Photoelectrochemical biosensor for detecting escherichia coli and preparation method thereof

Abstract

The invention discloses a photoelectrochemical biosensor for detecting escherichia coli and a preparation method of the photoelectrochemical biosensor, and belongs to the technical field of food safety analysis. According to the photoelectrochemical biosensor disclosed by the invention, Bi7O9I3-In2O3, AuNPs, an SH-DNA chain S1, a DNA chain S2 and Zr-MOF which are sequentially modified on the surface of a basic electrode are used as working electrodes. The nucleotide sequence of the SH-DNA chain S1 is as shown in SEQ ID NO. 3; the nucleotide sequence of the DNA chain S2 is as shown in SEQ ID NO. 6. According to the photoelectrochemical biosensor disclosed by the invention, the amplification of a photoelectric signal is realized by utilizing an energy band matching effect between Bi7O9I3 and In2O3; zr-MOF is connected to a phosphate group of a DNA chain S2 through metal-ligand coordination, p-nitrophenyl disodium phosphate in photoelectric detection liquid is catalyzed to be hydrolyzed to generate p-nitrophenol by utilizing the activity of Zr-MOF phosphatase, the p-nitrophenol is used as an electron acceptor to further reduce photocurrent signals, and sensitive detection on escherichia coli is realized by utilizing the difference value of the photocurrent signals.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety analysis, and particularly to a photoelectrochemical biosensor for detecting Escherichia coli and a preparation method thereof. Background Art

[0002] Foodborne pathogens are an important risk factor in the agricultural food industry, causing significant economic losses worldwide. Foodborne diseases remain prevalent, especially in the presence of hygiene problems or food and water contamination. The most common pathogens in food are Staphylococcus aureus, Escherichia coli, Campylobacter, etc. These pathogens can cause gastroenteritis after ingestion of contaminated food and water. Recent studies have confirmed that among other sources of contamination, bacteria still account for approximately 91% of the total number of foodborne diseases.

[0003] Escherichia coli is a bacterium that normally lives in the intestines of animals and humans and contributes to intestinal health. However, when contaminated food or beverages are consumed, certain strains can cause diarrhea. Most strains are harmless or cause transient diarrhea or urinary tract infections. However, certain strains, such as Escherichia coli O157:H7, can cause severe abdominal cramps, bloody diarrhea, and vomiting.

[0004] Currently, the most commonly used method for detecting Escherichia coli in food safety is cultivation in a specific medium based on colony-forming units. However, this method has complex sample preparation and a long time-consuming period (12 - 48h). To overcome these deficiencies, based on the specific binding of aptamers to Escherichia coli, a variety of rapid, sensitive, highly sensitive, and specific aptasensors have been developed using various detection techniques such as chemiluminescence, surface-enhanced Raman scattering, colorimetry, fluorescence, electrochemistry, and photoelectrochemistry for the accurate diagnosis of foodborne pathogens.

[0005] Among numerous detection methods, photoelectrochemical biosensors convert biological phenomena into photocurrent signals through biological recognition elements and signal converters, and utilize the changes in photocurrent signals to achieve quantitative and qualitative analysis of target substances. Compared with fluorescence, electrochemistry, and electrochemiluminescence techniques, it combines two completely separate energy forms of light as the excitation source and electricity as the detection signal, making it have the characteristics of high sensitivity, low background, and wide application in complex detection environments. It has become an analytical technology with great application value. Therefore, developing new photoelectrochemical biosensors for detecting Escherichia coli is of great significance. Summary of the Invention

[0006] Aiming at the above-mentioned prior art, the purpose of the present invention is to provide a photoelectrochemical biosensor for detecting Escherichia coli and a preparation method thereof.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions:

[0008] In a first aspect of the present invention, an optoelectrochemical biosensor is provided. The optoelectrochemical biosensor uses Bi7O9I3-In2O3, AuNPs, SH-DNA strand S1, DNA strand S2, and Zr-MOF sequentially modified on the surface of a base electrode as a working electrode.

