CN112432979B - Nanocomposite, ESAT-6 electrochemical aptamer sensor and preparation and detection methods thereof - Google Patents
Nanocomposite, ESAT-6 electrochemical aptamer sensor and preparation and detection methods thereof Download PDFInfo
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Abstract
The invention provides a nano composite material, which is a Polyethyleneimine (PEI) -functionalized nitrogen-doped graphene (NG) -modified HP-Uio-66-NH2-on-Ce-MOF nano-material, and the ESAT-6 electrochemical aptamer sensor prepared by using the nano-composite material is used for quantitative detection of ESAT-6. The invention finally forms PEI @ NG @ HP-Uio-66-NH after loading an electroactive substance toluidine blue (Tb)2-on-Ce-MOF @ Tb nanocomposite as a sensitive interface of a sensor; the signal amplification effect is carried out by utilizing the good conductivity of PEI @ NG, namely HP-Uio-66-NH2the-on-Ce-MOF has high porosity, large specific surface area and excellent biological activity, a large number of aptamers are loaded through hydrogen bonds, pi-pi accumulation and electrostatic interaction between the-on-Ce-MOF and the aptamers, and finally Tb signal response change is caused through specific binding of the aptamers and targets with different concentrations, so that quantitative detection of ESAT-6 is realized. The electrochemical aptamer sensor prepared by the invention is successfully used for the ultra-sensitive detection of ESAT-6.
Description
Technical Field
The invention relates to the technical field of electrochemical detection, in particular to a nano composite material, an ESAT-6 electrochemical aptamer sensor, a preparation method and a detection method thereof.
Background
Tuberculosis (TB) is a fatal infectious disease caused by Mycobacterium Tuberculosis (MTB), and more than 1000 million people become tuberculosis patients every year, which poses a serious challenge to international public health. MTB is a pathogen of tuberculosis, mainly infects the lung, but the infection can also affect other organs, such as bones, joints, kidneys, brains and the like, and has a huge threat to the life health of the public. Currently, asymptomatic infection and missed diagnosis of tuberculosis are the main causes of tuberculosis transmission and death, and therefore, there is a need to develop a sensitive and rapid tuberculosis detection method to discover tuberculosis as early as possible, thereby giving a correct treatment scheme and reducing further transmission of tuberculosis. Antigen target 6 secreted in early tuberculosis (ESAT-6) is an important secretory antigen of MTB and is also a virulence factor of MTB, is mainly coded by an Rv3875 gene, and ESAT-6 is secreted in early MTB infection, so that ESAT-6 protein can be used as a biomarker of tuberculosis.
Currently, the commonly used detection methods of ESAT-6 include MTB culture, sputum smear microscopy, chest X-ray imaging, immunology and the like. However, MTB culture takes a long time; the sputum smear microscopy has low positive rate and high requirement on the content of bacteria in sputum; chest X-ray imaging suffers from the inability to provide a causal diagnosis and to detect potential infections; immunological methods: such as tuberculin skin test, but it may have false positive results for people inoculated with BCG, and others such as diagnostic kit, and Anda-TB ELISA diagnostic kit, TB-SA antibody test kit, etc. are on the market, but the disadvantage is that it can not effectively distinguish active tuberculosis from latent infected people at present. Therefore, it is important to design a rapid, convenient, sensitive and specific method for detecting ESAT-6.
Aptamers (aptamers) are oligonucleotide fragments containing 10-50 variable bases synthesized in vitro by in vitro techniques, i.e., by exponential enrichment of ligands by systematic evolution (SELEX), and can be single-stranded dna (ssdna), double-stranded dna (dsdna), or RNA. Aptamers offer several advantages over antibodies, such as high affinity and high specificity for a wide range of targets (e.g., whole cells, proteins and low molecular weight organic or inorganic substrates), as well as low cost, good stability, ease of synthesis and modification by various chemical groups.
Disclosure of Invention
In order to solve the problems of the prior art, according to a first aspect of the present invention, there is provided a nanocomposite material comprising PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF @ Tb nanocomposite.
