carbon nanotube composite of the present invention.
FIG. 4 shows the Fourier transform infrared spectroscopy analysis of the multi-walled carbon nanotube composite of the present invention.
FIG. 5 shows the transmission electron microscope and the energy spectrum analysis results of the multi-walled carbon nanotube composite of the present invention, wherein: a) transmission electron microscope images of multi-walled carbon nanotubes; b & c) transmission electron microscope images of the multi-walled carbon nanotube composite; d) and (3) performing energy spectrum analysis on the multi-wall carbon nanotube composite.
FIG. 6 shows the result of the specificity of the fluorescent aptamer sensor of the invention.
FIG. 7 shows the sensitivity results of the fluorescent aptamer sensor of the invention, wherein: a) sensitivity of Escherichia coli O157 in pure culture of H7; b) the sensitivity of cow milk samples contaminated with E.coli O157H 7.
FIG. 8 is a reproduction result of the fluorescent-type aptamer sensor of the present invention.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present specification and which fall within the limits of the appended claims.
TABLE 1 information on the strains used in the following examples
Figure BDA0003489442630000041
Figure BDA0003489442630000051
TABLE 2 reagents used in the following examples
Figure BDA0003489442630000052
TABLE 3 Main apparatus used in the following examples
Figure BDA0003489442630000053
Figure BDA0003489442630000061
The nucleotide sequence of the aptamer of Escherichia coli O157: H7 is shown as SEQ ID NO. 1: 6-FAM-CGGACGCTT ATGCCTTGCCATCTACAGA GCAGGTGTGACGG.
EXAMPLE 1 preparation of Multi-walled carbon nanotube composites
(1) Weighing a proper amount of carboxylated multi-wall carbon nano tubes, adding the weighed carboxylated multi-wall carbon nano tubes into a MES (pH 4.0) glass bottle filled with 0.1mol/L, preparing the multi-wall carbon nano tubes into a concentration of 1mg/mL, and uniformly mixing the multi-wall carbon nano tubes by shaking.
(2) Adding 40mmol/L EDC and NHS 200 μ L, mixing, placing in a shaker at 25 deg.C and 180rpm, and reacting for 120 min.
(3) Adding carbonyl iron powder into the multi-wall carbon nano tube and the carbonyl iron powder in a ratio of 1:3, uniformly mixing, and reacting for 60min in a shaking table at 25 ℃ and 180rpm to obtain the nano carbon nano tube.
Example 2 characterization of multiwall carbon nanotube composites
1. zeta potential analysis
And respectively analyzing the surface charges of the multi-wall carbon nano tube, the carbonyl iron powder, the multi-wall carbon nano tube and the carbonyl iron powder compound by using a Malvern particle size analyzer. The detailed measurement procedure is as follows: cleaning a zeta potential determination cuvette special for a Malvern particle size analyzer for 3 times; selecting a function for measuring zeta potential in the tested software; adding the sample into the specified scale of the cuvette, and placing the sample in a corresponding position according to the correct direction; the sample is a black solution, so the parameter is adjusted to be the absorption rate for measurement; the assay was repeated 3 times, and the data were recorded and plotted for analysis.
zeta potential is generally the method used to achieve the characterization of the surface charge of the nanoparticles. As shown in FIG. 3, the surface of the multi-walled carbon nanotube showed a negative charge of 21.7mv, the surface of the carbonyl iron powder showed a positive charge of 3.4mv, and the surface of the multi-walled carbon nanotube and carbonyl iron powder composite showed a negative charge of 1.4mv, which is located between the two raw materials. This indicates that, under the optimal recombination reaction conditions, after the opposite charges on the surfaces of the two nanomaterials are activated or increased, the surface charge amount of the formed composite gradually decreases along with the progress of the redox reaction, and approaches to the uncharged state. The electrostatic repulsion between the nano particles is weakened due to the reduction of the same electric charge quantity, so that the nano particles can be promoted to be aggregated to a certain degree, the surface area of a single nano material after aggregation is increased, and the subsequent processes of connecting and magnetically separating the nucleic acid micromolecules on the surface of the compound can be facilitated.
