阅读说明：本技术 一种基于核酸外切酶辅助放大光电化学适配体传感器 (Electrochemical aptamer sensor based on exonuclease-assisted amplification ) 是由 郝洪顺 朱良良 赵一睿 李畅 丁超 甘寒薇 侯红漫 张公亮 毕景然 闫爽 于 2021-08-09 设计创作，主要内容包括：本发明公开的一种基于核酸外切酶辅助放大光电化学适配体传感器的制备方法及应用,水浴沉积的方法在与预处理过的FTO上沉积WO-(3),煅烧后冷却,将WO-(3)/FTO电极浸入HAuCl-(4)溶液,煅烧后得Au/WO-(3)/FTO电极。将活化的互补cDNA滴在Au/WO-(3)/FTO电极上,孵化过夜。用Tris-HCl缓冲液冲洗除去未连接的cDNA,然后用MCH封闭。Tris-HCl冲洗后将CdTe QDs-Ap偶联物滴在电极上孵育。用Tris-HCl溶液冲洗后得工作电极。将工作电极插入光电化学池中,再将饱和甘汞电极和铂对电极插入,组成三电极体系,外接电化学工作站,辅以模拟日光氙灯光源系统,组装成光电化学适配体传感器。滴加含Exo-I的LM溶液,孵育后用Tris-HCl溶液冲洗后即可进行检测。该传感器具有光电流响应能力强,检测灵敏度高,检测时间短,成本低廉,便携性的优势。(The invention discloses a preparation method and application of a photoelectrochemical aptamer sensor based on exonuclease-assisted amplification, and a water bath deposition method for depositing WO on pretreated FTO 3 Calcining and then cooling, and adding WO 3 Immersion of FTO electrode in HAuCl 4 The solution is calcined to obtain Au/WO 3 an/FTO electrode. Dropping the activated complementary cDNA to Au/WO 3 on/FTO electrode, incubate overnight. Unligated cDNA was removed by washing with Tris-HCl buffer and then blocked with MCH. After being washed by Tris-HCl, the CdTe QDs-Ap conjugate is dripped on an electrode for incubation. And washing with a Tris-HCl solution to obtain the working electrode. And inserting the working electrode into a photoelectrochemical cell, inserting the saturated calomel electrode and the platinum counter electrode into the photoelectrochemical cell to form a three-electrode system, connecting the three-electrode system with an external electrochemical workstation, and assembling the three-electrode system with a simulated daylight xenon lamp light source system to form the photoelectrochemical aptamer sensor. Dripping LM solution containing Exo-I, incubating and adding Tris-HClThe solution can be detected after being washed. The sensor has the advantages of strong photocurrent response capability, high detection sensitivity, short detection time, low cost and portability.)

1. A method for detecting Listeria monocytogenes based on an exonuclease-assisted amplification photoelectrochemical aptamer sensor is characterized by comprising the following steps:

step 1, preparation of WO₃/FTO electrode

SnO doped with fluorine₂And cleaning the conductive glass (FTO) by using acetone, sodium hydroxide and deionized water in sequence, and drying for later use. 0.4g of Na₂WO₄·2H₂O and 0.17g (NH)₄)₂C₂O₄·H₂O was dissolved in 33mL of deionized water, stirred for 10min, and then 9mL of HCl solution (37%) was added. Stirring for 10min, and adding 8mLH₂O₂(30%), stirring is continued for 20min, and then30mL of absolute ethanol was added and stirred for 30 min. Placing the pretreated FTO with the conductive surface facing downwards and inclined at 45 deg. to the wall of the beaker, placing in the above solution, and placing in a water bath for 200min at a certain temperature. Cooling to room temperature, washing with deionized water, and drying in a drying oven at 60 deg.C for 6 hr. Finally, calcining the mixture for 2 hours at a certain temperature by using a muffle furnace, cooling the mixture to room temperature, washing the cooled mixture by using deionized water and drying the washed mixture to obtain WO₃an/FTO electrode;

step 2, preparation of CdTe quantum dots (CdTe QDs)