Except for special description, the parts are parts by weight, and the percentages are mass percentages.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a nanocomposite characterized by: the nano composite material is
PEI@NG@HP-Uio-66-NH2-on-Ce-MOF @ Tb nanocomposite, prepared by the following steps: 1) preparing a PEI @ NG dispersion; 2) preparation of HP-Uio-66-NH2A material; 3) preparation of HP-Uio-66-NH2-on-Ce-MOF material; 4) preparation of PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF dispersion; 5) preparing Tb dispersion liquid; 6) adding the Tb dispersion liquid prepared in the step 5) into the PEI @ NG @ HP-Uio-66-NH prepared in the step 4)2Stirring, centrifuging and washing the-on-Ce-MOF dispersion liquid at room temperature, and dispersing the precipitate in ultrapure water to obtain PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF @ Tb nanocomposite solution.
In the technical scheme, the preparation method of the PEI @ NG dispersion in the step 1) comprises the following steps: dispersing 5mg of nitrogen-doped graphene (NG) in 10mL of absolute ethyl alcohol, performing ultrasonic dispersion uniformly, stirring at room temperature, then dropwise adding 4mL of 1 wt% Polyethyleneimine (PEI) aqueous solution into the nitrogen-doped graphene dispersion, and continuously stirring at room temperature for 1h to obtain a PEI @ NG dispersion; HP-Uio-66-NH in the step 2)2The preparation method of the nano material comprises the following steps: 80mg of ZrCl4And 2.4g of lauric acid were added to a 20ml N-Dimethylformamide (DMF) solution, sonicated at room temperature for 20min, and then 31mg of 2-aminoterephthalic acid (NH) was added to the solution2-BDC), continuing ultrasonic dispersion for 5min, transferring the obtained solution into a reaction kettle, reacting at 120 ℃ for 12h, cooling, centrifuging, washing with DMF and ethanol, and drying to obtain yellow HP-Uio-66-NH2A material; HP-Uio-66-NH in the step 3)2The preparation method of the-on-Ce-MOF material comprises the following steps: taking 40mg of HP-Uio-66-NH prepared in step 2)2The powder was mixed with 40mg of polyvinylpyrrolidone (PVP) in 5mL of ultrapure water, stirred for 12 hours, and then 108.5mg of Ce (NO) was added to the solution3)3·6H2O, stirring and heating in a water bath to 60 ℃ and adding 52.5mg of 1,3, 5-benzenetricarboxylic acid (H)3BTC) was dissolved in a water-ethanol mixture (v/v ═ 1:1) and then H was added3Adding BTC solution dropwise into the above 60 deg.C mixed solution, reacting for 1h, cooling, centrifuging, washing with water for 3 times, and drying at 60 deg.C to obtain light yellow HP-Uio-66-NH2-on-Ce-MOF material; PEI @ NG @ HP-Uio-66-NH in the step 4)2The preparation method of the-on-Ce-MOF dispersion liquid comprises the following steps: taking 10mg of HP-Uio-66-NH prepared in step 3)2Dispersing an-on-Ce-MOF material in 10mL of ultrapure water, ultrasonically dispersing for 3min, adding the material into the PEI @ NG dispersion liquid prepared in the step 1), and stirring for 12h to obtain PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF dispersion; the preparation method of the Tb dispersion liquid in the step 5) comprises the following steps: dissolving 15mg of toluidine blue (Tb) in 10mL of ultrapure water, and uniformly dispersing by ultrasonic to obtain Tb dispersion liquid; PEI @ NG @ HP-Uio-66-NH in the step 6)2The preparation method of the-on-Ce-MOF @ Tb nanocomposite solution comprises the following steps: dropwise adding the Tb dispersion prepared in the step 5) into the PEI @ NG @ HP-Uio-66-NH prepared in the step 4)2Stirring the-on-Ce-MOF dispersion liquid for 12 hours at room temperature, centrifuging, washing, and dispersing the precipitate in 10mL of ultrapure water to obtain PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF @ Tb nanocomposite solution.
The invention relates to a nitrogen-doped graphene (NG) modified HP-Uio-66-NH functionalized by Polyethyleneimine (PEI)2-on-Ce-MOF nanomaterial, and finally forming a nanocomposite PEI @ NG @ HP-Uio-66-NH after loading an electroactive substance toluidine blue (Tb)2the-on-Ce-MOF @ Tb further increases the active specific surface area and the conductivity of the nano material on the basis of maintaining the advantages of the original nano material, and realizes the effect of amplifying the sensor signal.