[0009] Preferably, the nucleotide sequence of the SH-DNA strand S1 is as shown in SEQ ID NO.3; the nucleotide sequence of the DNA strand S2 is as shown in SEQ ID NO.6.

[0010] Preferably, the base electrode is an ITO electrode.

[0011] Furthermore, the optoelectrochemical biosensor is a three-electrode system and further includes: a reference electrode and a counter electrode.

[0012] In a second aspect of the present invention, a method for preparing the above optoelectrochemical biosensor is provided, including the following steps:

[0013] (1) Drop a Bi7O9I3-In2O3 dispersion onto the surface of a pretreated base electrode and dry it to prepare a Bi7O9I3-In2O3 / ITO electrode;

[0014] (2) Drop an AuNPs dispersion onto the surface of the Bi7O9I3-In2O3 / ITO electrode and dry it to prepare an AuNPs / Bi7O9I3-In2O3 / ITO electrode;

[0015] (3) Modify the SH-DNA strand S1 onto the surface of the AuNPs / Bi7O9I3-In2O3 / ITO electrode through an Au-S bond to prepare an S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode;

[0016] (4) Drop a mixture containing the DNA strand S2 onto the surface of the S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode, and the DNA strand S2 is modified on the electrode surface by hybridizing with the SH-DNA strand S1 to prepare an S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode;

[0017] (5) Drop a Zr-MOF dispersion onto the surface of the S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode to prepare a Zr-MOF / S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode, which is used as a working electrode.

[0018] Preferably, in step (1), the Bi7O9I3-In2O3 dispersion is prepared by the following method:

[0019] Disperse Bi7O9I3 and In2O3 in deionized water, perform ultrasonic treatment, wash and centrifuge, and collect the solid to prepare Bi7O9I3-In2O3.

[0020] Then add Bi7O9I3-In2O3 to deionized water and perform ultrasonic dispersion to prepare a Bi7O9I3-In2O3 dispersion.

[0021] Preferably, in step (4), the mixture containing DNA strand S2 is prepared by the following method:

[0022] First, anneal the Escherichia coli aptamer, the aptamer complementary strand, primer DNA2, and primer DNA3 at 95 °C for 5 min, then mix the Escherichia coli aptamer and the aptamer complementary strand in equal volumes and react at 37 °C under humid conditions for 0.5 - 3 h, add an equal volume of Escherichia coli, and continue to react at 37 °C under humid conditions for 0.5 - 3 h; then add an equal volume of primer DNA2, primer DNA3, phi 29 DNA polymerase, Nt.BbvCI DNA nickase, and dNTPs solution, react under the same conditions for 0.5 - 3 h, and then heat at 80 °C for 20 min to inactivate the enzyme, and centrifuge to obtain the mixture containing DNA strand S2.

[0023] Furthermore, the nucleotide sequence of the Escherichia coli aptamer is shown as SEQ ID NO.1; the nucleotide sequence of the aptamer complementary strand is shown as SEQ ID NO.2.

[0024] Furthermore, the nucleotide sequence of primer DNA2 is shown as SEQ ID NO.4; the nucleotide sequence of primer DNA3 is shown as SEQ ID NO.5.

[0025] In the third aspect of the present invention, there is provided the application of the above optoelectrochemical biosensor in detecting Escherichia coli.

[0026] The optoelectrochemical biosensor of the present invention can be used to detect Escherichia coli in food, such as: meat products, ready-to-eat fruit and vegetable products, milk, etc.

[0027] In the fourth aspect of the present invention, there is provided a method for detecting Escherichia coli, including the following steps:

[0028] Perform nucleic acid isothermal amplification reaction on the Escherichia coli aptamer, the aptamer complementary strand, Escherichia coli with a series of concentrations, primer DNA2, primer DNA3, phi 29 DNA polymerase, Nt.BbvCI DNA nickase, and dNTPs to prepare a mixture containing DNA strand S2;

[0029] The working electrode was prepared by successively modifying Bi7O9I3-In2O3, AuNPs, SH-DNA strand S1, DNA strand S2, and Zr-MOF on the surface of the base electrode. A saturated calomel electrode was used as the reference electrode, and a Pt wire was used as the auxiliary electrode to form a three-electrode system. A Tris-HCl buffer solution containing pNPP was used as the electrode detection solution for photoelectrochemical signal detection. A standard curve between the current and the logarithm of the Escherichia coli concentration was established to detect the Escherichia coli content in the test solution.

