阅读说明：本技术 一种基于Au/CdS/CdIn2S4的光电化学适配体传感器检测沙门氏菌的方法 (Based on Au/CdS/CdIn2S4Method for detecting salmonella by using photoelectrochemical aptamer sensor ) 是由 郝洪顺 丁超 赵一睿 朱良良 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：本发明公开基于Au/CdS/CdIn-(2)S-(4)的光电化学适配体传感器检测沙门氏菌的方法,将CdS/CdIn-(2)S-(4)粉体超声溶于水中,滴于FTO上,煅烧后冷却,AuNPs溶液滴于修饰CdS/CdIn-(2)S-(4)的FTO上煅烧得Au/CdS/CdIn-(2)S-(4)/FTO电极,将活化致病菌适配体滴在CdS/CdIn-(2)S-(4)/Au/FTO电极上,孵化过夜用Tris-HCl缓冲液冲洗除去未连接的适配体,将适配体互补链(cDNA)与辣根过氧化物复合滴在电极上孵育,用Tris-HCl溶液冲洗后,得HRP-cDNA/Aptamer/Au/CdS/CdIn-(2)S-(4)/FTO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,即组装成光电化学适配体传感器,并提出了在一定检测条件下,利用该传感器对沙门氏菌的检测确定方法。本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 catalyst based on Au/CdS/CdIn 2 S 4 The method for detecting salmonella by using the photoelectrochemical aptamer sensor comprises the step of adding CdS/CdIn 2 S 4 Ultrasonically dissolving the powder in water, dripping the powder on FTO, cooling after calcining, dripping AuNPs solution in modified CdS/CdIn 2 S 4 Calcining on FTO to obtain Au/CdS/CdIn 2 S 4 FTO electrode, dropping activated pathogenic bacteria aptamer in CdS/CdIn 2 S 4 Washing with Tris-HCl buffer solution to remove unconnected Aptamer after incubation overnight, dropping Aptamer complementary chain (cDNA) and horse radish peroxide onto electrode for incubation, washing with Tris-HCl solution to obtain HRP-cDNA/Aptamer/Au/CdS/CdIn 2 S 4 an/FTO working electrode; inserting the working electrode into the photoelectrochemical cell, inserting the saturated calomel electrode and the platinum counter electrode to form a three-electrode system, and connecting the three-electrode system with external electricityThe chemical workstation is assisted with a simulated daylight xenon lamp light source system, namely a photoelectric chemical adapter sensor is assembled, and a method for detecting and determining salmonella by using the sensor under certain detection conditions is provided. The sensor has the advantages of strong photocurrent response capability, high detection sensitivity, short detection time, low cost and portability.)

1. Based on Au/CdS/CdIn₂S₄The method for detecting salmonella by using the photoelectrochemical aptamer sensor is characterized by comprising the following steps of:

step 1, preparing CdS/CdIn₂S₄Nanoparticles;

a. preparation of cadmium nitrate tetrahydrate (Cd (NO) in 50mL of ethylene glycol₃)₂·4H₂O) and thioacetamide, and mixed in a molar ratio of 1:1, followed by vigorous stirring for 40 minutes using a magnetic stirrer. The obtained homogeneous solution was transferred to a 100mL reaction vessel, kept at 160 ℃ for 12 hours, and naturally cooled to room temperature. The product was collected by centrifugation, washed 2 times with deionized water and ethanol to remove residual organic solvent and inorganic salts, then dried in air at 70 ℃ for 8 hours, and ground to obtain CdS powder.

b. Preparation of Cd (NO) in 70mL of ethylene glycol₃)₂·4H₂O，InCl₃.4H₂O and thioacetamide precursor solution, and mixing the precursor solution in a molar ratio of 1:2:4, then adding a certain amount of CdS powder prepared in the step 1a, and vigorously stirring the CdS powder for 40 minutes by using a magnetic stirrer. The obtained homogeneous phaseThe solution was transferred to a 100mL reaction vessel, kept at 160 ℃ for 12 hours, and then naturally cooled to room temperature. Collecting the product by centrifugation, washing with deionized water and ethanol for 2 times to remove residual organic solvent and inorganic salt, drying in air at 70 deg.C for 8 hr, grinding, calcining in 300 deg.C muffle furnace for 2 hr, naturally cooling to room temperature, and grinding again to obtain CdS/CdIn₂S₄And (3) powder.