According to a second aspect of the present invention, the present invention provides an electrochemical DNA aptamer sensor for ESAT-6 detection.
The electrochemical DNA aptamer sensor for detecting ESAT-6 is characterized in that: the PEI @ NG @ HP-Uio-66-NH described above was used2the-on-Ce-MOF @ Tb nanocomposite material increases the activity specific surface area and the conductivity of the DNA aptamer sensor.
According to a third aspect of the present invention, the present invention provides a method for preparing the above electrochemical DNA aptamer sensor for ESAT-6 detection.
The preparation method of the electrochemical DNA aptamer sensor for detecting ESAT-6 comprises the following steps:
1) the ESAT-6 binding aptamer was treated with 10mM TES (pH 7.4) buffer at room temperature and stored for future use;
2) piranha washing solution (98% H) for glassy carbon electrode2SO4/30％H2O2Soaking for 30min at a ratio of 3:1, v/v), and washing with ultrapure water for later use;
3) respectively using Al of 0.3 mu m and 0.05 mu m for the electrode obtained in the step 2)2O3Polishing the powder to form a mirror surface, then respectively carrying out ultrasonic treatment on the electrodes according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying for later use;
4) subjecting the electrode obtained in step 3) to a temperature of 0.5M H2SO4Performing electrochemical activation, washing with ultrapure water, and drying;
5) mu.L of the aforementioned PEI @ NG @ HP-Uio-66-NH2Dripping the-on-Ce-MOF @ Tb solution on the surface of the glassy carbon electrode cleaned in the step 4), and drying at room temperature;
6) dripping 10 mu L of the ESAT-6 binding aptamer prepared in the step 1) on the electrode prepared in the step 5) and incubating for 2h at room temperature;
7) and (3) dropwise adding 10 mu L of 3% BSA solution onto the electrode obtained in the step 6), and incubating for 1h at room temperature to obtain the electrochemical DNA aptamer sensor for ESAT-6 detection.
According to a fourth aspect of the present invention, the present invention provides a method for detecting ESAT-6 using the above-described electrochemical DNA aptamer sensor.
A method for detecting ESAT-6 by using the electrochemical DNA aptamer sensor comprises the following steps:
1) dripping target antigen 6 secreted in early tuberculosis stage of the target object with different concentrations on the electrode of the aptamer sensor;
2) the electrodes were characterized in 0.1M PBS (pH 7.0) solution and the current change was measured;
3) drawing a working curve according to the linear relation between the current change value obtained in the step 2) and the ESAT-6 concentration logarithm value;
4) and (3) detecting a sample to be detected by using the aptamer sensor, and calculating the obtained current value through the working curve prepared in the step 3) to obtain the ESAT-6 concentration of the sample to be detected.
Has the advantages that:
the invention provides a nano composite material, which is a Polyethyleneimine (PEI) -functionalized nitrogen-doped graphene (NG) -modified HP-Uio-66-NH2-on-Ce-MOF nano-material, and the ESAT-6 electrochemical aptamer sensor prepared by using the nano-composite material is used for quantitative detection of ESAT-6. The invention finally forms PEI @ NG @ HP-Uio-66-NH after loading an electroactive substance toluidine blue (Tb)2-on-Ce-MOF @ Tb nanocomposite as a sensitive interface of a sensor; the signal amplification effect is carried out by utilizing the good conductivity of PEI @ NG, namely HP-Uio-66-NH2the-on-Ce-MOF has high porosity, large specific surface area and excellent biological activity, a large number of aptamers are loaded through hydrogen bonds, pi-pi accumulation and electrostatic interaction between the-on-Ce-MOF and the aptamers, and finally Tb signal response change is caused through specific binding of the aptamers and targets with different concentrations, so that quantitative detection of ESAT-6 is realized. The prepared electrochemical aptamer sensor is successfully used for the ultra-sensitive detection of ESAT-6. Compared with the traditional ESAT-6 detection method, the invention has the advantages of high sensitivity, strong specificity, rapid detection, convenient operation, low equipment material price and no pollution, thereby providing a new analysis method for the detection of ESAT-6.