2. Fourier transform infrared spectroscopy
And respectively analyzing the surface functional groups of the multi-wall carbon nano tube, the carbonyl iron powder, the multi-wall carbon nano tube and the carbonyl iron powder compound by utilizing a Fourier transform infrared spectrometer. The detailed measurement procedure is as follows: grinding the nano material into superfine powder by using a mortar; pressing into round smooth slices; and (4) testing on a machine. And (4) carrying out functional group analysis according to the peak position, recording data and carrying out charting analysis again.
Fourier transform infrared spectroscopy is generally the method used to achieve characterization of nanoparticle surface functional groups. As shown in FIG. 4, the carbonyl iron powder showed no significant functional groups on the surface and only weak CO2The peak, and therefore the surface carbonyl content of the nanomaterial is extremely low, and it is not feasible to use carbonyl groups for the recombination reaction. The surface of the multi-wall carbon nano-tube presents two obvious functional group absorption peaks which are respectively in 3425cm-1And 1682cm-1The hydroxyl and the carbonyl are caused by more carboxyl on the surface of the raw material, and a large amount of carboxylated nano particles have a certain promotion effect on the occurrence of the complex reaction. 3425cm for the composite of multiwalled carbon nanotubes and carbonyl iron powder-1The peak intensity of hydroxyl group is obviously reduced, 1682cm-1The carbonyl peak of (a) is also present in the complex because in the redox reaction, hydrogen ions of carboxyl groups are ionized, and iron ions replace the positions of the hydrogen ions, thereby destroying hydroxyl groups on the surface of the complex. The very weak hydroxyl peak intensity of the compound reflects that the carboxyl sites on the surface of the nano material are almost completely occupied, and the hydroxyl groups are greatly damaged. Most importantly, the composite is at 610cm-1A very strong new peak appears, which is generated after the reaction of iron ions and carboxyl groups and is a characteristic absorption peak of the iron ions bonded to the carboxylated multi-wall carbon nano-tubes. The appearance of a new peak indicates that iron ions have undergone redox reaction with carboxyl groups, and a complex of multi-walled carbon nanotubes and carbonyl iron powder has been generated.
3. Transmission electron microscope and energy spectrum analysis
And respectively analyzing the surface morphology and the element content distribution of the multi-wall carbon nano tube, the carbonyl iron powder, the multi-wall carbon nano tube and the carbonyl iron powder compound by using a transmission electron microscope and a scanning electron microscope. The detailed measurement procedure is as follows: dispersing the nano material in the solution; performing ultrasonic treatment for 30 min; standing to enable the nano material to generate sedimentation; dropwise adding the upper liquid with good dispersibility to the net carrying film; and (4) after drying, putting the dried product into a transmission electron microscope to observe the surface appearance, carrying out energy spectrum analysis on the specific area by using a scanning electron microscope, and shooting and comparing the results.