Step 2, preparing nano gold solution

98mL of deionized water was placed in a two-necked flask, and 2mL of 50mmol/L HAuCl was added₄Solution of Final HAuCl₄The concentration of the solution is 1 mmol/L; respectively connecting a condenser and a glass plug to two necks of a double-neck flask, and stirring and refluxing in an oil bath kettle at the temperature of 120 ℃; when the solution starts to flow back, the glass stopper is taken down, 10mL of sodium citrate solution with the concentration of 38.8mmol/L is quickly added, the color of the solution is changed from light yellow to wine red within 1min, then the solution is continuously flowed back for 20min, finally, the heating source is closed, the solution is cooled to room temperature during stirring, and the solution is stored in a refrigerator with the temperature of 4 ℃ for standby;

step 3, preparing Horse Radish Peroxidase (HRP) -cDNA compound

mu.L of 100. mu. mol/L cDNA was taken and added to 1mL of 5mmol/L PBS buffer. To this PBS was added 50. mu.L of 1mg/mL HRP, incubated for 1h, centrifuged at 12000rpm to remove unbound HRP, the precipitate was washed with 5mmol/L PBS, and the washed precipitate was redispersed in 5mmol/L PBS containing 0.1% Bovine Serum Albumin (BSA) and stored at 4 ℃ until use.

Step 4, constructing a working electrode of the sensor

a. Weighing a certain amount of CdS/CdIn prepared in the step 1b₂S₄Adding 2mL of deionized water into a sample to prepare CdS/CdIn with a certain concentration₂S₄Magnetically stirring the solution at room temperature for 30min, ultrasonically dissolving at 40 deg.C for 30min, and grinding in mortar for 30min to obtain CdS/CdIn₂S₄The powder forms uniform suspension without fine granular feeling; dripping 25 μ L of the uniform suspension on FTO electrode, naturally drying at room temperature and no wind, and cooling to 300 deg.CCalcining for 2h under the condition, and naturally cooling the sample along with the furnace after the calcining is finished to obtain CdS/CdIn₂S₄an/FTO electrode;

b. 20 mu L of prepared nano Au solution is dripped to CdS/CdIn₂S₄The surface of the/FTO electrode is then uniformly coated by a glass rod and dried to obtain Au/CdS/CdIn₂S₄an/FTO electrode; then 20 mu L of salmonella aptamer solution with certain concentration is dripped on the obtained Au/CdS/CdIn₂S₄FTO electrode surface, incubating overnight at 4 deg.C; the Aptamer and the Au NPs are firmly fixed on the surface of the Au NPs through the strong interaction force of Au-S bonds, and Tris-HCl buffer solution is used for washing the surface of the electrode to remove the unfixed Aptamer molecules, so that the Aptamer/Au/CdS/CdIn is obtained₂S₄an/FTO electrode. Then, 20 mu L of HRP-cDNA compound solution prepared in the step 3 is dripped and coated on Aptamer/Au/CdS/CdIn₂S₄FTO electrode surface, incubating overnight at 4 deg.C; the HRP-cDNA compound and AuNPs are firmly fixed on the surface of Au NPs through the strong interaction force of Au-S bonds, the surface of an electrode is washed by Tris-HCl buffer solution, and the unfixed Aptamer molecules are removed to obtain HRP-cDNA/Aptamer/Au/CdS/CdIn₂S₄an/FTO electrode.

Step 5, constructing a photoelectrochemical aptamer sensor for detecting salmonella

HRP-cDNA/Aptamer/Au/CdS/CdIn prepared in the step 4b₂S₄the/FTO electrode is used as a working electrode, the platinum wire electrode is used as a counter electrode, the saturated calomel electrode is used as an auxiliary electrode, the three-electrode system is respectively arranged in a PBS solution to construct a three-electrode system, AA (ascorbic acid) is used as an electron donor, a xenon lamp is used as a light source, and the construction is based on Au/CdS/CdIn₂S₄The photoelectrochemical aptamer sensor of (1) is used for detecting salmonella.