According to the invention, through the polyethylene imine functionalized NG, the water solubility and stability of the NG are greatly improved, the aggregation of the NG is effectively avoided, and meanwhile, the HP-Uio-66-NH can be improved by the PEI @ NG2The inherent defect of poor conductivity of the-on-Ce-MOF is that the electron transfer capacity is improved, the signal amplification effect is realized, and the detection sensitivity is improved; double MOF material HP-Uio-66-NH2the-on-Ce-MOF has the advantages of large specific surface area, high porosity and the like, has better biological activity than a single MOF material, and is used as a loading platform of the aptamer through hydrogen bonds, pi-pi accumulation and electrostatic interaction. The aptamer prepared by the invention has high specificity when being used for identifying a target object, and can improve the selectivity of a sensor, thereby providing a new research direction and an analysis method for detecting trace ESAT-6. The materials involved in the invention can be synthesized under laboratory conditions, the operation is simple, the raw materials are low in price, the usage amount is very small each time, the experiment cost is reduced, and meanwhile, the steps of the whole detection analysis method are adoptedThe method is clear, simple and convenient, high in sensitivity and quick in signal response. The electrochemical aptamer sensor prepared by the method can provide a new method for detecting ESAT-6; the electrochemical aptamer sensor prepared by the method can also be applied to the aspects of measurement of other biological samples, monitoring of food, medicine and environment and the like.
Drawings
FIG. 1 is a schematic diagram of the construction and detection principle of the electrochemical aptamer sensor of the invention.
FIG. 2 is a plot of the cyclic voltammograms obtained at a sweep rate of 100mV/s for various modified electrodes at voltages ranging from-0.7 to 0.3V in 0.1M PBS (pH 7.0).
FIG. 3 is the results of detection by the sensor of the present invention of ESAT-6 at various concentrations, wherein panel A is the oxidation partial cyclic voltammogram of the sensor in 0.1M PBS (pH 7.0) for ESAT-6 scans at 0,0.0001,0.001,0.01,0.1, 1 and 10ng/mL, respectively (the inset is the cyclic voltammogram of the sensor scan); and the graph B is a calibration curve of the oxidation peak current change value of the sensor and different concentrations of ESAT-6 logarithmic values.
FIG. 4 shows the results of sensor stability measurements, wherein FIG. A is the cyclic voltammogram obtained after 50 consecutive scans of a sensor incubated with 1ng/mL ESAT-6; FIG. B is a long term stability result for a 1ng/mL ESAT-6 incubated sensor stored at 4 ℃ for 16 days and periodically tested.
FIG. 5 is a graph of the reproducibility of the results obtained after scanning the sensor obtained by incubating 1ng/mL ESAT-6 simultaneously with five different glassy carbon electrodes under the same conditions.
FIG. 6 is a specific assay diagram of an ESAT-6 aptamer sensor, where Blank is Blank, interferents are 0.9% NaCl (NS),5mM Glucose (GS), IFN-. gamma. (1pg/mL) and HSA (1 pg/mL).
Fig. 7 is a graph showing the influence of the amount of toluidine blue (Tb), the pH of Phosphate Buffered Saline (PBS), and the incubation time of aptamer (EBA) on the experimental results, where a is the influence of the amount of toluidine blue (Tb) on the experimental results during the material preparation process, B is the influence of the pH of Phosphate Buffered Saline (PBS) on the experimental results, and C is the influence of the incubation time of aptamer (EBA) on the experimental results.
Detailed Description
The present invention is described in detail below with reference to specific examples, which are given for the purpose of further illustrating the invention and are not to be construed as limiting the scope of the invention, and the invention may be modified and adapted by those skilled in the art in light of the above disclosure.