[0030] Preferably, the concentration of pNPP in the electrode detection solution is 10 mM.

[0031] Advantages of the present invention:

[0032] (1) The photoelectrochemical biosensor of the present invention utilizes the band matching effect between Bi7O9I3 and In2O3 to achieve the amplification of photoelectric signals. Then, AuNPs, SH-DNA strand S1, and DNA strand S2 are successively modified to reduce the photocurrent signal. Zr-MOF is connected to the phosphate group of DNA strand S2 through metal-ligand coordination. Utilizing the phosphatase-like activity of Zr-MOF, p-nitrophenyl phosphate disodium (pNPP) in the photoelectric detection solution is catalyzed to hydrolyze into p-nitrophenol (pNP), which serves as an electron acceptor to further reduce the photocurrent signal, increasing the difference in the photocurrent signal and achieving highly sensitive detection of Escherichia coli.

[0033] (2) The present invention utilizes an Escherichia coli aptamer, an aptamer complementary strand, primer DNA2, and primer DNA3 to achieve specific recognition of Escherichia coli in the test sample, improving the selectivity of detection. Description of the Drawings

[0034] Figure 1 : SEM image of the Bi7O9I3-In2O3 nanomaterial.

[0035] Figure 2 : Photocurrent response diagram of the construction process of the invented photoelectric sensor.

[0036] Figure 3 : Photocurrent response diagram of a series of concentrations of Escherichia coli.

[0037] Figure 4 : Linear fitting curve of the photocurrent intensity and the logarithm of the Escherichia coli concentration.

[0038] Figure 5 : Histogram of the change in photoelectrochemical response under different strain treatment conditions. Detailed Embodiments

[0039] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0040] Term Explanation:

[0041] In the invention, the "room temperature" ranges from 20 to 30 °C.

[0042] In the present invention, the humidity of the "humid condition" is 95 - 99%.

[0043] The "secondary water" in the present invention is water that has been distilled for the second time.

[0044] For the detection of Escherichia coli, the present invention constructs a new optoelectrochemical biosensor. The working electrode of the optoelectrochemical biosensor of the present invention is based on an ITO electrode, and Bi7O9I3-In2O3, AuNPs, SH-DNA strand S1, DNA strand S2, and Zr-MOF are sequentially modified onto the surface of the base electrode. Among them, Bi7O9I3-In2O3 can be used as an excellent photoactive material. Bi7O9I3 belongs to bismuth-based oxyhalides, has a layered structure and a suitable bandgap, has good visible light response, but the carrier recombination is relatively fast; In2O3 is a wide-bandgap semiconductor with a relatively low conduction band position, which is beneficial to electron migration; in the present invention, Bi7O9I3 is combined with In2O3, and the energy bands between the two match to form a heterojunction. The formation of the heterojunction improves the separation rate of photo-generated electron-hole pairs, accelerates the migration of photo-generated electrons, and increases the photocurrent intensity. AuNPs, as a water-insoluble nanomaterial, covers the surface of the photoactive material Bi7O9I3-In2O3 and serves as an intermediate for connecting DNA strands. After DNA strands S1 and S2 are connected to the surface of AuNPs, the photocurrent is reduced due to the steric hindrance effect, and the current is further reduced after connecting Zr-MOF.