2. Au/CdS/CdIn-based catalyst according to claim 1₂S₄The method for detecting salmonella by using the photoelectrochemical aptamer sensor is characterized in that in the step 1b, CdS nanoparticles are compounded in CdIn₂S₄CdS and CdIn on nanosheets₂S₄The optimal proportion is 1: 1.

3. Au/CdS/CdIn-based catalyst according to claim 1₂S₄The method for detecting salmonella by using the photoelectrochemical aptamer sensor is characterized in that in the step 4a, CdS/CdIn₂S₄The concentration is 20-40 mg/mL (namely that a certain amount of CdS/CdIn prepared in the step 1b is weighed₂S₄Adding 2mL of deionized water into a sample to prepare CdS/CdIn with a certain concentration₂S₄Concentration of solution "); in the step 4b, the concentration of the salmonella aptamer solution is 1-5 mu mol/L.

4. Au/CdS/CdIn-based catalyst according to claim 1₂S₄The method for detecting the salmonella by using the photoelectrochemistry aptamer sensor is characterized in that in the step 5, the concentration of AA is 0.05-0.2 mol/L, the pH value of a PBS solution is 5-11, and the incubation time is 30-80 min.

5. Au/CdS/CdIn-based catalyst according to claim 1₂S₄The method for detecting the salmonella by using the photoelectrochemistry aptamer sensor is characterized in that in the step 5, the detected food-borne pathogenic bacteria are the salmonella, but not limited to the salmonella.

6. Au/CdS/CdIn-based catalyst according to any one of claims 1 to 5₂S₄The method for detecting the salmonella by the photoelectrochemistry aptamer sensor is characterized in that the content of the salmonella is determined by the following mode:

△I＝11.31log C-14.843 (1)

wherein, Delta I is the photocurrent change value during detection in milliampere, C is the salmonella concentration in CFU/mL.

7. Au/CdS/CdIn-based catalyst according to claim 6₂S₄The method for detecting the salmonella by the photoelectrochemistry aptamer sensor is characterized in that the pH value of detection liquid is 7.2-7.8, the optimal pH value is 7.4, and the power of xenon lamp light source irradiation is 500W。

Technical Field

The invention belongs to the field of food safety detection, and particularly relates to a semiconductor nano biosensor for detecting food-borne pathogenic bacteria and a preparation method thereof.

Background

Worldwide, with the increasing dependence of people on processed foods, the number of global food-borne diseases is increased year by year. According to statistics, hundreds of millions of people worldwide cause diseases due to food pollution every year. Among them, the number of patients who are caused by microbial contamination is the first, and is the most prominent health and hygiene problem in the world at present. In recent years, the proportion of food-borne diseases in China is on the rise, and about one sixth of the population suffers from the food-borne diseases. Therefore, food poisoning and food-borne diseases caused by food-borne pathogenic bacteria become the first problems facing the food safety in China.

There are dozens of food-borne pathogenic bacteria, of which salmonella is the most common food-borne pathogenic bacteria, and diseases caused by salmonella are mainly caused by eating contaminated food, and are important sources of food poisoning in many countries. In view of the problems caused by salmonella, the development of rapid detection methods is urgently needed. As a novel detection means, the biosensor has the advantages of convenience, time saving, high precision, simple equipment, low price, easy miniaturization and the like, the collection and the processing of data are simple and convenient, no pollution is caused, and the detection of a large-flux sample on site can be realized. There are patents and literature reports that salmonella is detected by electrochemical immunosensors, optical fiber biosensors, fluorescence sensors, DNA sensors, etc., but at present, due to the diversity of detection environments and the inconsistency of detection standards, the detection sensitivity and specificity of various sensors fluctuate greatly, and there is no appropriate method and standard for determining the detection value.

Disclosure of Invention

In order to solve the technical problems, the invention establishes a semiconductor nano biosensor method for quickly detecting food-borne pathogenic bacteria by combining the rapidity of the sensor and the specificity of an aptamer. Provides a CdS composite CdIn modified based on nano-gold₂S₄The nano material modified biosensor converts optical signals into electric signals by utilizing the action of a photoelectric conversion material, and is used for quickly detecting pathogenic bacteria with high sensitivity and high specificity by detecting current change caused by pathogenic bacteria, particularly salmonella. The invention integrates the advantages of electrochemistry and photoelectrochemistry, adopts different forms of excitation and detection signals, has lower background signal and higher sensitivity, and is expected to be well applied in the field of rapid detection of salmonella.