The main chemical reagents used in the examples of the present invention are as follows:
the early tuberculosis secretory target antigen 6(ESAT-6) standard was purchased from Prospec-Tany Technogene (Israel); polyethyleneimine (PEI) was purchased from Alfa Aeasar (USA); nitrogen-doped graphene (NG) was purchased from nanjing pioneer nano ltd (nanjing, china); ZrCl4、Ce(NO3)3·6H2O and 1,3, 5-benzenetricarboxylic acid (H)3BTC) was purchased from alatin biochemical technology, inc (shanghai, china); 2-amino terephthalic acid (NH)2BDC) from sigma (usa); n, N-Dimethylformamide (DMF) was purchased from Michelin Biochemical technology Ltd (Shanghai, China); bovine Serum Albumin (BSA) and toluidine blue (Tb) were purchased from J&K Scientific Ltd (Beijing, China);
DNA oligonucleotide sequence of ESAT-6 binding aptamer (EBA): 5'-GGGAGCTCAGAATAAACGCTCAACCTCCCTGGGTCACCATAGACTCCATCTAAGATGCTTCGACATGAGGCCCGGATC-3' was synthesized by Shanghai Producer, Inc.
The equipment and technical parameters used are as follows:
the instrument comprises the following steps: cyclic Voltammetry (CV) measurements were performed using a Metrohm Autolab b.v. electrochemical workstation (switzerland Modular instrument). The electrochemical detection adopts a three-electrode system: the modified glassy carbon electrode (diameter 4mm) is used as a working electrode, a platinum wire is used as a counter electrode, and a Saturated Calomel Electrode (SCE) is used as a reference electrode. The pH meter monitors the pH value (S210SevenCompact, mettler-toledo, shanghai, china). A three electrode system was used to generate Cyclic Voltammetry (CV) from-0.7 to 0.3V at a scan rate of 100mV/s in 0.1M PBS (pH 7.0).
Example 1
Preparation of PEI @ NG @ HP-Uio-66-NH2on Ce-MOF @ Tb nanocomposite
The method comprises the following steps:
1) dispersing 5mg of nitrogen-doped graphene (NG) in 10mL of absolute ethyl alcohol, performing ultrasonic dispersion uniformly, stirring at room temperature, then dropwise adding 4mL of 1 wt% Polyethyleneimine (PEI) aqueous solution into the nitrogen-doped graphene dispersion, and continuously stirring at room temperature for 1h to obtain a PEI @ NG dispersion;
2) 80mg of ZrCl4And 2.4g of lauric acid were added to a 20ml N-Dimethylformamide (DMF) solution, sonicated at room temperature for 20min, and then 31mg of 2-aminoterephthalic acid (NH) was added to the solution2-BDC), continuing ultrasonic dispersion for 5min, transferring the obtained solution into a reaction kettle, reacting at 120 ℃ for 12h, cooling, centrifuging, washing with DMF and ethanol, and drying to obtain yellow HP-Uio-66-NH2A material;
3) taking 40mg of HP-Uio-66-NH prepared in step 2)2The powder was mixed with 40mg of polyvinylpyrrolidone (PVP) in 5mL of ultrapure water, stirred for 12 hours, and then 108.5mg of Ce (NO) was added to the solution3)3·6H2O, stirring and heating in a water bath to 60 ℃ and adding 52.5mg of 1,3, 5-benzenetricarboxylic acid (H)3BTC) was dissolved in a water-ethanol mixture (v/v ═ 1:1) and then H was added3Adding BTC solution dropwise into the above 60 deg.C mixed solution, reacting for 1h, cooling, centrifuging, washing with water for 3 times, and drying at 60 deg.C to obtain light yellow HP-Uio-66-NH2-on-Ce-MOF material;
4) taking 10mg of HP-Uio-66-NH prepared in step 3)2Dispersing an-on-Ce-MOF material in 10mL of ultrapure water, ultrasonically dispersing for 3min, adding the material into the PEI @ NG dispersion liquid prepared in the step 1), and stirring for 12h to obtain PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF dispersion;
5) dissolving 15mg of toluidine blue (Tb) in 10mL of ultrapure water, and uniformly dispersing by ultrasonic to obtain Tb dispersion liquid;
6) dropwise adding the Tb dispersion prepared in the step 5) into the PEI @ NG @ HP-Uio-66-NH prepared in the step 4)2Stirring the-on-Ce-MOF dispersion liquid for 12 hours at room temperature, centrifuging, washing, and dispersing the precipitate in 10mL of ultrapure water to obtain PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF @ Tb nanocomposite solution.