0.2mmol of CdCl₂·2.5H₂O was dissolved in 50mL of deionized water, then 18. mu.L of thioglycolic acid was added and stirred for 10min, then the pH was adjusted with 2M NaOH. 0.04mmol of K₂TeO₃Dissolved in 50mL of deionized water, stirred well and added to the above solution, and stirred for 20 min. Then 80mg NaBH was added₄Fully reacting for 5 min. The flask was then connected to a condenser for condensation at 100c for a period of time. Cooling to room temperature, centrifuging, washing, adding equivalent deionized water, and storing in a 4 deg.C refrigerator;

step 3, preparing CdTe QDs-aptamer (CdTe QDs-Ap) conjugate

400 μ L of CdTe QDs were activated with 40 μ L of a solution containing 40mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 10mM N-hydroxysuccinimide (NHS) at room temperature for 1h, then added to 300 μ L of a concentrated Listeria Monocytogenes (LM) aptamer (Ap) solution and stirred continuously overnight. Centrifuging the obtained solution at 4 ℃ to remove excessive Ap to obtain a CdTe QDs-Ap conjugate;

step 4, constructing a working electrode of the photoelectrochemical aptamer sensor

Firstly, WO₃/FTO electrode immersion 0.01M HAuCl₄Calcining the solution for 50min at 300 ℃ for 2h to form gold nanoparticles to obtain Au/WO (gold-loaded gold nanoparticles) (pH 4.5)₃an/FTO electrode.

mu.M complementary DNA (cDNA) to the LM aptamer was activated with tris (2-carboxyethyl) phosphine (TCEP) (0.6. mu.L, 10mM) for 1h and dropped onto Au/WO₃On the/FTO electrode. Incubate overnight at 4 ℃ and wash with Tris-HCl (pH 7.4, 10mM) buffer, then block with 30 μ L6-hydroxy-1-hexanethiol (MCH) for 1 h. Tris-HCAfter L wash, 30 μ L of CdTe QDs-Ap conjugate was dropped onto the electrode and incubated at 37 ℃ for 1h to allow hybridization of Ap to cDNA. Washing with Tris-HCl solution to obtain the working electrode;

step 5, constructing a photoelectric chemical aptamer sensor for detecting LM

And (4) inserting the working electrode prepared in the step (4) into an electrolytic bath, then inserting a saturated calomel electrode and a platinum counter electrode to form a three-electrode system, externally connecting an electrochemical workstation, and simulating sunlight by using a xenon lamp light source system, so that the photoelectrochemistry aptamer sensor is assembled and used for photoelectrochemical detection of the LM.

2. The method for detecting listeria monocytogenes based on the exonuclease-assisted amplified photoelectrochemical aptamer sensor as claimed in claim 1, wherein in the step 1, the temperature of the water bath is 70-90 ℃, and the temperature of the muffle furnace is 300-800 ℃.

3. The method for preparing a photoelectrochemical aptamer sensor according to claim 1, wherein in the step 2, the pH value of the solution is 10-11.5, and the condensation time is 1-10 h.

4. The method of claim 1, wherein in step 3, the LM aptamer concentration is 1-5 μ M.

5. The method for detecting listeria monocytogenes with photoelectrochemical aptamer sensor as claimed in claim 1, wherein the method for determining the content of listeria monocytogenes comprises:

△I＝9.76logC-4.44

in the formula, delta I is the current change amount before and after modifying the pathogenic bacteria during detection, and is milliampere, and C is the salmonella concentration, and is CFU/mL.

6. The method for detecting listeria monocytogenes based on the exonuclease assisted amplification photoelectrochemical aptamer sensor according to claim 5, wherein the photoelectrochemical detection is performed at room temperature with Phosphate Buffered Saline (PBS) containing 0.1M Ascorbic Acid (AA), pH 7.4, 0.1M. During the test, a simulated daylight xenon lamp system provides a light source, and the light source is switched on and off once every 20 s. The applied voltage was 0.0V.

7. The photoelectrochemical aptamer sensor prepared by the method according to any one of claims 1 to 6, which is used for rapid detection of Listeria monocytogenes and is also suitable for rapid detection of other food-borne pathogenic bacteria.

