CN114518453B - Multi-wall carbon nano tube compound and preparation method and application thereof - Google Patents
Multi-wall carbon nano tube compound and preparation method and application thereof Download PDFInfo
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- CN114518453B CN114518453B CN202210091643.6A CN202210091643A CN114518453B CN 114518453 B CN114518453 B CN 114518453B CN 202210091643 A CN202210091643 A CN 202210091643A CN 114518453 B CN114518453 B CN 114518453B
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Abstract
The invention discloses a multi-wall carbon nano tube compound, a preparation method and application thereof. The invention also discloses a fluorescent aptamer sensor prepared from the multi-wall carbon nano tube compound and a preparation method thereof. Compared with the prior art, the method for synthesizing the multi-wall carbon nano tube compound by adopting the one-step method is simple, safe and effective, the obtained multi-wall carbon nano tube compound has magnetism, can adsorb the nucleic acid aptamer marked by frame fluorescence, and can be used for rapidly detecting escherichia coli, in particular to the rapid detection of escherichia coli O157:H27.
Description
Technical Field
The invention belongs to the technical field of escherichia coli detection, and particularly relates to a multiwall carbon nanotube compound and a preparation method and application thereof.
Background
The multiwall carbon nanotube can be connected with biomolecules such as nucleic acid aptamers with different targeting functions under covalent or non-covalent actions due to the special structure, surface functional group modification and pi bond accumulation. Gu et al used polyethylene glycol as an adhesive to covalently link hydroxylated multiwall carbon nanotubes to amino-modified aptamer for diagnosis of prostate cancer disease, and validated both in cell and animal experiments. Barbosa et al uses ultrasound as the primary treatment means to non-covalently link multiwall carbon nanotubes to aptamer for detection of human colonCancer cells gave good results in cytotoxicity assays. Although single multi-wall carbon nanotubes have better connection characteristics, the increasing demands are not satisfied, and if the multi-wall carbon nanotubes are endowed with magnetism, the separation process of materials can be simplified, and the applicability of nano materials can be enhanced. The solvothermal method and the chemical coprecipitation method are the two most commonly used methods, and iron ions can be reduced on the surface of the multi-wall carbon nanotube to obtain the multi-wall carbon nanotube attached with the magnetic particles, so that the multi-wall carbon nanotube is applied to more fields. Deng et al used solvothermal method to attach Fe on the surface of multiwall carbon nanotubes 3 O 4 And the composite material is proved to have good activity of catalyzing acid orange II. Liu Ke et al prepared magnetic multiwall carbon nanotubes by chemical coprecipitation and explored the effect of removing methylene blue and copper from water. Therefore, after magnetism is given to the multi-wall carbon nano tube, 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 are needed to be adopted, corresponding protective measures are needed to be taken in the test process, otherwise, personal injury is possibly caused by misoperation. This obviously does not adapt to the trend of green chemistry and puts a certain limit on non-professional workers, so that an effective compounding method which is simple, safe, convenient and highly adaptable is of great importance.
Disclosure of Invention
The invention aims to: in order to solve the problems in the prior art, the invention provides a multi-wall carbon nano tube compound, a preparation method and application thereof, and the multi-wall carbon nano tube compound can be used for preparing a fluorescence type aptamer sensor and can be used for rapidly detecting escherichia coli.
The technical scheme is as follows: in order to achieve the aim of the invention, the invention adopts the following technical scheme:
a multi-wall carbon nano-tube compound is prepared from multi-wall carbon nano-tube and carbonyl iron powder.
Preferably, the ratio of the multiwall carbon nanotubes to carbonyl iron powder is 1: (1-6), more preferably 1:3.
Preferably, the multiwall carbon nanotubes are selected from carboxylated multiwall carbon nanotubes.
The preparation method of the multi-wall carbon nano tube compound comprises the following steps:
(1) Adding the multiwall carbon nanotubes into a buffer solution, uniformly mixing, and then adding EDC and NHS for reaction;
(2) And (3) after the reaction in the step (1), adding carbonyl iron powder, uniformly mixing, and continuing the reaction to obtain the product.