Example 2
Preparation of electrochemical DNA aptamer sensor for ESAT-6 detection
The method comprises the following steps: (the construction principle is shown in FIG. 1)
1) ESAT-6 binding aptamer (EBA) was treated with 10mM TES (pH 7.4) buffer at room temperature and left at 4 ℃ until use;
2) piranha washing solution (98% H) for glassy carbon electrode2SO4/30％H2O2Soaking for 30min at a ratio of 3:1, v/v), and washing with ultrapure water for later use;
3) respectively using Al of 0.3 mu m and 0.05 mu m for the electrode obtained in the step 2)2O3Polishing the powder to form a mirror surface, then respectively carrying out ultrasonic treatment on the electrodes for 5min according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use.
4) Subjecting the electrode obtained in step 3) to a temperature of 0.5M H2SO4Carrying out electrochemical activation, scanning the potential to be-0.3-1.55V until a stable cyclic voltammogram is obtained, then washing with ultrapure water again and drying in the air.
5) mu.L of PEI @ NG @ HP-Uio-66-NH prepared in example 12Dropwise adding the-on-Ce-MOF @ Tb solution onto the surface of the glassy carbon electrode cleaned in the step 4), and drying the glassy carbon electrode in air at room temperature;
6) mu.L of 5. mu.M EBA prepared in step 1) was added dropwise to the electrode prepared in step 5) and incubated for 2h at room temperature.
7) And (3) dropwise adding 10 mu L of 3% BSA solution onto the electrode obtained in the step 6), and incubating for 1h at room temperature to block the remaining non-specific binding sites, so as to obtain the electrochemical DNA aptamer sensor for ESAT-6 detection. The resulting aptamer sensors and all materials and reagents were stored at 4 ℃ when not in use.
Example 3
Detection of ESAT-6 using electrochemical DNA aptamer sensors
The electrochemical DNA aptamer sensor constructed in example 2 is used for detecting ESAT-6, and the following steps are carried out:
drawing a working curve
1) The modified electrodes obtained in the steps 4) to 7) of example 2 were placed in a solution containing 0.1M Na2HPO4,0.1M KH2PO410mM KCl and 2mM MgCl2Was characterized in 0.1M PBS (pH 7.0), and its electrochemical response signal was measured, with the results shown in fig. 2: (a) a bare glassy carbon electrode; (b) dropwise adding PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF @ Tb nanocomposite; (c) binding to EBA; (d) blocking with BSA.
2) To the electrode of the aptamer sensor prepared in example 2, 10 μ L of target antigen 6 secreted from the early stage of tuberculosis at different concentrations was added dropwise, and the current change was measured.
3) According to the linear relation between the obtained current change value (shown in figure 3A) and the logarithm value of the concentration of the target antigen 6 secreted in the early stage of tuberculosis, a working curve (shown in figure 3B) is drawn. The measurement result shows that the current response value and the early tuberculosis secretion target antigen 6 have a good linear relation in a concentration log-value range of 100 fg/mL-10 ng/mL, the linear correlation coefficient is 0.9930, and the detection limit is 12.39 fg/mL.
Example 4 sensor stability testing
After the sensor prepared in example 2 was subjected to continuous CV measurement for 50 cycles under the optimum conditions, the response current was reduced by only about 7.20% (as shown in fig. 4A), and when the sensor prepared in example 2 was stored at 4 ℃ and periodically tested, the response current was reduced by 2.1% after 4 days of storage, 3.1% after 8 days of storage, and 91% of the response current was still obtained after 16 days of storage (as shown in fig. 4B), indicating that the sensor had good stability.
Example 5 sensor reproducibility test
After CV measurement was performed on the sensor prepared in example 2 by incubating ESAT-6(1ng/mL) at the same concentration using five different glassy carbon electrodes (as shown in FIG. 5), the Relative Standard Deviation (RSD) was 1.65%, indicating that the sensor was highly reproducible.
Example 6 sensor specificity test
To study the specificity of the proposed adaptive sensors, several interferents present in serum were used: the current response values of different interferents, measured at the same concentration and conditions with NaCl, Glucose, gamma interferon (INF-gamma) and Human Serum Albumin (HSA), in 0.1M PBS (pH 7.0). The results show (as shown in FIG. 6), that the aptamer sensor based on the high specificity reaction of ESAT-6 to EBA has good specificity.