Technical Field

The invention belongs to the field of electrochemical detection, and relates to a photoelectrochemical aptamer sensor and a preparation method thereof, which are used for rapidly detecting food-borne pathogenic bacteria.

Background

Listeria Monocytogenes (LM) is the most virulent strain of Listeria species that is pathogenic to humans among 8 species of Listeria. LM is a gram-positive bacilli, which causes food-borne listeriosis relatively rare but very serious, and is a food-borne pathogenic bacterium with very high lethality rate.

LM can survive at 0-45 ℃, has very tenacious vitality and exists in milk, dairy products, eggs, poultry and meat. The LM can still grow and propagate in large quantities at the refrigerating temperature of 4-6 ℃ of the refrigerator, which means that the LM cannot be placed in a dead place even if food is refrigerated in the refrigerator, and the LM is also an important characteristic of the bacterium different from other food-borne pathogenic bacteria.

The LM rapid detection technology which is the most studied at present mainly comprises a PCR technology, a real-time fluorescence quantitative PCR technology, a loop-mediated isothermal amplification technology, an enzyme-linked immunosorbent assay technology and an immunochromatographic test strip. The PCR technology and the real-time fluorescent quantitative PCR technology have complicated operation processes, require operators with professional techniques, and have expensive required equipment and reagents. The LAMP method has high sensitivity, strong specificity, rapidness and high efficiency, but the detection process is easily polluted to cause false positive. Enzyme-linked immunosorbent assay has the same advantages in detection efficiency and specificity, but has certain limitations due to the long preparation time and high cost of LM antibody. Previously, due to the diversity of detection environments and the inconsistency of detection standards, the detection sensitivity and specificity of various sensors generate large fluctuation, and no proper detection value determination method and standard exist.

Disclosure of Invention

In order to solve the technical problems, the invention provides a rapid, simple and convenient photoelectrochemical aptamer sensor with higher sensitivity, which is used for LM detection. The invention integrates the technologies of nano material preparation, quantum dot sensitization, aptamer molecule recognition, exonuclease I auxiliary circulation and the like, constructs a photoelectrochemical aptamer sensor, and establishes a rapid, simple, accurate and sensitive LM detection method.

The complete technical scheme of the invention comprises the following steps:

a photoelectrochemical aptamer sensor based on exonuclease auxiliary amplification and a method for detecting Listeria monocytogenes thereof comprise the following steps:

step 1, preparation of WO₃/FTO electrode

And cleaning the FTO electrode by using acetone, sodium hydroxide and deionized water in sequence, and drying for later use. 0.4g of Na₂WO₄·2H₂O and 0.17g (NH)₄)₂C₂O₄·H₂O was dissolved in 33mL of deionized water, stirred for 10min, and then 9mL of HCl solution (37%) was added. Stirring for 10min and adding 8mL of H₂O₂(30%) stirring was continued for 20min, and 30mL of absolute ethanol was added and stirred for 30 min. And (3) placing the pretreated FTO conductive glass with the conductive surface facing downwards and inclined at 45 degrees to be close to the wall of the beaker, placing the FTO conductive glass into the solution, and placing the solution into a water bath kettle at a certain temperature for 200 min. Cooling to room temperature, washing with deionized water, and drying in a drying oven at 60 deg.C for 6 hr. Finally, calcining the mixture for 2 hours at a certain temperature by using a muffle furnace, cooling the mixture to room temperature, washing the cooled mixture by using deionized water and drying the washed mixture to obtain WO₃an/FTO electrode.

Step 2, preparation of CdTe QDs

0.2mmol of CdCl₂·2.5H₂O was dissolved in 50mL of deionized water, then 18. mu.L of thioglycolic acid was added and stirred for 10min, then the pH was adjusted with 2M NaOH. 0.04mmol of K₂TeO₃Dissolved in 50mL of deionized water, stirred well and added to the above solution, and stirred for 20 min. Then 80mg NaBH was added₄Fully reacting for 5 min. The flask was then connected to a condenser for condensation at 100c for a period of time. Cooling to room temperature, centrifuging, washing, adding equivalent deionized water, and storing in a refrigerator at 4 deg.C.