Preferably, in step (1), the buffer is buffer MES having a pH of 4.0-6.0, more preferably pH 5.0.
Preferably, in the step (1), the reaction time is 110-130min; the reaction was carried out at 25.+ -. 2 ℃ with shaking at 160-200 rpm.
Preferably, in step (2), the reaction time is 20 to 70min, more preferably 60min; the reaction was carried out at 25.+ -. 2 ℃ with shaking at 160-200 rpm.
The multi-wall carbon nano tube compound is applied to the rapid detection of escherichia coli, in particular to the rapid detection of escherichia coli O157:H27.
A fluorescent aptamer sensor is prepared from the multi-wall carbon nanotube composite.
The preparation method of the fluorescent aptamer sensor comprises the following steps:
(1) Adding deionized water into the multi-wall carbon nano tube compound, dispersing, and then mixing with an escherichia coli aptamer;
(2) Taking the mixture, carrying out ultrasonic dispersion for 50-70min, and standing at 4+/-2 ℃;
(3) And (3) washing 3-5 times by using deionized water after magnetic separation, and collecting the solution after resuspension.
Preferably, the escherichia coli aptamer is selected from escherichia coli O157:H27 aptamer, a 6-FAM group is modified at the 5' end of the escherichia coli aptamer, and the nucleotide sequence of the escherichia coli aptamer is shown as SEQ ID NO. 1: 6-FAM-CGGACGCTTATGCCTTGCCATCTACAGAGCAGGTGTGACGG; the ratio of the multi-wall carbon nano tube compound to the escherichia coli aptamer is 1:1.
Preparation of the multiwall carbon nanotube composite of the inventionThe composite material forming process mainly comprises the hydrolysis reaction of carboxylated multiwall carbon nanotubes, the displacement reaction of carbonyl iron powder, the combination reaction of the carbonyl iron powder and the multiwall carbon nanotubes and the oxidation reaction of the carbonyl iron powder. EDC and NHS are used to activate and stabilize carboxyl groups on the surface of the multiwall carbon nanotubes while acting as cross-linking agents. With the addition of carbonyl iron powder, the carbonyl iron can react with H in the solution + A displacement reaction occurs to generate Fe with positive charge 2+ And the carboxylated multiwall carbon nano tube with negative charges is combined with carbonyl iron particles with positive charges under the electrostatic attraction action of opposite charges, and the carboxylated multiwall carbon nano tube and the carbonyl iron particles are closely adsorbed to form a carbon nano tube compound. The chemical reaction process and examples of the preparation of the multi-walled carbon nanotube composite of the present invention are shown in FIG. 1.
The optical properties of the multi-wall carbon nanotube composite are applied to the detection process of a fluorescence aptamer sensor, and the detection principle is shown in figure 2. Adding fluorescent group-marked escherichia coli O157:H27 aptamer into a uniformly dispersed solution of the multi-wall carbon nano tube compound, and non-covalently adsorbing the aptamer on the surface of the compound under the pi bond effect of the surface of the multi-wall carbon nano tube compound, wherein fluorescence resonance energy transfer can occur in the adsorption of the aptamer and the aptamer, so that the fluorescent group marked by the aptamer has a quenching effect, thereby obtaining the novel fluorescent aptamer sensor. When the target bacteria exist in the sample to be detected, the aptamer marked with the fluorescent group can be separated from the surface of the multi-wall carbon nano tube compound, the target bacteria are bound, and after the compound is separated under the action of an external magnetic field, the fluorescent group in the solution can recover a fluorescent signal. Otherwise, if the sample to be detected does not have target bacteria, the aptamer still adheres to the surface of the multi-wall carbon nano tube compound under the action of non-covalent adsorption, the signal of the fluorescent group still remains quenched at the moment, and the solution is separated along with the multi-wall carbon nano tube compound under the action of an external magnetic field, so that the fluorescent signal cannot be detected in the solution. Thereby realizing the indication of the Escherichia coli O157 to H7 with different concentrations in the sample to be detected according to the intensity of the fluorescence signal of the solution after magnetic separation.