Example 7 consideration of influence factors
For optimal aptamer sensor performance, the effect of toluidine blue (Tb) amount, Phosphate Buffered Saline (PBS) pH, and aptamer (EBA) incubation time on the experimental results was investigated. The results show that: the influence of the toluidine blue amount (Tb) on the experimental results in the preparation process is shown in FIG. 7A, the current value increases with the increase of Tb, but starts to decrease when the Tb amount reaches 1.5mg, therefore, the optimal amount of Tb is 1.5 mg/mL; the results of the effect of the pH value of Phosphate Buffered Saline (PBS) on the experiment are shown in FIG. 7B, wherein the current value of PBS is continuously increased when the pH value is 5-7, and the current response is obviously reduced when the pH value is 8, so that the optimal pH value of PBS is 7; the effect of aptamer (EBA) incubation time on the experiment is shown in fig. 7C, where the peak current changes within 1-5 h of the aptamer EBA incubation time, and it is known that the current value is slightly decreased by a longer incubation time, and therefore, the optimal incubation time for EBA is 2 h.
Example 8 actual sample analysis application
To evaluate the practical applicability and accuracy of the proposed aptamer sensor, ESAT-6 (shown in Table 1) was added at various concentrations to a 50-fold diluted sample of healthy human serum, and then detected using the electrochemical aptamer sensor prepared in example 2 with reference to example 3. The results are shown in Table 1, with relative standard deviation ranging from 0.89% to 3.39% and recovery rates of 91.59% to 94.15%.
Table 1 ESAT-6(n ═ 3) of human healthy serum samples measured using the electrochemical aptamer sensor prepared according to the invention
. The result shows that the aptamer sensor prepared by the invention is feasible for detecting ESAT-6 and can meet the requirement of practical analysis.
Claims (4)
1. A nanocomposite material, its characteristicsCharacterized in that: the nano composite material is PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF @ Tb nanocomposite, prepared by the following steps: 1) preparing a PEI @ NG dispersion; 2) preparation of HP-Uio-66-NH2A material; 3) preparation of HP-Uio-66-NH2-on-Ce-MOF material; 4) preparation of PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF dispersion; 5) preparing Tb dispersion liquid; 6) adding the Tb dispersion liquid prepared in the step 5) into the PEI @ NG @ HP-Uio-66-NH prepared in the step 4)2Stirring, centrifuging and washing the-on-Ce-MOF dispersion liquid at room temperature, and dispersing the precipitate in ultrapure water to obtain PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF @ Tb nanocomposite solution; the preparation method of the PEI @ NG dispersion in the step 1) comprises the following steps: dispersing 5mg of nitrogen-doped graphene (NG) in 10mL of absolute ethyl alcohol, performing ultrasonic dispersion uniformly, stirring at room temperature, then dropwise adding 4mL of 1 wt% Polyethyleneimine (PEI) aqueous solution into the nitrogen-doped graphene dispersion, and continuously stirring at room temperature for 1h to obtain a PEI @ NG dispersion; HP-Uio-66-NH in the step 2)2The preparation method of the nano material comprises the following steps: 80mg of ZrCl4And 2.4g of lauric acid were added to a 20ml N-Dimethylformamide (DMF) solution, sonicated at room temperature for 20min, and then 31mg of 2-aminoterephthalic acid (NH) was added to the solution2-BDC), continuing ultrasonic dispersion for 5min, transferring the obtained solution into a reaction kettle, reacting at 120 ℃ for 12h, cooling, centrifuging, washing with DMF and ethanol, and drying to obtain yellow HP-Uio-66-NH2A material; HP-Uio-66-NH in the step 3)2The preparation method of the-on-Ce-MOF material comprises the following steps: taking 40mg of HP-Uio-66-NH prepared in step 2)2The powder was mixed with 40mg of polyvinylpyrrolidone (PVP) in 5mL of ultrapure water, stirred for 12 hours, and then 108.5mg of Ce (NO) was added to the solution3)3·6H2O, stirring and heating in a