[0045] When detecting Escherichia coli, Escherichia coli aptamer, complementary strand of aptamer, Escherichia coli, primer DNA2, primer DNA3, phi 29 DNA polymerase, Nt.BbvCI DNA nickase, and dNTPs are successively added into a centrifuge tube for nucleic acid isothermal amplification reaction. The Escherichia coli aptamer and the complementary strand of aptamer form a stable double-stranded structure due to their unique nucleotide sequences. After adding Escherichia coli, the Escherichia coli aptamer specifically captures Escherichia coli, thereby releasing the complementary strand of aptamer. The complementary strand of aptamer, as a target primer, forms a stable three-way junction structure with primer DNA2 and primer DNA3. After adding phi 29 DNA polymerase, Nt.BbvCI DNA nickase, and dNTPs, the nucleic acid isothermal amplification reaction is initiated. After a period of reaction, a large number of DNA single strands with phosphate groups are obtained, which are named DNA strand S2; the product of the nucleic acid isothermal amplification reaction is a mixed solution containing DNA strand S2.

[0046] The SH-DNA strand S1 is modified onto the surface of the Bi7O9I3-In2O3 / Au electrode through Au-S bonds. The mixed solution containing DNA strand S2 is dropped onto the electrode surface. DNA strand S2 is modified onto the electrode surface by hybridizing with the SH-DNA strand S1. A Zr-MOF dispersion is dropped onto the above electrode surface, and it is connected to the phosphate group of DNA strand S2 through metal-ligand coordination. Utilizing the phosphatase-like activity of Zr-MOF, p-nitrophenyl phosphate disodium (pNPP) in the photoelectric detection solution is catalyzed to hydrolyze into p-nitrophenol (pNP), which further reduces the photocurrent signal as an electron acceptor. Within the same reaction time, the number of Escherichia coli in the centrifuge tube determines the number of released complementary strands of aptamer, thus determining the number of cut DNA strand S2. Since Zr-MOF and DNA strand S2 are connected one-to-one, the amount of Zr-MOF is determined by the number of Escherichia coli; if there is no Escherichia coli, DNA strand S2 will not be produced, and subsequent Zr-MOF cannot be connected to the electrode surface. Therefore, the presence or absence of Escherichia coli in the system, as well as the level of Escherichia coli concentration, will affect the intensity of the photocurrent of the electrode. Using the linear relationship between the logarithm of Escherichia coli concentration and the photocurrent intensity, quantitative detection of Escherichia coli can be achieved.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with specific embodiments.

[0048] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. The experimental methods without specifying detailed conditions are carried out according to conventional test methods or according to the operation manuals recommended by the suppliers. Among them:

[0049] Bismuth oxyiodide (Bi7O9I3) used in the present invention is prepared by the following method:

[0050] Dissolve 5 mmol of bismuth nitrate pentahydrate in 50 mL of ethylene glycol. After ultrasonic treatment, add 15 mmol of potassium iodide. Stir for half an hour and then gradually add 1 M sodium hydroxide solution to adjust the pH to 10. Continue stirring for half an hour. After the reaction is completed, wash and centrifuge, and collect the solid precursor material; place the obtained solid precursor material in a muffle furnace and calcine at 350 °C for one hour. Wash and centrifuge again, and collect the solid to prepare Bi7O9I3.

[0051] Indium oxide (In2O3) used in the present invention is prepared by the following method:

[0052] Dissolve 6 mmol of indium nitrate monohydrate in 30 mL of N,N-dimethylformamide. After ultrasonic treatment, add 8 mmol of terephthalic acid. After ultrasonic homogenization, stir until the solution becomes clear, and carry out hydrothermal reaction at 100 °C for 24 h. After the reaction is completed, wash and centrifuge, and collect the solid MIL-68(In) nanomaterial; place the obtained MIL-68(In) nanomaterial in a muffle furnace and calcine at 450 °C for 2 h. Wash and centrifuge again, and collect the solid to prepare In2O3.

[0053] AuNPs used in the present invention are prepared by the following method:

[0054] Add 96 mL of secondary water and 4 mL of 0.05 mol / L chloroauric acid solution to a two-necked flask and reflux. When the solution starts to reflux, add 20 mL of 38.8 mmol / L sodium citrate solution. After refluxing for 40 minutes, cool naturally to obtain a 2 mmol / L AuNPs solution.