The invention mainly utilizes oxidation-reduction reaction to synthesize the compound of the multiwall carbon nanotube and carbonyl iron powder by a one-step method, and the multiwall carbon nanotube is easy to agglomerate under the self adsorption action and can not be dispersed in a solvent generally, so that the uniform dispersion in the solvent is not easy to realize by simply utilizing the natural property of the multiwall carbon nanotube. EDC and NHS are added into the acidic MES buffer solution of the carboxylated multiwall carbon nanotube, so that the effects of activating the carboxyl on the surface of the multiwall carbon nanotube and improving the stability of the nanomaterial can be achieved, and the dispersibility of the multiwall carbon nanotube in an aqueous solution can be 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 proper conditions for the replacement reaction of iron particles. The research gets rid of the traditional means for preparing the magnetic composite carbon nano material, and obtains a simple, safe and effective composite method, the diameter of the adopted multi-wall carbon nano tube is very small (less than 8 nm), and the composite method is proved to be capable of effectively combining the magnetic particles on the surface of the multi-wall carbon nano tube. Therefore, the invention provides possibility for synthesizing the magnetic carbon composite nano material, and the composite method can be popularized to multi-wall carbon nano tubes with larger diameter or nano materials of other types, and the unique application of various composite nano materials can be fully developed.
The multi-wall carbon nano tube adopted by the invention has larger specific surface area, and the multi-wall carbon nano tube has magnetism after the carbonyl iron powder is compounded. Under the action of non-covalent adsorption on the surface of the multiwall carbon nanotube, the fluorescent-labeled aptamer can be adsorbed, the fluorescent group is quenched under the transfer of fluorescence resonance energy, and fluorescent signals can be recovered to different degrees when targets with different concentrations exist, so that the fluorescent aptamer sensor constructed based on the multiwall carbon nanotube composite is obtained and applied to an actual cow milk sample.
The beneficial effects are that: compared with the prior art, the method for synthesizing the multi-wall carbon nano tube compound by adopting the one-step method is simple, safe and effective, the obtained multi-wall carbon nano tube compound has magnetism, can adsorb the nucleic acid aptamer marked by frame fluorescence, and can be used for rapidly detecting escherichia coli, in particular to the rapid detection of escherichia coli O157:H27.
Drawings
FIG. 1 is a schematic diagram of the chemical reaction process and principle of the present invention for preparing multi-walled carbon nanotube composite.
FIG. 2 is a schematic diagram of the detection principle of the multi-walled carbon nanotube composite of the present invention applied to a fluorescence type aptamer sensor.
FIG. 3 shows the zeta potential analysis results of the multi-walled carbon nanotube composite according to the present invention.
FIG. 4 shows the result of Fourier transform infrared spectrum analysis of the multi-walled carbon nanotube composite of the present invention.
FIG. 5 shows the result of transmission electron microscope and energy spectrum analysis of the multi-walled carbon nanotube composite of the present invention, wherein: a) A multi-wall carbon nano tube transmission electron microscope image; b & c) a multi-walled carbon nanotube composite transmission electron microscopy; d) And (5) multi-wall carbon nano tube compound energy spectrum analysis results.
FIG. 6 shows the specificity results of the fluorescent aptamer sensor of the invention.
FIG. 7 is a sensitivity result of the fluorescence type aptamer sensor of the invention, wherein: a) Sensitivity of E.coli O157H 7 in pure culture; b) E.coli O157H 7 pollutes the sensitivity of cow milk samples.
FIG. 8 shows the reproducibility of the fluorescence type aptamer sensor of the invention.
Detailed Description
The present invention is further illustrated below in conjunction with specific embodiments, it being understood that these embodiments are meant to be illustrative of the invention only and not limiting the scope of the invention, and that modifications of the invention, which are equivalent to those skilled in the art to which the invention pertains, will fall within the claims appended hereto.
