阅读说明：本技术 纳米复合材料、lps电化学适体传感器的制备方法及检测方法 (Nano composite material, preparation method of LPS electrochemical aptamer sensor and detection method ) 是由 母昭德 白丽娟 田江漫 于 2019-09-09 设计创作，主要内容包括：本发明公开了一种P-rGO-TNT-Ag纳米复合材料,还公开了一种用于LPS检测的电化学DNA适体传感器,由以下方法制备得到：1)用Tris-HCl缓冲液处理LPS适体备用；2)将金电极抛光成镜面,处理电极,干燥备用；3)将电极清洗,电化学活化,水冲洗,干燥；4)将P-rGO-TNT-Ag溶液滴加到金电极表面上,干燥；5)将LPS特异性结合适体滴加在电极上室温孵育；6)将BSA孵育在电极表面,即得。还公开了采用该传感器检测LPS的方法。(The invention discloses a P-rGO-TNT-Ag nano composite material and an electrochemical DNA aptamer sensor for LPS detection, which is prepared by the following method: 1) processing the LPS aptamer by using Tris-HCl buffer solution for later use; 2) polishing the gold electrode into a mirror surface, processing the electrode, and drying for later use; 3) cleaning the electrode, performing electrochemical activation, washing with water, and drying; 4) dripping the P-rGO-TNT-Ag solution on the surface of a gold electrode, and drying; 5) dropping the LPS specific binding aptamer on an electrode for incubation at room temperature; 6) and (3) incubating BSA on the surface of the electrode to obtain the BSA-activated protein. Methods of detecting LPS using the sensor are also disclosed.)

1. A P-rGO-TNT-Ag nano composite material is characterized by being prepared by the following steps: 1) preparing a rGO dispersion; 2) preparing a P-rGO dispersion; 3) preparation of TiO₂Dispersions of nanotubes (TNTs); 4) preparing TNT-Ag dispersion liquid; 5) adding the TNT-Ag dispersion liquid prepared in the step 4) into the P-rGO dispersion liquid prepared in the step 2), stirring for 24 hours at room temperature, centrifuging, washing, and dispersing the precipitate into ultrapure water to obtain the P-rGO-TNT-Ag nano composite material solution.

2. The P-rGO-TNT-Ag nanocomposite material of claim 1, wherein the preparation method of the rGO dispersion in step 1) is: dispersing 10mg GO in 25mL of ultrapure water, ultrasonically homogenizing, and then dropwise addingAdjusting the pH value to be about 8-9 by 20 mu L of ammonia water, adding 250mg of Ascorbic Acid (AA), stirring at room temperature for 0.5h, continuing stirring for 1h under an oil bath at 95 ℃, centrifuging, washing, and dispersing the precipitate in 10mL of ultrapure water to obtain an rGO dispersion liquid; the preparation method of the PDDA-rGO dispersion liquid in the step 2) comprises the following steps: dripping 20 mu L of 20 wt% polydiallyldimethylammonium chloride (PDDA) solution into 1mL of the rGO dispersion liquid prepared in the step 1), and stirring for 0.5h to obtain a P-rGO dispersion liquid; the preparation method of the TNTs dispersion liquid in the step 3) comprises the following steps: 1g of TiO₂Adding the powder into 20mL of 10mol/L NaOH solution, stirring at room temperature for 1h, transferring the solution into a reaction kettle, reacting at 130 ℃ for 10h, cooling, centrifuging, washing with 1M HCl solution until the pH value is 1, washing with ultrapure water until the pH value is 7, and dispersing the precipitate in 10mL of ultrapure water to obtain a TNTs dispersion liquid; the preparation method of the TNT-Ag dispersion liquid in the step 4) comprises the following steps: diluting 1mL of the TNTs dispersion prepared in step 3) to 5mL with ultrapure water, and adding 1mL of 8.5mg/mL AgNO₃The solution was stirred for 5min, to which was added 1mL of 3mg/mL NaBH₄And continuously stirring the solution for 1h, centrifuging, washing, and dispersing the precipitate in 5mL of ultrapure water to obtain the TNT-Ag dispersion liquid.