Transmission electron microscopy is generally one of the most intuitive characterization methods used to observe the surface topography of nanomaterials. Energy spectroscopy is a characterization method commonly used to determine the type of elements contained in nanoparticles. As shown in fig. 5, under a transmission electron microscope (fig. 5 (a) and (b)) with a 500nm observation wavelength, the morphology and diameter of the multi-walled carbon nanotube before and after the recombination reaction are well preserved, the original multi-walled carbon nanotube has a longer length, and exhibits significant bending and overlapping, which is not favorable for fully exerting the adsorption effect on the surface. The composite material after oxidation-reduction reaction has shorter length, only has slight bending and less overlapping, and can fully utilize the surface area of the composite material. Under a transmission electron microscope (fig. 5 (c)) observing 50nm, it can be clearly seen that carbonyl iron powder is attached to the surface of the multi-walled carbon nanotube, which imparts magnetism to the composite material. Further qualitative analysis was made on the element types of the composite material (fig. 5 (d)), and the nanomaterial after the composite reaction had both C and Fe elements, indicating that the composite material was a combination of multi-walled carbon nanotubes and carbonyl iron powder. Therefore, the redox recombination method adopted in the research can combine the magnetic carbonyl iron powder on the surface of the multiwalled carbon nanotube with extremely small diameter (less than 8nm), and can also be popularized to the multiwalled carbon nanotube material with other larger diameter range
Example 3 construction of fluorescent aptamer sensor
(1) Weighing a proper amount of the multi-walled carbon nanotube composite prepared in the example 1, adding the multi-walled carbon nanotube composite into a 2mL centrifuge tube filled with deionized water to prepare the multi-walled carbon nanotube composite with the concentration of 1mg/mL, and performing ultrasonic dispersion for 60 min.
(2) And mixing the multi-wall carbon nano tube compound solution after ultrasonic treatment with an Escherichia coli O157: H7 aptamer with the concentration of 100nmol/L, wherein the ratio of the two aptamers is 1: 1.
(3) The mixture was ultrasonically dispersed for 60min and placed in a refrigerator at 4 ℃ overnight.
(4) And (5) after magnetic separation, washing with deionized water, and collecting the solution after heavy suspension to obtain the magnetic-separation-type magnetic-separation material.
Example 4 Performance verification and application of fluorescent aptamer sensor
1. Specificity verification of fluorescent aptamer sensor
The specificity of the fluorescent aptamer sensor is verified by selecting a food-borne pathogenic bacterial strain purchased or isolated from a laboratory (see table 1). Adjusting the pure culture of all test strains to the same concentration by adopting sterile deionized water, then fully mixing the pure culture of the strains and a fluorescent aptamer sensing system in a lightproof brown centrifuge tube according to the volume ratio of 10:1, replacing bacteria by adopting the sterile deionized water in a negative control group, performing the same operation as that of a positive test group in the rest operation, performing vortex oscillation, and then incubating in an incubator at 37 ℃ for 10 min. And finally, separating the multi-walled carbon nanotube compound on a magnet frame, transferring the solution to a black 96-well plate, and performing fluorescence measurement on each sample under the conditions of excitation wavelength of 492nm and emission wavelength of 532nm of an enzyme marker to perform three parallel tests. And (4) recording the fluorescence intensity of a sample of each strain, and comparing and analyzing the specificity of the fluorescent aptamer sensor.
In the specificity verification of the fluorescent aptamer sensor, the recovery value of fluorescence intensity is larger for the detection of 4 strains of escherichia coli O157: H7, but the recovery of fluorescence intensity is not more obvious for the detection of blank control and 23 strains of non-escherichia coli O157: H7, and the result is shown in FIG. 6.
2. Sensitivity test of fluorescent aptamer sensor
Escherichia coli O157: H7 is used as a research object for sensitivity test of a fluorescent aptamer sensor, and the sensitivity of target bacteria under pure culture and actual milk sample pollution is respectively researched. In the pure culture detection of Escherichia coli O157H 7, the target bacteria are washed by sterile deionized water and diluted by 10 times in a gradient manner, and the concentrations of different bacteria liquids are determined by a dilution coating plate method. And then, respectively and fully mixing the bacterial liquid with each concentration gradient and the fluorescent aptamer sensing system in a dark brown centrifugal tube according to the volume ratio of 10:1, replacing bacteria with sterile deionized water in a negative control group, performing the same operation as the positive test group in the rest operations, performing vortex oscillation, and then incubating in an incubator at 37 ℃ for 10 min. And finally, separating the multi-wall carbon nanotube compound on a magnet frame, transferring the solution to a black 96-well plate, and performing fluorescence measurement on each sample under the conditions of excitation wavelength of 492nm and emission wavelength of 532nm of a microplate reader to perform three parallel tests. And (3) recording the fluorescence intensity of the sample of the target bacteria with each concentration gradient, and analyzing the sensitivity of the fluorescent aptamer sensor for detecting pure culture of Escherichia coli O157: H7.