water bath to 60 ℃ and adding 52.5mg of 1,3, 5-benzenetricarboxylic acid (H)3BTC) was dissolved in a v/v ═ 1:1 water-ethanol mixture, then H was added3Adding BTC solution dropwise into the above 60 deg.C mixed solution, reacting for 1h, cooling, centrifuging, washing with water for 3 times, and drying at 60 deg.C to obtain light yellow HP-Uio-66-NH2-on-Ce-MOF material; PEI @ NG @ HP-Uio-66-NH in the step 4)2The preparation method of the-on-Ce-MOF dispersion liquid comprises the following steps: get10mg of HP-Uio-66-NH prepared in step 3)2Dispersing an-on-Ce-MOF material in 10mL of ultrapure water, ultrasonically dispersing for 3min, adding the material into the PEI @ NG dispersion liquid prepared in the step 1), and stirring for 12h to obtain PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF dispersion; the preparation method of the Tb dispersion liquid in the step 5) comprises the following steps: dissolving 15mg of toluidine blue (Tb) in 10mL of ultrapure water, and uniformly dispersing by ultrasonic to obtain Tb dispersion liquid; PEI @ NG @ HP-Uio-66-NH in the step 6)2The preparation method of the-on-Ce-MOF @ Tb nanocomposite solution comprises the following steps: dropwise adding the Tb dispersion prepared in the step 5) into the PEI @ NG @ HP-Uio-66-NH prepared in the step 4)2Stirring the-on-Ce-MOF dispersion liquid for 12 hours at room temperature, centrifuging, washing, and dispersing the precipitate in 10mL of ultrapure water to obtain PEI @ NG @ HP-Uio-66-NH2-on-Ce-MOF @ Tb nanocomposite solution.
2. An electrochemical DNA aptamer sensor for ESAT-6 detection, which is characterized in that: use of the PEI @ NG @ HP-Uio-66-NH of claim 12the-on-Ce-MOF @ Tb nanocomposite material increases the activity specific surface area and the conductivity of the DNA aptamer sensor.
3. The electrochemical DNA aptamer sensor for ESAT-6 detection as claimed in claim 2, prepared by the steps of:
1) the ESAT-6 binding aptamer was treated with 10mM TES buffer pH 7.4 at room temperature and stored for future use;
2) the volume ratio v/v of a glassy carbon electrode is 98% H2SO4/30％H2O2Soaking the piranha washing liquor with the ratio of 3:1 for 30min, and then washing the piranha washing liquor with ultrapure water for later use;
3) respectively using Al of 0.3 mu m and 0.05 mu m for the electrode obtained in the step 2)2O3Polishing the powder to form a mirror surface, then respectively carrying out ultrasonic treatment on the electrodes according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying for later use;
4) subjecting the electrode obtained in step 3) to a temperature of 0.5M H2SO4Performing electrochemical activation, washing with ultrapure water, and drying;
5) 10 μ L of the PEI @ NG @ HP-Uio-66 of claim 1NH2Dripping the-on-Ce-MOF @ Tb solution on the surface of the glassy carbon electrode cleaned in the step 4), and drying at room temperature;
6) dripping 10 mu L of the ESAT-6 binding aptamer prepared in the step 1) on the electrode prepared in the step 5) and incubating for 2h at room temperature;
7) and (3) dropwise adding 10 mu L of 3% BSA solution onto the electrode obtained in the step 6), and incubating for 1h at room temperature to obtain the electrochemical DNA aptamer sensor for ESAT-6 detection.
4. A method for detecting ESAT-6 using the electrochemical DNA aptamer sensor of claim 2 or 3, comprising the steps of:
1) dripping different concentrations of target early tuberculosis secretory target antigens 6 onto the electrode of the aptamer sensor as claimed in claim 2 or 3;
2) the electrode is placed in a PBS solution with the pH value of 7.0 at 0.1M for characterization, and the current change value is measured;
3) drawing a working curve according to the linear relation between the current change value obtained in the step 2) and the ESAT-6 concentration logarithm value;
4) and (3) detecting a sample to be detected by using the aptamer sensor, and calculating the obtained current value through the working curve prepared in the step 3) to obtain the ESAT-6 concentration of the sample to be detected.
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