3. An electrochemical DNA aptamer sensor for LPS detection, which is prepared by the following method, comprising the following steps:

1) treating the amino-labeled LPS-binding aptamer with 20mM Tris-HCl (pH 7.4) buffer at room temperature and storing for future use;

2) carassius auratus washing liquid (98% H) for gold 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) dripping 10 μ L of the P-rGO-TNT-Ag solution of claim 1 onto the surface of the gold electrode cleaned in step 4), and drying at room temperature;

6) 20 mu L of the LPS combined aptamer prepared in the step 1) is added dropwise on the electrode prepared in the step 5) and incubated for 15h at room temperature;

7) and (3) dropwise adding 20 mu L of 1% BSA solution onto the electrode obtained in the step 6), and incubating at room temperature for 40min to obtain the electrochemical DNA aptamer sensor for LPS detection.

4. A method for detecting LPS by using an electrochemical DNA aptamer sensor is characterized by comprising the following steps:

1) dripping endotoxin as a target substance at different concentrations onto an electrode of the aptamer sensor as claimed in claim 3;

2) the electrodes were characterized in 0.1M PBS (pH7.0) 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 log value of the LPS concentration;

4) detecting a sample to be detected by using the aptamer sensor as claimed in claim 3, and calculating the obtained current value through the working curve prepared in the step 3) to obtain the concentration of LPS in the sample to be detected.

Technical Field

The invention relates to the technical field of electrochemical detection, in particular to a nano composite material, a preparation method of an LPS electrochemical aptamer sensor and a detection method.

Background

Bacterial endotoxins are characteristic structures of the outer membrane of the cell wall of gram-negative bacteria, and their main component is Lipopolysaccharide (LPS), also known as a "pyrogen". Endotoxin is released after the bacteria die or autolyzed, and after the endotoxin enters blood, fever, endotoxemia and microcirculation disturbance can be caused, and septic shock, disseminated intravascular coagulation and the like are caused in severe cases. Preparations such as biological products, injection medicaments, chemical medicaments, radiopharmaceuticals, antibiotics, vaccines, dialyzates and medical instruments (such as disposable syringes and implantable biomaterials) can be used after being qualified through bacterial endotoxin detection tests, and the limit of bacterial endotoxin of the medicines and the biological products is clearly specified in the 'Chinese pharmacopoeia' 2015 edition.

Currently, the conventional LPS detection methods include rabbit pyrogen method, limulus test (LAL), High Performance Liquid Chromatography (HPLC), flow cytometry, enzyme-linked immunoassay, and the like. However, the rabbit pyrogen method has individual difference, low sensitivity and time consumption; the limulus test method is based on enzymatic reaction, the detection result is easily affected by enzyme and impurities, the false positive rate is high, and the reaction condition is harsh; operations such as HPLC, flow cytometry and the like are complicated, equipment is expensive, and operators need professional training; the enzyme-linked immunoassay is based on antigen and antibody, is easily influenced by the environment, and has large matrix interference of complex samples. The above disadvantages limit its use for rapid detection of trace amounts of LPS. Therefore, it is necessary to design a highly selective, accurate, rapid and convenient method for detecting LPS.

Aptamers (aptamers), which are a stretch of oligonucleotide sequences that can bind to a target molecule with very high affinity and specificity, are repeatedly screened from a random pool of oligonucleotide sequences synthesized in vitro by the exponential enrichment of ligand systems evolution (SELEX) technique, and can be RNA, single-stranded dna (ssdna), or double-stranded dna (dsdna). 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. Therefore, as an ideal identification element, the aptamer is always used in the construction of an electrochemical sensor, and the obtained electrochemical aptamer sensor has the characteristics of high sensitivity, quick response and low cost.

The application of the novel nano composite material is an effective strategy for amplifying sensor signals, and the nano composite material can further increase the active specific surface area and the conductivity on the basis of maintaining the advantages of the original nano material. The P-rGO-TNT-Ag nano composite material finally formed by the poly (diallyldimethylammonium chloride) (PDDA) functionalized reduced graphene oxide (rGO) modified TiO2 nano tubes (TNTs)/Ag nano combination has good electrocatalytic capacity. Firstly, the water solubility of hydrophobic rGO can be improved by the water-dispersible PDDA, the aggregation of the rGO is avoided through the electrostatic repulsion effect, and the excellent physical and chemical properties of the rGO are fully exerted; secondly, oxygen-containing groups exist on the surface of rGO and can be used as binding sites for fixing metals; finally, the P-rGO and the TNTs have larger specific surface area and stronger adsorption capacity, and the synergistic effect of the P-rGO and the TNTs can improve the loading capacity of the electroactive material Ag on a unit area and further improve the electron transfer efficiency.