In the detection of the actual milk sample polluted by Escherichia coli O157: H7, the commercial ultra-high temperature sterilized milk adopted in the test is detected by the national standard GB4789.36-2016, and no target bacteria exist. And (3) washing and gradient diluting the Escherichia coli O157H 7 by using sterile deionized water, and judging the concentration of different bacteria liquid by adopting a dilution coating plate method. And respectively polluting the liquid cow milk with the bacteria liquid with each gradient concentration, replacing bacteria with sterile deionized water in a negative control group, and placing the negative control group and the positive control group in a shaking table at 37 ℃ and 180rpm for incubation for 1h, wherein the rest operations are the same as those of the positive test group. And (3) treating the incubated sample on the liquid cow milk sample by adopting the method of International GB 4789.18-2010, fully mixing the treated sample and a fluorescent aptamer sensing system in a dark brown centrifugal tube according to the volume ratio of 10:1, performing vortex oscillation, and then placing the mixture in an incubator at 37 ℃ for incubation for 10 min. And finally, separating the multi-wall carbon nanotube compound on a magnet frame, transferring the solution to a black 96-well plate, and performing fluorescence measurement on each sample under the conditions of excitation wavelength of 492nm and emission wavelength of 532nm of a microplate reader to perform three parallel tests. And (3) recording the fluorescence intensity of the sample of the target bacteria with each concentration gradient, and analyzing the sensitivity of the fluorescence type aptamer sensor for detecting the cow milk sample polluted by the Escherichia coli O157: H7.
The results of the sensitivity test of the fluorescent aptamer sensor are shown in FIG. 7. In pure culture of Escherichia coli O157H 7, the concentration of the fluorescent aptamer sensor in Escherichia coli O157H 7 is 104-107The cfu/mL has good linear relation, the fluorescence intensity is increased along with the increase of the concentration of Escherichia coli O157H 7, and the detection limit is 7.15 multiplied by 103cfu/mL (S/N3). The actual milk is polluted by Escherichia coli O157H 7In the sample, after pre-incubation for 1H, the concentration of the fluorescent aptamer sensor in Escherichia coli O157: H7 is 103-106The cfu/mL has good linear relation, the fluorescence intensity is increased along with the increase of the concentration of Escherichia coli O157H 7, and the detection limit is 3.15 multiplied by 102cfu/mL (S/N3). Therefore, the fluorescent aptamer sensor has higher sensitivity to Escherichia coli O157: H7.
3. Reproducibility analysis of fluorescent aptamer sensor
And respectively randomly selecting 8 groups of test systems with the same or different preparation batches, and carrying out reproducibility evaluation on the fluorescent aptamer sensor so as to analyze test differences between groups. The pure culture of Escherichia coli O157: H7 and the fluorescent aptamer sensing system of each test group at the same concentration were added into a light-shielding brown centrifuge tube at a volume ratio of 10: 1. After mixing well under a vortex shaker, the mixture was incubated in an incubator at 37 ℃ for 10 min. And then, placing the brown centrifugal tube on a magnet frame, aggregating and separating the multi-wall carbon nano tube compound, putting the solution into a black 96-well plate, and measuring the fluorescence signal intensity under an enzyme-linked immunosorbent assay (ELIAS), wherein the excitation wavelength of the selected sample is 492nm, and the emission wavelength is 532 nm. The fluorescence intensity of each sample was recorded, triplicate experiments were performed, plotted, and the relative standard deviation between and between groups was calculated.