TABLE 1 information on strains used in the following examples
TABLE 2 reagents used in the following examples
TABLE 3 Main instruments used in the following examples
The nucleotide sequence of the Escherichia coli O157H 7 aptamer is shown as SEQ ID NO. 1: 6-FAM-CGGACGCTTATGCCTTGCCATCTACAGA GCAGGTGTGACGG.
EXAMPLE 1 preparation of multiwall carbon nanotube composite
(1) Weighing a proper amount of carboxylated multi-wall carbon nanotubes, adding the carboxylated multi-wall carbon nanotubes into a MES (pH 4.0) glass bottle filled with 0.1mol/L, preparing the multi-wall carbon nanotubes into the concentration of 1mg/mL, and shaking and uniformly mixing.
(2) 40mmol/L of EDC and 200. Mu.L of NHS were added, mixed and placed in a shaker at 25℃and 180rpm for 120min.
(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 in a shaking table at 25 ℃ and 180rpm for 60min to obtain the nano-carbon nano tube.
Example 2 characterization of multiwall carbon nanotube complexes
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 Markov particle size analyzer. The detailed measurement steps are as follows: washing a special zeta potential measurement cuvette of the Markov particle size analyzer for 3 times; selecting a function for measuring the zeta potential in the tested software; adding the sample into the specified scale of the cuvette, and placing the sample in a corresponding place according to the correct direction; the sample is black solution, so the adjustment parameter is the absorption rate for measurement; the assay was repeated 3 times, data recorded and plotted.
zeta potential is typically the method used to achieve surface charge characterization of nanoparticles. As shown in fig. 3, the material surface of the multiwall carbon nanotube showed a negative charge of 21.7mv, the material surface of the carbonyl iron powder showed a positive charge of 3.4mv, and the surface of the multiwall carbon nanotube and carbonyl iron powder composite showed a negative charge of 1.4mv, which is intermediate to the two raw materials. This indicates that under the optimal composite reaction condition, after the charges with opposite electric properties on the surfaces of the two nano materials are activated or increased, the surface charge quantity of the composite is gradually reduced and approaches to a state without charges along with the progress of the oxidation-reduction reaction. The reduction of the electric charge quantity with the same electric property also weakens electrostatic repulsive force among the nano particles, which possibly promotes the nano particles to generate a certain degree of aggregation, and the surface area of the single nano material after aggregation is increased, which is more beneficial to the subsequent process of connecting and magnetically separating the nucleic acid small molecules on the surface of the compound.
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 extremely fine powder by using a mortar; pressing into round smooth slices; and (5) performing test on the machine. Functional group analysis was performed according to the peak positions, data were recorded and the mapping analysis was repeated.
Fourier transform infrared spectroscopy is typically used to achieve a method of characterizing the functional groups on the surface of the nanoparticle. As shown in FIG. 4, the surface of carbonyl iron powder has no obvious functional group and only has weaker CO 2 The peak, and thus the surface carbonyl content of the nanomaterial, is extremely low and complex reactions using carbonyl groups are not feasible. The surface of the multiwall carbon nanotube presents two obvious functional group absorption peaks at 3425cm respectively -1 And 1682cm -1 The hydroxyl and carbonyl groups of the nano-particles are caused by the fact that the surfaces of the raw materials have more carboxyl groups, and a large number of carboxylated nano-particles have certain promotion effect on the occurrence of a composite reaction. For the composite of multi-walled carbon nanotubes and carbonyl iron powder, 3425cm -1 The hydroxyl group peak intensity of (C) is obviously reduced by 1682cm -1 The carbonyl peaks of (2) are also present in the complex because in the oxidation-reduction reaction, hydrogen ions of the carboxyl group 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 composite reflects the surface carboxyl sites of the nanomaterial, which are almost completely occupied and the hydroxyl groups have been largely destroyed. Most importantly, the complex is at 610cm -1 A very strong new peak appears, which is generated after the reaction of iron ions and carboxyl groups, and is a marked absorption peak of the iron ions bound to the carboxylated multiwall carbon nanotubes. The appearance of a new peak indicates that the iron ions have undergone a redox reaction with the carboxyl groups, and a complex of multi-walled carbon nanotubes and carbonyl iron powder has been formed.