[0055] Zr-MOF used in the present invention is prepared by the following method:

[0056] Dissolve 2 mmol of zirconium tetrachloride in a mixed solution of 800 mmol of acetic acid and 80 mL of N,N-dimethylformamide to form solution A, and place it under stirring at 50 °C; dissolve 2 mmol of 2-aminoterephthalic acid in a mixed solution composed of 30 mL of N,N-dimethylformamide and 0.125 mL of deionized water to form solution B. Add solution B to solution A, stir evenly, and then place it in an oil bath at 120 °C for reaction for 12 h. After the reaction is completed, wash and centrifuge, and collect the Zr-MOF nanomaterial.

[0057] The electrode cleaning buffer used in the present invention has the following composition: 3-15 mM Tris-HCl, pH = 7.4, and the solvent is sterilized water.

[0058] The E. coli strain used in the present invention is E. coli O157:H7.

[0059] The base sequence (5'→3') of the E. coli aptamer used in the present invention is:

[0060] TGGTCGTGGTGAGGTGCGTGTATGGGTGGTGGATGAGTGTGTGGC. (SEQ ID NO.1)

[0061] The sequence of the complementary strand of the aptamer (5'→3') is:

[0062] GCCACACACTCATCCACCACCCATACACGCACCTCACCACGACCA. (SEQ ID NO.2)

[0063] The sequence of the SH-DNA strand S1 (5'→3') is:

[0064] SH-ATAATAAGCTGA. (SEQ ID NO.3)

[0065] The sequence of the primer DNA2 (5'→3') is:

[0066] TGGTCGTGGTGAGGTGCGTGCTC. (SEQ ID NO.4)

[0067] The sequence of the primer DNA3 (5'→3') is:

[0068] ATAATAAGCTGAGGGAGCGTATGGGTGGTGGATGAGTGTGTGGC. (SEQ ID NO.5)

[0069] The sequence of the DNA strand S2 (5'→3') is:

[0070] PO4-TCAGCTTATTAT. (SEQ ID NO.6)

[0071] Example 1: Construction of a photoelectrochemical biosensor

[0072] The photoelectrochemical biosensor of the present invention includes: a working electrode, a reference electrode, and a counter electrode. Among them: a saturated calomel electrode is used as the reference electrode, and a Pt electrode is used as the counter electrode;

[0073] The working electrode is prepared by the following method:

[0074] 1. Electrode pretreatment:

[0075] The ITO conductive glass is cut into 5×1 cm 2, acetone, aqueous solution of 1M NaOH in alcohol (V 无水乙醇 :V 二次水 = 1:1), and deionized water were used to ultrasonically treat the ITO electrode for 20 min, then it was rinsed with deionized water and air-dried for later use.

[0076] 2. Immobilization of Bi7O9I3-In2O3:

[0077] 1 g of Bi7O9I3 and 1 g of In2O3 were weighed and dispersed in 10 mL of deionized water. After ultrasonic treatment for 1 hour, they were washed and centrifuged, and the solid was collected to prepare Bi7O9I3-In2O3.

[0078] The prepared Bi7O9I3-In2O3 was subjected to structural characterization, and its SEM image is as Figure 1 shown.

[0079] 20 mg of the prepared Bi7O9I3-In2O3 was weighed and added to 5 mL of deionized water, and ultrasonic dispersion was carried out for 30 min to prepare a Bi7O9I3-In2O3 dispersion.

[0080] 40 μL of the Bi7O9I3-In2O3 dispersion was dropped onto the surface of the pretreated ITO electrode, and it was dried under infrared lamp irradiation; then it was washed 3 times with electrode cleaning buffer and dried with nitrogen. The prepared electrode was labeled as Bi7O9I3-In2O3 / ITO electrode.

[0081] 3. Immobilization of AuNPs:

[0082] The AuNPs solution was diluted 10 times with deionized water to obtain an AuNPs dispersion; 20 μL of the AuNPs dispersion was dropped onto the surface of the Bi7O9I3-In2O3 / ITO electrode, and it was dried under infrared lamp irradiation; then it was washed 3 times with electrode cleaning buffer and dried with nitrogen. The prepared electrode was labeled as AuNPs / Bi7O9I3-In2O3 / ITO electrode.