Step 3, preparing CdTe QDs-Ap conjugate

400 μ L of CdTe QDs are activated with 40 μ L of a solution containing 40mM EDC and 10mM NHS for 1h at room temperature, then added to 300 μ L of a concentrated solution of LM aptamer (Ap) and stirred overnight. Centrifuging the obtained solution at 4 ℃ to remove excessive Ap to obtain the CdTe QDs-Ap conjugate.

Step 4, constructing a working electrode of the photoelectrochemical aptamer sensor

Firstly, WO₃/FTO electrode immersion 0.01M HAuCl₄Calcining the solution for 50min at 300 ℃ for 2h to form gold nanoparticles to obtain Au/WO (gold-loaded gold nanoparticles) (pH 4.5)₃an/FTO electrode.

mu.M complementary DNA (cDNA) to the LM aptamer was activated with tris (2-carboxyethyl) phosphine (TCEP) (0.6. mu.L, 10mM) for 1h and dropped onto Au/WO₃On the/FTO electrode. Incubate overnight at 4 ℃ and wash with Tris-HCl (pH 7.4, 10mM) buffer, then block with 30 μ L6-hydroxy-1-hexanethiol (MCH) for 1 h. After Tris-HCl wash, 30 μ L of CdTe QDs-Ap conjugate was dropped onto the electrode and incubated at 37 ℃ for 1h to hybridize Ap to cDNA. And washing with Tris-HCl solution to obtain the working electrode.

Step 5, constructing a photoelectrochemical aptamer sensor for detecting Listeria monocytogenes

And (4) inserting the working electrode prepared in the step (4) into an electrolytic bath, then inserting a saturated calomel electrode and a platinum counter electrode to form a three-electrode system, externally connecting an electrochemical workstation, and simulating sunlight by using a xenon lamp light source system to assemble the photoelectric chemical aptamer sensor for the photoelectric chemical detection of the listeria monocytogenes.

In the step 1, the temperature of the water bath is 70-90 ℃, and the calcining temperature of the muffle furnace is 300-800 ℃.

In the step 2, the pH value of the solution is 10-11.5, and the condensation time is 1-10 h.

In step 3, the concentration of LM aptamer is 1-5 μ M.

The photoelectrochemical aptamer sensor for detecting LM constructed by the invention amplifies signals based on CdTe quantum dot sensitization and Exo-I auxiliary circulation, and has the following advantages and characteristics:

(1)Au/WO₃the composite structure is a substrate material of the working electrode, can absorb simulated sunlight to generate photocurrent, and Au nano particles can modify an aptamer complementary chain on the electrode by virtue of an Au-S bond and can also increase the photocurrent intensity.

(2) Sensitizing Au/WO by using CdTe QDs as sensitizer₃And signal amplification is realized, and further signal amplification is realized by utilizing the auxiliary circulation effect of Exo-I. When the incubation is not carried out, the quantum dots generate sensitization, and the photocurrent intensity response is enhanced. And after the aptamer sensor is incubated in LM and Exo-I, the quantum dots are far away from the surface of the electrode, the sensitization is greatly weakened, and the photocurrent intensity is also obviously reduced.

(3) The accuracy and specificity of the experiment are greatly improved by the specific combination of the LM thalli and the aptamer of the LM thalli. The provided detection standard method can quickly, sensitively and inexpensively determine the concentration content of LM bacteria, and has the advantages of fast detection, good linear response characteristic and good specificity compared with the prior art.

(4) The LM detection time is short, the cost is low, and the potential portability is realized.

(5) As a novel detection means, compared with the traditional detection means, the biosensor has the advantages that the background signal is separated from the detection signal, the biosensor has good sensitivity, simplicity and rapidness, and the rapid detection on site can be realized. The photoelectric active material and the biological recognition probe are basic elements for constructing the photoelectrochemical sensor. Compared with detection elements such as antibodies, the aptamer has the characteristics of good affinity, low cost, strong specificity and the like.