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

based on Au/CdS/CdIn₂S₄The method for detecting salmonella by using the photoelectrochemical aptamer sensor is characterized by comprising the following steps of:

step 1, preparing CdS/CdIn₂S₄Nanoparticles;

a. preparation of cadmium nitrate tetrahydrate (Cd (NO) in 50mL of ethylene glycol₃)₂·4H₂O) and thioacetamide, and mixed in a molar ratio of 1:1, followed by vigorous stirring for 40 minutes using a magnetic stirrer. The obtained homogeneous solution was transferred to a 100mL reaction vessel, kept at 160 ℃ for 12 hours, and naturally cooled to room temperature. The product was collected by centrifugation, washed 2 times with deionized water and ethanol to removeRemoving residual organic solvent and inorganic salt, drying in air at 70 deg.C for 8 hr, and grinding to obtain CdS powder.

b. Preparation of Cd (NO) in 70mL of ethylene glycol₃)₂·4H₂O，InCl₃.4H₂O and thioacetamide precursor solution, and mixing the precursor solution in a molar ratio of 1:2:4, then adding a certain amount of CdS powder prepared in the step 1a, and vigorously stirring the CdS powder for 40 minutes by using a magnetic stirrer. The obtained homogeneous solution was transferred to a 100mL reaction vessel, kept at 160 ℃ for 12 hours, and then naturally cooled to room temperature. Collecting the product by centrifugation, washing with deionized water and ethanol for 2 times to remove residual organic solvent and inorganic salt, drying in air at 70 deg.C for 8 hr, grinding, calcining in 300 deg.C muffle furnace for 2 hr, naturally cooling to room temperature, and grinding again to obtain CdS/CdIn₂S₄And (3) powder.

Step 2, preparing nano gold solution

98mL of deionized water was placed in a two-necked flask, and 2mL of 50mmol/L HAuCl was added₄Solution of Final HAuCl₄The concentration of the solution is 1 mmol/L; respectively connecting a condenser and a glass plug to two necks of a double-neck flask, and stirring and refluxing in an oil bath kettle at the temperature of 120 ℃; when the solution starts to flow back, the glass stopper is taken down, 10mL of sodium citrate solution with the concentration of 38.8mmol/L is quickly added, the color of the solution is changed from light yellow to wine red within 1min, then the solution is continuously flowed back for 20min, finally, the heating source is closed, the solution is cooled to room temperature during stirring, and the solution is stored in a refrigerator with the temperature of 4 ℃ for standby;

step 3, preparing Horse Radish Peroxidase (HRP) -cDNA compound

mu.L of 100. mu. mol/L cDNA was taken and added to 1mL of 5mmol/L PBS buffer. To this PBS was added 50. mu.L of 1mg/mL HRP, incubated for 1h, centrifuged at 12000rpm to remove unbound HRP, the precipitate was washed with 5mmol/L PBS, and the washed precipitate was redispersed in 5mmol/L PBS containing 0.1% Bovine Serum Albumin (BSA) and stored at 4 ℃ until use.

Step 4, constructing a working electrode of the sensor

a. Weighing a certain amount of CdS/CdIn prepared in the step 1b₂S₄Adding 2mL of deionized water into a sample to prepare CdS/CdIn with a certain concentration₂S₄Magnetically stirring the solution at room temperature for 30min, ultrasonically dissolving at 40 deg.C for 30min, and grinding in mortar for 30min to obtain CdS/CdIn₂S₄The powder forms uniform suspension without fine granular feeling; dripping 25 microliter of the uniform suspension on an FTO electrode, naturally drying at normal temperature and no wind, calcining for 2 hours at 300 ℃, and naturally cooling a sample along with a furnace after calcining to obtain CdS/CdIn₂S₄an/FTO electrode;