[0083] 4. Immobilization of SH-DNA strand S1:

[0084] 20 μL of 1 μM SH-DNA strand S1 was modified onto the surface of the AuNPs / Bi7O9I3-In2O3 / ITO electrode through Au-S bonds, and it was washed 3 times with electrode cleaning buffer and dried with nitrogen. 20 μL of mercaptohexanol solution was dropped onto the surface of this electrode to block the electrode, and it was washed 3 times with electrode cleaning buffer and dried with nitrogen. The prepared electrode was labeled as S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode.

[0085] 5. Immobilization of DNA strand S2:

[0086] Sequentially add Escherichia coli aptamer, aptamer complementary strand, Escherichia coli, primer DNA2, primer DNA3, phi 29 DNA polymerase, Nt.BbvCI DNA nickase, and dNTPs into a centrifuge tube for nucleic acid isothermal amplification reaction to prepare a mixed solution containing DNA strand S2.

[0087] Drop 20 μL of the mixed solution containing DNA strand S2 onto the surface of the S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode. DNA strand S2 is modified on the electrode surface by hybridizing with SH-DNA strand S1, and then washed 3 times with electrode washing buffer and dried with nitrogen. The prepared electrode is labeled as the S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode.

[0088] 6. Immobilization of Zr-MOF:

[0089] Weigh 10 mg of Zr-MOF nanomaterials and add them to 5 mL of deionized water, and ultrasonically disperse for 30 min to prepare a Zr-MOF dispersion.

[0090] Drop 40 μL of the Zr-MOF dispersion onto the surface of the S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode, wash 3 times with electrode washing buffer, and dry with nitrogen. The prepared electrode is labeled as the Zr-MOF / S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode, which is the working electrode of the photoelectrochemical biosensor of the present invention.

[0091] For the electrodes prepared in the above steps 2 - 6, using the electrode prepared without Escherichia coli (Without E.coli) in step 5 as a control, the photocurrent response test is carried out under the same conditions, and the results are as Figure 2 shown. The results show that Bi7O9I3-In2O3 can accelerate the migration of photo-generated electrons and increase the photocurrent intensity; AuNPs cover the surface of the photoactive material Bi7O9I3-In2O3 and act as an intermediate for connecting DNA strands; after DNA strand S1 is connected to the surface of AuNPs, the photocurrent is reduced due to the steric hindrance effect. After connecting DNA strand S2 and Zr-MOF, the photocurrent is further reduced, making the difference (-ΔI) between the photocurrent after connecting DNA strand S2 and the photocurrent after connecting DNA strand S1 increase; while for the electrode prepared without Escherichia coli (Without E.coli), since it does not produce DNA strand S2, it cannot connect Zr-MOF subsequently, and there is no significant difference in the photocurrent value compared with the photocurrent after connecting DNA strand S1.

[0092] Example 2: Photoelectrochemical detection of Escherichia coli

[0093] 1. Construct a standard curve between the photocurrent intensity and the logarithm of the Escherichia coli concentration:

[0094] First, anneal the Escherichia coli aptamer, the complementary strand of the aptamer, primer DNA2, and primer DNA3 at 95 °C for 5 min. Then, mix 30 μL of the Escherichia coli aptamer (concentration: 1 μM) with 30 μL of the complementary strand of the aptamer (concentration: 1 μM) and react at 37 °C under humid conditions for 100 min. Add 30 μL of Escherichia coli with a series of concentrations to centrifuge tubes respectively, react at 37 °C under humid conditions for 100 min, then add 30 μL of primer DNA2 (concentration: 1 μM), 30 μL of primer DNA3 (concentration: 1 μM), and 30 μL of the mixture of phi 29 DNA polymerase and dNTPs (concentration: 2 U / μL), react at 37 °C under humid conditions for 100 min, then add 20 μL of Nt.BbvCI DNA nickase (concentration: 1 U / μL), react at 37 °C under humid conditions for 100 min, and then heat at 80 °C for 20 min to inactivate the enzyme, and centrifuge to obtain a mixed solution containing DNA strand S2 prepared based on different Escherichia coli concentrations.