3. Transmission electron microscope and energy spectrum analysis
And analyzing the surface morphology and 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 respectively. The detailed measurement procedure is as follows: dispersing the nanomaterial in a solution; ultrasonic treatment for 30min; standing to enable the nano material to be settled; dripping the liquid with good dispersibility on the carrier net film; and (3) after drying, placing the obtained product into a transmission electron microscope to observe the surface morphology, carrying out energy spectrum analysis on a specific area by using a scanning electron microscope, shooting the result, and comparing and analyzing.
Transmission electron microscopy is generally one of the most intuitive characterization methods used to observe the surface topography of nanomaterials. Spectroscopy is typically a characterization method used to determine the type of element contained in a nanoparticle. The analysis result is shown in fig. 5, and under the observation of a 500nm transmission electron microscope (fig. 5 (a) and (B)), the morphology and the diameter of the multiwall carbon nanotubes before and after the composite reaction are well preserved, and the original multiwall carbon nanotubes have longer lengths and show obvious bending and overlapping, which is not beneficial to fully playing the adsorption function of the surface. The length of the composite material after oxidation-reduction reaction is shorter, only slight bending and less overlapping are realized, and the surface area of the composite material can be fully utilized. Under observation of a 50nm transmission electron microscope (fig. 5 (C)), it can be clearly seen that carbonyl iron powder is attached to the surface of the multiwall carbon nanotubes, which imparts magnetism to the composite. Further, qualitative analysis is performed on the element types of the composite material (fig. 5 (D)), and the composite reaction nano material has both elements of C and Fe, which indicates that the composite material is a combination of multi-walled carbon nanotubes and carbonyl iron powder. Therefore, the redox composite method adopted in the research can combine the carbonyl iron powder with magnetism on the surface of the multiwall carbon nanotube with extremely small diameter (less than 8 nm), and can also be popularized to the multiwall carbon nanotube materials with other larger diameter ranges
Example 3 construction of fluorescent aptamer sensor
(1) Weighing a proper amount of the multi-wall carbon nanotube compound prepared in the example 1, adding the multi-wall carbon nanotube compound into a 2mL centrifuge tube filled with deionized water, and preparing the multi-wall carbon nanotube compound into a solution with the concentration of 1mg/mL, and performing ultrasonic dispersion for 60min.
(2) Mixing the ultrasonic multi-wall carbon nano tube compound solution with Escherichia coli O157/H7 aptamer with the concentration of 100nmol/L, wherein the ratio of the two is 1:1.
(3) The above mixture was subjected to ultrasonic dispersion for 60min and then placed in a refrigerator at 4℃overnight.
(4) And after magnetic separation, washing with deionized water, and collecting the resuspended solution to obtain the magnetic separation material.
Example 4 Performance verification and application of fluorescence aptamer sensor
1. Specificity verification of fluorescent aptamer sensor
The food-borne pathogenic bacteria strains purchased or isolated and stored in the laboratory (see table 1) are selected for the specificity verification of the fluorescent aptamer sensor. Pure culture of all test strains is adjusted to the same concentration by adopting sterile deionized water, then the pure culture of the strains and a fluorescent aptamer sensing system are fully mixed in a brown centrifuge tube protected from light according to the volume ratio of 10:1, bacteria are replaced by a negative control group by adopting the sterile deionized water, other operations are the same as those of a positive test group, and the bacteria are incubated for 10min in a 37 ℃ incubator after vortex oscillation. Finally, after the multi-wall carbon nano tube compound is separated on a magnet frame, the solution is removed to a black 96-well plate, and fluorescence measurement of each sample is carried out under the conditions of 492nm excitation wavelength and 532nm emission wavelength of an enzyme-labeled instrument, so that three parallel experiments are carried out. The fluorescence intensity of the samples of each strain was recorded, and the specificity of the fluorescence type aptamer sensor was compared and analyzed.