b. 20 mu L of prepared nano Au solution is dripped to CdS/CdIn₂S₄The surface of the/FTO electrode is then uniformly coated by a glass rod and dried to obtain Au/CdS/CdIn₂S₄an/FTO electrode; then 20 mu L of salmonella aptamer solution with certain concentration is dripped on the obtained Au/CdS/CdIn₂S₄FTO electrode surface, incubating overnight at 4 deg.C; the Aptamer and the Au NPs are firmly fixed on the surface of the Au NPs through the strong interaction force of Au-S bonds, and Tris-HCl buffer solution is used for washing the surface of the electrode to remove the unfixed Aptamer molecules, so that the Aptamer/Au/CdS/CdIn is obtained₂S₄an/FTO electrode. Then, 20 mu L of HRP-cDNA compound solution prepared in the step 3 is dripped and coated on Aptamer/Au/CdS/CdIn₂S₄FTO electrode surface, incubating overnight at 4 deg.C; the HRP-cDNA compound and AuNPs are firmly fixed on the surface of Au NPs through the strong interaction force of Au-S bonds, the surface of an electrode is washed by Tris-HCl buffer solution, and the unfixed Aptamer molecules are removed to obtain HRP-cDNA/Aptamer/Au/CdS/CdIn₂S₄an/FTO electrode.

Step 5, constructing a photoelectrochemical aptamer sensor for detecting salmonella

HRP-cDNA/Aptamer/Au/CdS/CdIn prepared in the step 4b₂S₄the/FTO electrode is used as a working electrode, the platinum wire electrode is used as a counter electrode, the saturated calomel electrode is used as an auxiliary electrode, the three-electrode system is respectively arranged in a PBS solution to construct a three-electrode system, AA (ascorbic acid) is used as an electron donor, a xenon lamp is used as a light source, and the construction is based on Au/CdS/CdIn₂S₄The photoelectrochemical aptamer sensor of (1) is used for detecting salmonella.

In step 1b, CdS nanoparticles are composited in CdIn₂S₄CdS and CdIn on nanosheets₂S₄The optimal proportion is 1: 1.

In step 4a, CdS/CdIn₂S₄The concentration is 20-40 mg/mL (namely that a certain amount of CdS/CdIn prepared in the step 1b is weighed₂S₄Adding 2mL of deionized water into a sample to prepare CdS/CdIn with a certain concentration₂S₄Concentration of solution "); in the step 4b, the concentration of the salmonella aptamer solution is 1-5 mu mol/L.

In step 5, the detected food-borne pathogenic bacteria are salmonella, but not limited to salmonella.

Has the advantages that: the invention prepares nano-gold modified CdS composite CdIn₂S₄The nano material is modified to FTO to construct a semiconductor nano biosensor for detecting food-borne pathogenic bacteria, and the advantages and the characteristics are as follows:

(1) CdS composite CdIn with a certain proportion₂S₄The composite effect widens the spectrum range and improves the photocurrent response capability.

(2) Through the specific combination of the food-borne pathogenic bacteria and the aptamer thereof, the accuracy and specificity of the experiment are greatly improved.

(3) The provided detection standard method can quickly, sensitively and inexpensively determine the concentration content of the salmonella, and has the advantages of fast detection, good linear response characteristic and good specificity compared with the prior art.

(4) By using the nano material, the aptamer and the signal amplification strategy modified by the HRP, the photocurrent response signal is greatly improved.

(5) The photoelectrochemical aptamer sensor based on the semiconductor nano material integrates the advantages of electrochemistry and photoelectrochemistry, adopts excitation and detection signals in different forms, and has lower background signal and higher sensitivity.

(6) The semiconductor nano biosensor has the advantages of high detection speed, low cost, potential portability and the like, and the detection technology is expected to be well applied to the field of rapid detection of food-borne pathogenic bacteria.

Drawings

FIG. 1 is a schematic diagram of a construction process of a working electrode of a sensor according to the present invention.

FIG. 2 is a diagram of the photocurrent of a working electrode for modifying different substances, wherein a: CdIn₂S₄/FTO，b： CdS/CdIn₂S₄/FTO，c：Au/CdS/CdIn₂S₄/FTO，d：aptamer/Au/CdS/CdIn₂S₄/FTO，e：HRP- cDNA/Aptamer/Au/CdS/CdIn₂S₄/FTO，f：FPB/HRP-cDNA/Aptamer/Au/CdS/CdIn₂S₄/FTO。

FIG. 3(a) is a graph of photocurrent response of different concentrations of Salmonella (FPB); FIG. 3(b) shows the result of verification for FPB detection (a → f: 2.4X 10 in the figure)²→2.4×10⁷CFU/mL)。

FIG. 4 is based on Au/CdS/CdIn₂S₄The specific result graph of the photoelectrochemistry aptamer sensor on the salmonella detection is shown.