Drawings

FIG. 1 shows the process of constructing the working electrode of the photoelectrochemical aptamer sensor of the invention.

FIG. 2 shows a sample WO₃XRD pattern of/FTO.

FIG. 3 is an XRD pattern of sample CdTe QDs.

Fig. 4 is a graph of photocurrent for different modified electrodes. (a: WO)₃/FTO,b:Au/WO₃/FTO,c:MCH/cDNA/Au/WO₃/FTO,d:QDS-Ap/MCH/cDNA/Au/WO₃/FTO,e:LM-ExoⅠ/QDS-Ap/MCH/cDNA/Au/WO₃/FTO)

FIG. 5 is a detection response curve of a photoelectrochemical aptamer sensor.

FIG. 6 shows the result of a specificity test of a photoelectrochemical aptamer sensor.

Detailed Description

The invention is further described with reference to the following figures and detailed description. As shown in FIG. 1, the working electrode of the photoelectrochemical aptamer sensor of the invention is constructed by the following steps:

step (1) preparation of WO₃/FTO electrode

And cleaning the FTO electrode by using acetone, sodium hydroxide and deionized water in sequence, and drying for later use. 0.4g of Na₂WO₄·2H₂O and 0.17g (NH)₄)₂C₂O₄·H₂O was dissolved in 33mL of deionized water, stirred for 10min, and then 9mL of HCl solution (37%) was added. Stirring for 10min and adding 8mL of H₂O₂(30%) stirring was continued for 20min, and 30mL of absolute ethanol was added and stirred for 30 min. Placing the pretreated FTO conductive glass with the conductive surface facing downwards and inclined at 45 degrees to the wall of the beaker, placing the FTO conductive glass into the solution, and placing the FTO conductive glass into a water bath kettle to keep the temperature at 85 ℃ for 200 min. Cooling to room temperature, washing with deionized water, and drying in a drying oven at 60 deg.C for 6 hr. Finally, calcining the mixture for 2 hours at 500 ℃ in a muffle furnace, cooling the mixture to room temperature, washing the cooled mixture with deionized water and drying the washed mixture to obtain WO₃an/FTO electrode.

Step (2) preparation of CdTe QDs

0.2mmol of CdCl₂·2.5H₂O was dissolved in 50mL of deionized water, then 18. mu.L of thioglycolic acid was added and stirred for 10min, then the pH was adjusted to 10.5 with 2M NaOH. 0.04mmol of K₂TeO₃Dissolved in 50mL of deionized water, stirred well and added to the above solution, and stirred for 20 min. Then 80mg NaBH was added₄Fully reacting for 5 min. The flask was then connected to a condenser and condensed at 100 ℃ for 5 h. Cooling to room temperature, centrifuging, washing, adding equivalent deionized water, and storing in a refrigerator at 4 deg.C.

Step (3) preparation of CdTe QDs-Ap conjugate

400 μ L of CdTe QDs are activated with 40 μ L of a solution containing 40mM EDC and 10mM NHS for 1h at room temperature, then added to 300 μ L of a 2 μ M solution of LM aptamer (Ap) and stirred overnight. Centrifuging the obtained solution at 4 ℃ to remove excessive Ap to obtain the CdTe QDs-Ap conjugate.

Step (4) constructing a working electrode of a photoelectrochemical aptamer sensor

Firstly, WO₃/FTO electrode immersion 0.01M HAuCl₄Calcining the solution for 50min at 300 ℃ for 2h to form gold nanoparticles to obtain Au/WO (gold-loaded gold nanoparticles) (pH 4.5)₃an/FTO electrode.

mu.M complementary DNA (cDNA) to the LM aptamer was activated with tris (2-carboxyethyl) phosphine (TCEP) (0.6. mu.L, 10mM) for 1h and dropped onto Au/WO₃On the/FTO electrode. Incubate overnight at 4 ℃ and wash with Tris-HCl (pH 7.4, 10mM) buffer, then block with 30 μ L6-hydroxy-1-hexanethiol (MCH) for 1 h. After Tris-HCl wash, 30 μ L of CdTe QDs-Ap conjugate was dropped onto the electrode and incubated at 37 ℃ for 1h to hybridize Ap to cDNA. And washing with Tris-HCl solution to obtain the working electrode.