In the specificity verification of the fluorescent aptamer sensor, the recovery value of the fluorescence intensity is larger for the detection of 4 strains of escherichia coli O157:H27 strains, but no obvious recovery of the fluorescence intensity is generated for the detection of blank control and 23 strains of non-escherichia coli O157:H27 strains, and the colorimetric aptamer sensor is judged to have better specificity for escherichia coli O157:H27 by comparison with the results of the blank control, and the results are shown in figure 6.
2. Sensitivity test of fluorescent aptamer sensor
In the detection of the E.coli O157H 7 polluted actual cow milk sample, the commercial ultrahigh temperature sterilized cow milk adopted in the test is detected by national standard GB4789.36-2016, and no target bacteria exist. And (3) washing and gradient diluting the escherichia coli O157 to H7 by using sterile deionized water, and judging the concentration of different bacterial solutions by adopting a dilution coating flat-plate method. And then the bacterial solutions with the gradient concentrations are respectively polluted by the liquid cow milk, bacteria are replaced by sterile deionized water in a negative control group, and other operations are the same as those of a positive test group, and the negative control group is placed in a shaking table at 37 ℃ and 180rpm for incubation for 1h. And (3) treating the incubated sample by using a method of International GB 4789.18-2010 to obtain a liquid cow milk sample, fully mixing the treated sample and a fluorescent aptamer sensing system in a light-resistant brown centrifuge tube according to a volume ratio of 10:1, and placing the mixture in an incubator at 37 ℃ for incubation for 10min after vortex oscillation. Finally, separating the multi-wall carbon nanotube composite on a magnet frame, transferring the solution to a black 96-well plate, and carrying out fluorescence measurement on each sample under the conditions of 492nm excitation wavelength and 532nm emission wavelength of an enzyme-labeled instrument to carry out three parallel experiments. The fluorescence intensity of the samples of the target bacteria with various concentration gradients is recorded, and the sensitivity of the fluorescent aptamer sensor for detecting the E.coli O157H 7 polluted cow milk samples is analyzed.
The sensitivity test results of the fluorescence type aptamer sensor are shown in fig. 7. In the pure culture of the Escherichia coli O157H 7, the concentration of the fluorescent aptamer sensor in the Escherichia coli O157H 7 is 10 4 -10 7 The cfu/mL has good linear relation, and the fluorescence intensity is increased along with the increase of the concentration of the Escherichia coli O157:H27, and the detection limit is 7.15X10 3 cfu/mL (S/n=3). In E.coli O157H 7 contaminated actual cow milk samples, after pre-incubation for 1H, the fluorescence aptamer sensor was measured to have a concentration of 10 in E.coli O157H 7 3 -10 6 The cfu/mL has good linear relation, and the fluorescence intensity is increased along with the increase of the concentration of the Escherichia coli O157:H7, and the detection limit is 3.15X10 2 cfu/mL (S/n=3). Therefore, the fluorescence type aptamer sensor shows higher sensitivity to Escherichia coli O157:H7.
3. Reproducibility analysis of fluorescence aptamer sensor
8 groups of test systems with the same preparation batch and different preparation batches are randomly selected respectively, and the reproducibility evaluation of the fluorescence type aptamer sensor is carried out so as to analyze the test differences in the groups and among the groups. Coli O157: H7 pure cultures of the same concentration and the fluorescent aptamer sensing system of each test group were added to a dark brown centrifuge tube at a volume ratio of 10:1. After thoroughly mixing under vortex shaker, the mixture was incubated in an incubator at 37℃for 10min. Then, the brown centrifuge tube is placed on a magnet rack to gather and separate the multi-wall carbon nano tube compound, the solution is taken in a black 96-well plate, the fluorescence signal intensity is measured under an enzyme-labeling instrument, and the excitation wavelength of the selected sample is 492nm and the emission wavelength is 532nm. The fluorescence intensity of each sample was measured by recording, performing three parallel experiments, performing a mapping analysis, and calculating the relative standard deviation within and between groups.