Detailed Description

The invention is further described with reference to the following figures and detailed description. As shown in FIG. 1, the construction process of the working electrode of the photoelectrochemical aptamer sensor comprises the step of sequentially modifying CdIn on the surface of an FTO electrode₂S₄CdS, Au, Aptamer, cDNA-HRP and FPB (the specific embodiment takes salmonella as an example), and obtaining the working electrode of the biosensor, which comprises the following specific steps:

step 1, preparing CdS/CdIn₂S₄Nanoparticles

a. Preparation of Cd (NO) in 50mL of ethylene glycol₃)₂·4H₂O and thioacetamide in a molar ratio of 1:1, and then vigorously stirred for 40 minutes using a magnetic stirrer. The obtained homogeneous solution was transferred to a 100ml reaction vessel, reacted at 160 ℃ for 12 hours, and then naturally cooled to room temperature. The product was collected by centrifugation, washed 2 times with deionized water and ethanol to remove residual organic solvents and inorganic salts, and then dried in air at 70 ℃ for 8 hours. And grinding to obtain CdS powder.

b. Preparation of Cd (NO) in 70mL of ethylene glycol₃)₂·4H₂O，InCl₃.4H₂O and thioacetamide precursor solution, mixing the precursor solution with the thioacetamide precursor solution according to the molar ratio of 1:2:4, adding CdS powder with the equal molar ratio, and stirring vigorously for 40 minutes by using a magnetic stirrer. The obtained homogeneous solution was transferred to a 100ml reaction vessel, reacted at 160 ℃ for 12 hours, and then naturally cooled to room temperature. Collecting the product by centrifugation, washing with deionized water and ethanol for 2 times to remove residual organic solvent and inorganic salt, drying in air at 70 deg.C for 8 hr, grinding, calcining in 300 deg.C muffle furnace for 2 hr, naturally cooling to room temperature, and grinding again to obtain CdS/CdI_n2S₄And (3) powder.

Step 2, preparing nano gold solution

98mL of deionized water was placed in a two-necked flask, and 2mL of 50mmol/L HAuCl was added₄Solution of Final HAuCl₄The concentration of the solution is 1 mmol/L; respectively connecting a condenser and a glass plug to two necks of a double-neck flask, and stirring and refluxing in an oil bath kettle at the temperature of 120 ℃; when the solution started to reflux, the glass stopper was removed and 10mL of 38.8mmol/L sodium citrate solution was added quickly to change the color of the solution from pale yellow to wine-red within 1min, followed by continued reflux for 20min, and finally the heating source was turned off to cool it to room temperature while stirring and stored in a refrigerator at 4 ℃ until ready for use.

Step 3, preparing HRP-cDNA compound

mu.L of 100. mu. mol/L cDNA was taken and added to 1mL of 5mmol/L PBS. To this PBS was added 50. mu.L of 1mg/mL HRP, incubated for 1h, centrifuged at 12000rpm to remove unbound HRP, the precipitate was washed with 5mmol/L PBS, and the washed precipitate was redispersed in 5mmol/L PBS containing 0.1% BSA and stored at 4 ℃ until use.

Step 4, constructing a working electrode of the biosensor and detecting salmonella by using the working electrode

(1) 0.08g CdS/CdIn were weighed out₂S₄Adding 2mL of deionized water into the powder to prepare 40mg/mL CdS/CdIn₂S₄Magnetically stirring the solution at room temperature for 30min, ultrasonically dissolving at 40 deg.C for 30min, and grinding in mortar for 30min to obtain CdS/CdIn₂S₄The powder forms uniform suspension without fine granular feeling; dripping 25 microliter of the uniform suspension on an FTO electrode, naturally drying at normal temperature and no wind, calcining for 2 hours at 300 ℃, and naturally cooling a sample along with a furnace after calcining to obtain CdS/CdIn₂S₄an/FTO electrode;