Step (5) constructing a photoelectrochemical biosensor for detecting listeria monocytogenes

And (4) inserting the working electrode prepared in the step (4) into an electrolytic bath, then inserting a saturated calomel electrode and a platinum counter electrode to form a three-electrode system, externally connecting an electrochemical workstation, and simulating sunlight by using a xenon lamp light source system to assemble the photoelectric chemical aptamer sensor for the photoelectric chemical detection of the listeria monocytogenes.

The invention also discloses a method for detecting the concentration and the content of the listeria monocytogenes by using the prepared photoelectrochemistry biosensor, in the detection process, by carrying out big data acquisition and analysis on detection sensitivity and specificity results under various different detection conditions, the inventor discovers that the concentration of a bacterium solution, the pH value of a detection solution and the current illumination condition have different degrees of influence on the detection sensitivity during detection, and after analyzing and calculating all factors, in order to ensure the balance of the detection sensitivity and the detection cost, the invention provides the following method for determining the content of the listeria monocytogenes:

△I＝9.76logC-4.44

in the formula, delta I is the current change amount before and after modifying the pathogenic bacteria during detection, and is milliampere, and C is the salmonella concentration, and is CFU/mL.

In addition, in order to obtain better detection results, the invention also limits the detection to be carried out at room temperature by using PBS buffer solution (pH 7.4, 0.1M) containing 0.1M Ascorbic Acid (AA). During the test, a simulated daylight xenon lamp system provides a light source, and the light source is switched on and off once every 20 s. The applied voltage was 0.0V.

Verifying the detection result of the photoelectrochemistry sensitivity:

photoelectrochemical sensitivity detection is carried out to photoelectrochemical aptamer sensor based on above detection listeria monocytogenes

The photoelectrochemical aptamer sensor constructed in the above way is used for sensitivity detection of Listeria monocytogenes, and the steps are as follows:

(1) preparing listeria monocytogenes bacterial liquids with different concentration gradients; the concentration is 10CFU/mL, 100CFU/mL, 10 respectively³CFU/mL、10⁴CFU/mL、10⁵CFU/mL、10⁶CFU/mL、10⁷CFU/mL。

(2) Dropwise adding the listeria monocytogenes bacterial liquid prepared in the step (1) to the surface of a working electrode;

(3) the photoelectrochemical assay was performed at room temperature with 0.1M Ascorbic Acid (AA) in PBS buffer (pH 7.4, 0.1M). During the test, a simulated daylight xenon lamp system provides a light source, and the light source is switched on and off once every 20 s. The applied voltage was 0.0V.

The I-t image is studied, the current change delta I before and after modification of pathogenic bacteria is taken as a vertical coordinate, and the logarithm of the concentration of pathogenic bacteria is taken as an abscissa to draw electricityFlow-concentration standard curve. As shown in FIG. 5, when the content concentration of Listeria monocytogenes is determined by the method of 9.76log C-4.44, the change response of the photocurrent has a good linear dependence on the logarithm of the concentration of Salmonella, especially when the concentration of Salmonella is 10-10⁷Between the CFU/mL range, the limit of detection (LOD, minimum sensitivity of detection) is 45CFU/mL, and both the accuracy and sensitivity of the detection are significantly higher than the prior art.

And (3) verifying a specificity experiment:

the prepared listeria monocytogenes aptamer sensor is used for specificity experiments: the dropwise addition of listeria monocytogenes in (2) is changed into the dropwise addition of sterile water, escherichia coli, staphylococcus aureus and salmonella bacteria liquid, as shown in fig. 6, wherein the diagram is as follows from left to right: a Listeria monocytogenes, b is blank, c is Escherichia coli, d is Staphylococcus aureus, and e is Salmonella. The experimental result shows that other bacteria liquid except the listeria monocytogenes can not generate obvious change on the photocurrent. The specificity of the detection is significantly higher than in the prior art.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalent structural changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.