Disclosure of Invention

In order to solve the problems, the invention provides a P-rGO-TNT-Ag nano composite material which is prepared by the following steps: 1) preparing a rGO dispersion; 2) preparing a P-rGO dispersion; 3) preparation of TiO₂Dispersions of nanotubes (TNTs); 4) preparing TNT-Ag dispersion liquid; 5) adding the TNT-Ag dispersion liquid prepared in the step 4) into the P-rGO dispersion liquid prepared in the step 2), stirring for 24 hours at room temperature, centrifuging, washing, and dispersing the precipitate into ultrapure water to obtain the P-rGO-TNT-Ag nano composite material solution.

In the above technical scheme, the preparation method of the rGO dispersion in step 1) is: dispersing 10mg of GO in 25mL of ultrapure water, performing ultrasonic homogenization, then dropwise adding about 20 mu L of ammonia water to enable the pH to be about 8-9, then adding 250mg of Ascorbic Acid (AA), stirring at room temperature for 0.5h, then continuously stirring for 1h under an oil bath at 95 ℃, centrifuging, washing, and then dispersing the precipitate in 10mL of ultrapure water to obtain a rGO dispersion liquid; the preparation method of the P-rGO dispersion liquid in the step 2) comprises the following steps: dripping 20 mu L of 20 wt% polydiallyldimethylammonium chloride solution into 1mL of the rGO dispersion liquid prepared in the step 1), and stirring for 0.5h to obtain a P-rGO dispersion liquid; the preparation method of the TNTs dispersion liquid in the step 3) comprises the following steps: 1g of TiO₂Adding the powder into 20mL of 10mol/L NaOH solution, stirring at room temperature for 1h, transferring the solution into a reaction kettle, reacting at 130 ℃ for 10h, cooling, centrifuging, washing with 1M HCl solution until the pH value is 1, washing with ultrapure water until the pH value is 7, and dispersing the precipitate in 10mL of ultrapure water to obtain a TNTs dispersion liquid; the preparation method of the TNT-Ag dispersion liquid in the step 4) comprises the following steps: diluting 1mL of the TNTs dispersion prepared in step 3) to 5mL with ultrapure water, and adding 1mL of 8.5mg/mL AgNO₃The solution was stirred for 5min, to which was added 1mL of 3mg/mL NaBH₄Solutions ofAnd continuously stirring for 1h, centrifuging, washing, and dispersing the precipitate in 5mL of ultrapure water to obtain the TNT-Ag dispersion liquid.

In another aspect, the present invention provides an electrochemical DNA aptamer sensor for LPS detection, prepared by the following method, comprising the following steps:

1) treating the amino-labeled LPS-binding aptamer with 20mM Tris-HCl (pH 7.4) buffer at room temperature and storing for future use;

2) carassius auratus washing liquid (98% H) for gold 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) dripping 10 mu L of the P-rGO-TNT-Ag solution on the surface of the gold electrode cleaned in the step 4), and drying at room temperature;

6) 20 mu L of the LPS combined aptamer prepared in the step 1) is added dropwise on the electrode prepared in the step 5) and incubated for 15h at room temperature;

7) and (3) dropwise adding 20 mu L of 1% BSA solution onto the electrode obtained in the step 6), and incubating at room temperature for 40min to obtain the electrochemical DNA aptamer sensor for LPS detection.

In another aspect, the present invention provides a method for detecting LPS using an electrochemical DNA aptamer sensor, comprising the steps of:

1) dropwise adding target substance endotoxin with different concentrations to the electrode of the aptamer sensor;

2) the electrodes were characterized in 0.1M PBS (pH7.0) 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 log value of the LPS concentration;

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 LPS concentration of the sample to be detected.