(2) 20 μ L of the prepared Au NPs solution was applied dropwise to CdS/CdIn₂S₄The surface of the/FTO electrode is then uniformly coated by a glass rod and dried to obtain Au/CdS/CdIn₂S₄an/FTO electrode;

(3) 20 mu L of salmonella aptamer solution with the concentration of 3 mu mol/L is dripped on the obtained Au/CdS/CdIn₂S_4/Incubating the surface of the FTO electrode at 4 ℃ overnight; the Aptamer and AuNPs are firmly fixed on the surface of the Au NPs through the strong interaction force of Au-S bonds, Tris-HCl buffer solution is used for washing the surface of the electrode to remove the unfixed Aptamer molecules, and the Aptamer/Au/CdS/CdIn is obtained₂S₄an/FTO electrode;

(4) dripping 20 mu L of HRP-cDNA compound solution on the obtained Aptamer/Au/CdS/CdIn₂S₄FTO electrode surface, incubating overnight at 4 deg.C; the HRP-cDNA compound and AuNPs are firmly fixed on the surface of Au NPs through the strong interaction force of Au-S bonds, Tris-HCl buffer solution is used for washing the surface of an electrode to remove unfixed Aptamer molecules, and HRP-cDNA/Aptamer/Au/CdS/CdIn is obtained₂S₄an/FTO electrode.

(5) Finally in HRP-cDNA/Aptamer/Au/CdS/CdIn₂S₄Respectively dripping 20 mu L of salmonella with different concentrations on the FTO electrode, incubating for 1 hour at 4 ℃, washing with PBS buffer solution, inserting the working electrode into an electrolytic cell to ensure that the concentration of AA in the electrolytic cell is 0.13mol/L and the pH value of the PBS solution is 7.4, and carrying out photocurrent detection.

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

△I＝11.31logC-14.843

wherein, Delta I is the photocurrent change value during detection in milliampere, C is the salmonella concentration in CFU/mL, and the preferred range is 2.4 × 10²-2.4×10⁷CFU/mL。

In addition, in order to obtain a better detection result, the pH value of the detection liquid is limited to 7.2-7.8, preferably 7.4, and the power of the xenon lamp light source during irradiation is 500W.

Verifying the sensitivity detection result of the photoelectrochemistry aptamer sensor:

the prepared biosensor is used for sensitivity detection of salmonella, and the steps are as follows:

(1) formulation 10²-10⁷CFU/mL salmonella bacterial liquid with concentration gradient;

(2) dropwise adding the salmonella bacterial liquid prepared in the step (1) to the surface of a working electrode;

(3) the prepared electrode is used as a working electrode, a platinum wire electrode is used as a counter electrode, a saturated calomel electrode is used as an auxiliary electrode, the electrodes are respectively placed in a PBS solution to construct a three-electrode system, the pH value is 7.4, 0.13M AA is added, a 500W xenon lamp light source is used for irradiation for photoelectrochemical analysis, and an I-t image is used as an experimental basis and is shown in figure 3. When the method of delta I ═ 11.31logC-14.843 is used for determining the concentration of the salmonella, the change response of the photocurrent has good linear dependence on the logarithm of the concentration of the salmonella, especially when the concentration of the salmonella is 2.4 x 10²-2.4×10⁷Between the CFU/mL range, the limit of detection (LOD, minimum sensitivity of detection) is 25CFU/mL, and both the accuracy and sensitivity of the detection are significantly higher than in the prior art.

And (3) verifying a specificity experiment result:

the prepared photoelectrochemical aptamer sensor is used for specificity experiments: the drop adding of the salmonella is changed into the drop adding of the listeria monocytogenes (b), the staphylococcus aureus (c) and the enterobacter (d), and the physiological saline (a) is used as a blank control. As shown in FIG. 4, 2.4X 10 as compared with the blank solution⁴The photocurrent intensity of the CFU/mL salmonella (e) solution was significantly reduced, while the photocurrent intensities of listeria, staphylococcus aureus and enterobacter were substantially not much different from the blank solution. It can be seen that the aptamer sensor constructed has selectivity and specificity for detecting FPB over 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.