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 results are shown in fig. 8, the measured values of the samples in the groups are more concentrated, the measured values of the samples between the groups are more divergent, and the reproducibility 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 a better reproducibility for both the intra-group and inter-group test samples.
4. Practical evaluation of fluorescence aptamer sensor
To further evaluate the performance of the fluorescent aptamer sensor in detecting Escherichia coli O157:H2 7, 30 parts of liquid cow milk on the market were randomly selected as test samples, and 10 parts of liquid cow milk were used after detecting Escherichia coli-free O157:H2 7 according to national standard GB4789.36-2016 4 The concentration of cfu/mL pollutes 20 test samples as a positive group and the other 10 test samples as a negative group, and after the test samples are simultaneously placed on a shaking table for incubation, all liquid milk samples are treated according to International GB 4789.18-2010, and then the detection method of the actual samples in 2.2.6.2 is adopted to complete the detection of the escherichia coli O157:H27.
The practicality of the fluorescent aptamer sensor was evaluated by using 30 liquid milk samples as test samples. Wherein, the target bacteria can be detected in 20 positive liquid milk samples of artificially contaminated escherichia coli O157:H7, and the target bacteria can not be detected in 10 negative liquid milk samples of artificially contaminated escherichia coli O157:H7. The fluorescence type aptamer sensor of the test has good practicability, and the detection standard rate is 100%.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (9)
1. The preparation method of the multi-wall carbon nano tube compound is characterized in that the multi-wall carbon nano tube compound is mainly prepared from multi-wall carbon nano tubes and carbonyl iron powder; the ratio of the multiwall carbon nanotube to carbonyl iron powder is 1: (1-6);
the preparation method comprises the following steps:
(1) Adding the multiwall carbon nanotubes into a buffer solution, uniformly mixing, and then adding EDC and NHS for reaction;
(2) After the reaction of the step (1), adding carbonyl iron powder, uniformly mixing, and continuing the reaction to obtain the catalyst;
the application of the multi-wall carbon nano tube compound in the rapid detection of escherichia coli;
the multiwall carbon nanotubes are selected from carboxylated multiwall carbon nanotubes.
2. The method of claim 1, wherein the ratio of the multiwall carbon nanotubes to carbonyl iron powder is 1:3.
3. The method of claim 1, wherein in step (1), the buffer is a buffer MES having a pH of 4.0 to 6.0.
4. The method of claim 1, wherein in step (1), the buffer is a buffer MES having a pH of 5.0.
5. The method of preparing a multi-walled carbon nanotube composite according to claim 1, wherein in the step (1), the reaction time is 110 to 130min; the reaction is carried out at 25+/-2 ℃ and shaking at 160-200 rpm; in the step (2), the reaction time is 20-70min; the reaction was carried out at 25.+ -. 2 ℃ with shaking at 160-200 rpm.
6. The method of claim 5, wherein in the step (2), the reaction time is 60 minutes.
7. A fluorescent aptamer sensor made from the multiwall carbon nanotube composite of any of the methods of preparation of claims 1-6.
8. The method for preparing the fluorescent aptamer sensor of claim 7, comprising the steps of:
(1) Adding deionized water into the multi-wall carbon nano tube compound, dispersing, and then mixing with an escherichia coli aptamer;
(2) Taking the mixture, carrying out ultrasonic dispersion for 50-70min, and standing at 4+/-2 ℃;
(3) And (3) washing 3-5 times by using deionized water after magnetic separation, and collecting the solution after resuspension.
9. 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, a 6-FAM group is modified at the 5' end of the aptamer, and the nucleotide sequence of the aptamer is shown as SEQ ID NO. 1; the ratio of the multi-wall carbon nano tube compound to the escherichia coli aptamer is 1:1.
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| 碳纳米生物传感检测金黄色葡萄球菌;杨丽霞等;《食品安全质量检测学报》;20171025(第10期);摘要,第3988页第1栏第3段 * |
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