Reproducibility is an important factor in the evaluation of microbiological detection techniques. The test samples in the groups and between the groups are respectively selected for 8 times, the analysis result is shown in fig. 8, the measured values of the samples in the groups are gathered, the measured values of the samples between the groups are dispersed, and the repeatability of the samples in the groups is better than that of the samples between the groups. Further calculation of the relative standard deviation, 3.57% for the intra-group samples and 4.43% for the inter-group samples, both less than 5%, indicates that there is good reproducibility for both the intra-and inter-group test samples.
4. Evaluation of practicability of fluorescent aptamer sensor
In order to further evaluate the performance of the fluorescent aptamer sensor for detecting Escherichia coli O157: H7, 30 parts of liquid milk in the market are randomly selected as test samples, 10 parts of liquid milk is used for detecting Escherichia coli-free O157: H7 according to the national standard GB4789.36-2016, and then4cfu/mL, 20 test samples as positive groups and the other 10 test samples as negative groups, and after incubation on a shaker, all liquid milk samples were processed according to International GB 4789.18-2010, and then the determination of E.coli O157: H7 was completed by the method of actual sample detection in 2.2.6.2.
The practicability of the fluorescent aptamer sensor was evaluated using 30 parts of the liquid milk sample as a test sample. Wherein the target bacteria can be detected in 20 parts of positive liquid milk samples artificially contaminated by Escherichia coli O157: H7, and the target bacteria can not be detected in 10 parts of negative liquid milk samples not artificially contaminated by Escherichia coli O157: H7. The fluorescent aptamer sensor for the test has good practicability, and the detection standard reaching rate is 100%.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (10)

1. A multi-wall carbon nanotube composite is characterized by being mainly prepared from multi-wall carbon nanotubes and carbonyl iron powder.
2. The multi-walled carbon nanotube composite of claim 1, wherein the ratio of the multi-walled carbon nanotubes to carbonyl iron powder is 1: (1-6), preferably 1: 3.
3. The multi-walled carbon nanotube composite of claim 1, wherein the multi-walled carbon nanotubes are selected from carboxylated multi-walled carbon nanotubes.
4. The method of preparing a multi-walled carbon nanotube composite as claimed in any one of claims 1 to 3, comprising the steps of:
(1) adding the multi-walled carbon nanotube into a buffer solution, uniformly mixing, then adding EDC and NHS, and reacting;
(2) and (2) after the reaction in the step (1) is finished, adding carbonyl iron powder, uniformly mixing, and continuing to react to obtain the iron-based catalyst.
5. The method for preparing a multi-walled carbon nanotube composite according to claim 4, wherein in step (1), the buffer is MES buffer with pH 4.0-6.0, preferably pH 5.0.
6. The method as claimed in claim 4, wherein the reaction time in step (1) is 110-130 min; the reaction is carried out at 25 +/-2 ℃ and in shaking at 160-; in the step (2), the reaction time is 20-70min, preferably 60 min; the reaction was carried out at 25. + -. 2 ℃ with shaking at 160-.
7. Use of the multi-walled carbon nanotube composite of any of claims 1-3 for the rapid detection of E.
8. A fluorescent aptamer sensor made from the multi-walled carbon nanotube composite of any of claims 1-3.
9. The method of preparing a fluorescent aptamer sensor of claim 8, comprising the steps of:
(1) adding deionized water into the multi-walled carbon nanotube compound, dispersing, and mixing with the escherichia coli aptamer;
(2) taking the mixture, ultrasonically dispersing for 50-70min, and standing at 4 +/-2 ℃;
(3) and (4) after magnetic separation, washing the mixture for 3 to 5 times by using deionized water, and collecting the solution after heavy suspension to obtain the magnetic separation material.
10. The method for preparing the fluorescent aptamer sensor according to claim 8, wherein the Escherichia coli aptamer is selected from Escherichia coli O157: H7 aptamer, the 5' end of which is modified with 6-FAM group, and the nucleotide sequence of which is shown in SEQ ID No. 1; the ratio of the multi-walled carbon nanotube compound to the escherichia coli aptamer is 1: 1.
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