[0095] Construct a Zr-MOF / S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode according to the method of Example 1.

[0096] Using an electrochemical workstation as the signal acquisition instrument, a 3W LED lamp as the light source, Zr-MOF / S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO as the working electrode, a saturated calomel electrode as the reference electrode, a Pt electrode as the counter electrode, and a Tris-HCl buffer solution containing 10 mM pNPP as the electrode detection solution, use the i-t technique to conduct the detection research of the analyte, and establish a standard curve between the photocurrent intensity and the logarithm of the Escherichia coli concentration.

[0097] The photocurrent response diagrams of the working electrodes constructed with the mixed solutions containing DNA strand S2 prepared from Escherichia coli with different concentrations are as Figure 3 shown; the standard curve between the constructed photocurrent intensity difference (-ΔI) and the logarithm of the Escherichia coli concentration is as Figure 4 shown. -ΔI = I2 - I1, where I1 is the electrode photocurrent value of the AuNPs / Bi7O9I3-In2O3 / ITO electrode treated with the SH-DNA strand S1, and I2 is the photocurrent value after the reaction with different concentrations of Escherichia coli as the target molecule and the final electrode treatment.

[0098] The results show that the photoelectrochemical biosensor of the present invention has a detection range for Escherichia coli of 10 0.3 -107.2 CFU / mL. According to the detection limit calculation formula: LOD = 3.3*δ / S, the calculated detection limit is 0.11 CFU / mL.

[0099] 2. Detection of actual samples:

[0100] For the detection of Escherichia coli in actual samples, prepare a mixed solution containing DNA strand S2 according to the above method. Then construct a Zr-MOF / S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode. Then, using an electrochemical workstation as the signal acquisition instrument, a 3W LED lamp as the light source, Zr-MOF / S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO as the working electrode, a saturated calomel electrode as the reference electrode, a Pt electrode as the counter electrode, and Tris-HCl(pNPP) buffer solution as the electrode detection solution. Using -0.1V voltage as the working voltage, the i-t technique is used to measure the photocurrent intensity, and the content of Escherichia coli in the actual sample is calculated using the constructed standard curve.

[0101] Example 3: Investigation of detection selectivity

[0102] Selectivity is an important indicator of the performance of a photoelectrochemical sensor. To study the specificity of the constructed sensor, Salmonella, Pseudomonas aeruginosa, Citrobacter freundii, and Staphylococcus aureus with a concentration of 10 3 CFU / mL were selected as interferents, and a mixed strain (Mixture) of Salmonella, Pseudomonas aeruginosa, Citrobacter freundii, Staphylococcus aureus and Escherichia coli mixed in equal proportions was used as an interferent to study the selectivity of the photoelectrochemical sensor prepared in Example 1. The change values of the photocurrent (-ΔI = I2 - I1, where I1 is the electrode photocurrent value of the AuNPs / Bi7O9I3-In2O3 / ITO electrode treated with SH-DNA strand S1, and I2 is the photocurrent value after different interferents participated in the reaction as target molecules and the electrode treatment was carried out to the last step) of the sensor constructed in Example 1 with different interferents were compared.

[0103] The results show that the change in the current value of the sensor constructed with interferents is significantly lower than the change in the current value of the sensor constructed with Escherichia coli, indicating that the sensor constructed by the present invention has good specificity ( Figure 5 ).

[0104] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various modifications and variations can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A photoelectrochemical biosensor, characterized in that: The photoelectrochemical biosensor uses Bi7O9I3-In2O3, AuNPs, SH-DNA chain S1, DNA chain S2 and Zr-MOF which are sequentially modified on the surface of the base electrode as working electrodes.

2. The photoelectrochemical biosensor according to claim 1, characterized in that: The nucleotide sequence of the SH-DNA chain S1 is shown in SEQ ID NO.3; the nucleotide sequence of the DNA chain S2 is shown in SEQ ID NO.