The P-rGO-TNT-Ag nano composite material finally formed by modifying a TiO2 nano tube (TNTs)/Ag nano conjugate by polydiallyl dimethyl ammonium chloride (PDDA) functionalized reduced graphene oxide (rGO) is used as a sensitive interface of a sensor; the characteristics of large specific surface area and strong adsorption capacity of P-rGO and TNTs are utilized to synergistically improve the loading capacity of an electroactive material Ag in a unit area, so that the electron transfer between the Ag and an electrode is promoted; then the amino-labeled single-chain LPS aptamer is immobilized on an electrode through an Ag-NH2 bond for indication, and finally, different changes of electrochemical signals are caused through specific binding of the aptamer and targets with different concentrations, so that quantitative detection of LPS is realized. The prepared electrochemical aptamer sensor is successfully used for the ultra-sensitive detection of LPS. Compared with the traditional LPS detection method, the method has the advantages of high sensitivity, strong specificity, quick detection, convenient operation, low equipment material price and no pollution, thereby providing a new analysis method for detecting the endotoxin.

The invention has the beneficial effects that:

1) through polydiallyldimethylammonium chloride functionalized rGO, the water solubility of the rGO can be greatly improved, the aggregation of the rGO is effectively avoided, the rGO can be uniformly spread on the surface of an electrode, and the excellent physical and chemical properties of the rGO can be fully exerted

2) The P-rGO and the TNTs have larger specific surface area and stronger adsorption capacity, and the synergistic effect of the P-rGO and the TNTs can improve the immobilization amount of Ag and promote the electron transfer between the Ag and an electrode, so that the signal amplification of an electrochemical sensor can be realized, and the detection sensitivity can be improved.

3) The aptamer 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 LPS.

4) The related materials can be synthesized under the laboratory condition, the operation is simple, the raw materials are low in price, the using amount is very small each time, and the experiment cost is reduced.

5) The whole detection and analysis method has clear and simple steps, high sensitivity and rapid signal response.

6) The electrochemical aptamer sensor prepared by the method can provide a new method for detecting endotoxin; the electrochemical aptamer sensor prepared by the method can also be applied to the aspects of analysis and detection of biological products, food and drugs, medical equipment 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-0.3 to 0.5V in 0.1M PBS (pH 7.0).

FIG. 3 is the results of the detection of LPS at different concentrations by the sensor of the present invention, wherein Panel A is a cyclic voltammogram of the sensor for LPS scans at 0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100ng/mL in 0.1M PBS (pH7.0), respectively; graph B is a calibration curve of the oxidation peak current response of the sensor versus the log of LPS at different concentrations.

FIG. 4 is a graph of sensor stability measurements, wherein A is a cyclic voltammogram of a 50ng/mL LPS incubated sensor after 60 consecutive scans; panel B is a long term stability result for a 50fg/mL LPS incubated sensor stored at 4 ℃ for 25 days and periodically tested.

FIG. 5 is a graph of the reproducibility of the results obtained from scanning the sensor under identical conditions with five different gold electrodes incubated simultaneously with 50ng/mL LPS.

FIG. 6 is a specific assay graph of an LPS aptamer sensor, wherein the interferents are blank, 1ng/mL NaCl, NaH₂PO₄，NaHSO₃EDTA-2Na and Glucose.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting.

The main chemical reagents used in the examples of the present invention are as follows:

endotoxin standards and polydiallyldimethylammonium chloride (PDDA) were purchased from sigma (usa); TiO2₂Biochemical technology of Shanghai MielinLimited (shanghai, china); AgNO₃Purchased from shanghai reagent factory (shanghai, china); graphene Oxide (GO) was purchased from Nanjing pioneer nanometer Limited (Nanjing, China); bovine Serum Albumin (BSA) and Ascorbic Acid (AA) were purchased from J&K Scientific Ltd (Beijing, China);

DNA oligonucleotide sequence of LPS-binding aptamer (LBA): 5' -NH₂-(CH₂)₆-CTTCTGCCCGCCTCCTTCCTAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT-3' was synthesized by Shanghai Biotech Ltd.

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 gold electrode (diameter 4mm) was used as the working electrode, the platinum wire as the counter electrode, and the Saturated Calomel Electrode (SCE) as the reference electrode. The pH meter monitors the pH value (PHS-3C, Reye, Shanghai, China). A three electrode system was used to generate Cyclic Voltammetry (CV) from-0.3 to 0.5V at a scan rate of 100mV/s in 0.1M PBS (pH 7.0).