6.

3. The photoelectrochemical biosensor according to claim 1, characterized in that: The base electrode is an ITO electrode.

4. The method for preparing the photoelectrochemical biosensor according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) dropping a Bi7O9I3-In2O3 dispersion onto the pretreated surface of a base electrode and drying the mixture to prepare a Bi7O9I3-In2O3 / ITO electrode; (2) dropping the AuNPs dispersion onto the surface of the Bi7O9I3-In2O3 / ITO electrode and drying it to prepare the AuNPs / Bi7O9I3-In2O3 / ITO electrode; (3) The SH-DNA chain S1 was modified onto the surface of the AuNPs / Bi7O9I3-In2O3 / ITO electrode through Au-S bonds to prepare the S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode; (4) adding a mixed solution containing DNA chain S2 to the surface of S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode, and the DNA chain S2 is modified on the electrode surface by hybridization with the SH-DNA chain S1, thereby preparing S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode; (5) The Zr-MOF dispersion was dropped onto the surface of the S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode to prepare the Zr-MOF / S2 / S1 / AuNPs / Bi7O9I3-In2O3 / ITO electrode, which was used as the working electrode.

5. The preparation method according to claim 4, characterized in that: In step (1), the Bi7O9I3-In2O3 dispersion is prepared by the following method: Bi7O9I3 and In2O3 are dispersed in deionized water, subjected to ultrasonic treatment, washed, centrifuged, and the solid is collected to prepare Bi7O9I3-In2O3; Then, Bi7O9I3-In2O3 is added into deionized water and dispersed by ultrasonic to prepare Bi7O9I3-In2O3 dispersion.

6. The preparation method according to claim 4, characterized in that: In step (4), the mixed solution containing the DNA chain S2 is prepared by the following method: First, the E. coli aptamer, aptamer complementary chain, primer DNA2, and primer DNA3 are annealed at 95°C for 5 minutes respectively, then the E. coli aptamer and aptamer complementary chain are mixed in equal volumes, reacted at 37°C humid conditions for 0.5-3 hours, an equal volume of E. coli is added, and the reaction is continued at 37°C humid conditions for 0.5-3 hours; then an equal volume of primer DNA2, primer DNA3, phi29 DNA polymerase, Nt.BbvcI DNA nicking enzyme, and dNTPs solution are added, and the reaction is carried out under the same conditions for 0.5-3 hours, and then the enzyme is inactivated by heating at 80°C for 20 minutes, and centrifuged to obtain a mixed solution containing DNA chain S2.

7. The preparation method according to claim 6, characterized in that: The nucleotide sequence of the E. coli aptamer is shown in SEQ ID NO.1; the nucleotide sequence of the aptamer complementary chain is shown in SEQ ID NO.

2.

8. The preparation method according to claim 6, characterized in that: The nucleotide sequence of primer DNA2 is shown in SEQ ID NO.4; the nucleotide sequence of primer DNA3 is shown in SEQ ID NO.

5.

9. Use of the photoelectrochemical biosensor according to any one of claims 1 to 3 in detecting Escherichia coli.

10. A method for detecting Escherichia coli, characterized in that: The following steps are involved: The E. coli aptamer, the aptamer complementary chain, E. coli of a series of concentrations, primer DNA2, primer DNA3, phi29 DNA polymerase, Nt.BbvcI DNA nicking enzyme and dNTPs are subjected to nucleic acid isothermal amplification reaction to prepare a mixed solution containing DNA chain S2; Bi7O9I3-In2O3, AuNPs, SH-DNA chain S1, DNA chain S2 and Zr-MOF were modified on the surface of the base electrode in sequence as working electrodes. A saturated calomel electrode was used as the reference electrode and a Pt wire was used as the auxiliary electrode to form a three-electrode system. Tris-HCl buffer solution containing pNPP was used as the electrode detection solution for photoelectrochemical signal detection. A standard curve between the current and the logarithm of the Escherichia coli concentration was established to detect the Escherichia coli content in the test solution.

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