CN113999820B

CN113999820B - Salmonella enteritidis phage SEP37 and electrochemical impedance spectrum sensor and detection method thereof

Info

Publication number: CN113999820B
Application number: CN202111197364.XA
Authority: CN
Inventors: 王小红; 王吉; 李慧慧; 丁一峰; 朱文娟
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-10-14
Filing date: 2021-10-14
Publication date: 2023-08-11
Anticipated expiration: 2041-10-14
Also published as: CN113999820A

Abstract

The application discloses salmonella enteritidis phage SEP37, an electrochemical impedance spectrum sensor and a detection method thereof, and the preservation number is as follows: cctccc NO: m20211127; the sensor takes the bacteriophage as a biological identification element, and consists of a measuring device with three electrodes, a nitrogen supply device and an electrochemical analyzer, wherein the electrochemical analyzer is communicated with a superior computer. The phage SEP37 has the characteristic of high specificity, the electrochemical impedance spectrum sensor obtained by utilizing the phage SEP37 is a potential tool capable of rapidly and accurately detecting salmonella in various samples, and the detection method of the sensor has the characteristics of low detection limit, high specificity, good stability and high detection speed; the sensor is used for detecting the marked lake water, lettuce and chicken samples, and the result shows that the sensor can rapidly and accurately detect and quantify the concentration of bacteria in the samples.

Description

Salmonella enteritidis phage SEP37 and electrochemical impedance spectrum sensor and detection method thereof

Technical Field

The application relates to the technical field of biosensors, in particular to salmonella enteritidis phage SEP37, an electrochemical impedance spectrum sensor and a detection method thereof.

Background

Food-borne diseases caused by salmonella are serious public health problems worldwide, and bring heavy burden to human health and economic development, while the existing detection methods have more or less defects, such as time and labor consumption in microbial culture and physiological and biochemical identification, the immunological method needs to obtain high-affinity antibodies, and the molecular biological method cannot distinguish live cells from dead cells and is easy to generate false positive results, so that rapid and accurate detection of salmonella is always a research hotspot. In recent years, phage-based biosensors are considered as efficient and simple tools for detecting food-borne pathogens, and are attracting more and more attention from researchers, and are expected to be a detection tool with good selectivity, high sensitivity, high analysis speed and low cost.

Phage (bacteriophage or phage) as the most abundant organism on earth (10 ³¹ Individual) is a virus capable of specifically recognizing and infecting bacteria, and thus, can be used as a biological recognition element based on a specific recognition relationship between phage and bacteria. The use of phage as a biological recognition element has several advantages:

firstly, they are ubiquitous in nature and are resistant to harsh environments;

second, they are highly specific for bacterial strains and harmless to humans;

third, they are susceptible to genetic and chemical modification and thus possess more stable and controllable properties, while they are also less costly to produce and survive longer.

In recent years, on the one hand, electrochemical biosensors based on phage have become research hotspots due to their inherent advantages, such as strong specificity, low detection limit, good stability, etc.; on the other hand, in electrochemical biosensors, since an electrochemical impedance spectroscopy (Electrochemical Impedance Spectroscopy, EIS) detection method is extremely sensitive to modification of electrode surfaces and a biological recognition process, it is widely used to accurately detect a change in minute charge transfer impedance (Charge Transfer Resistance, rct) caused by an analyte bound to a biological recognition element.

Disclosure of Invention

The application aims to overcome the defects of the prior art and provides a salmonella enteritidis phage SEP37, an electrochemical impedance spectrum sensor and a detection method thereof.

To achieve the above object, the present application relates to a salmonella enteritidis phage (Salmonella enteritidis bacteriophage) SEP37, the preservation number of which is: cctccc NO: m20211127.

The salmonella enteritidis phage (Salmonella enteritidis bacteriophage) SEP37 is preserved in China center for type culture collection, and the preservation number is: cctccc NO: m2021127, storage date 2021, 9, 1, address: university of martial arts in chinese.

The salmonella enteritidis phage (Salmonella enteritidis bacteriophage) SEP37 is obtained by laboratory separation and purification, has strong specificity, can identify salmonella with different serotypes, and does not identify bacteria of other species; observing its morphology using a transmission electron microscope, said phage SEP37 belonging to the family sarcodaceae (Myoviridae family) phages; it has the characteristic of rapid adsorption of Salmonella (25 min can reach the maximum adsorption rate); it has high pH stability (3-12) and thermal stability (30-60 ℃).

The application also provides an electrochemical impedance spectrum biosensor for detecting salmonella based on phage, which takes the phage as a biological recognition element, and consists of a measuring device comprising three electrodes, a nitrogen supply device and a P4000A-PARSTAT 4000A electrochemical analyzer, wherein the electrochemical analyzer is communicated with a superior computer.

Further, the three-electrode measuring device comprises a reactor containing a redox probe and three electrodes arranged in the redox probe, wherein the three electrodes are a working electrode, a platinum wire counter electrode and an Ag/AgCl reference electrode respectively (the working electrode has the dual functions of specifically identifying and capturing salmonella and conducting electric signals; the platinum wire counter electrode is used for forming a closed loop, and the Ag/AgCl is used as a reference of standard potential).

Still further, the working electrode is manufactured by the following steps:

1) Mechanically polishing, cleaning and activating the gold plate electrode;

2) Physically depositing gold nanoparticles on the working surface of a gold disk electrode to obtain GDE-AuNPs;

3) Then immersing GDE-AuNPs in a mercaptoethylamine solution at 4 ℃ for 12 hours; obtaining GDE-AuNPs-Cys

4) Immersing GDE-AuNPs-Cys in the activated phage SEP37 suspension for 4h to obtain GDE-AuNPs-Cys-PhageSEP37

5) Finally, the GDE-AuNPs-Cys-PhageSEP37 is incubated with bovine serum albumin solution for 30min, and washed to obtain a working electrode (GDE-AuNPs-Cys-PhageSEP 37-BSA).

Still further, the gold disk electrode (Gold Disk Electrode, GDE) has a diameter of 2mm,

in the step 2) of the above-mentioned process,

gold nanoparticles (AuNPs) with a diameter of 25-30 nm,

mercaptoethylamine (Cys) was used at a molar concentration of 1mmol/L,

the use titer of salmonella enteritidis phage SEP37 was: 5X 10 ¹⁰ PFU/mL，

The mass fraction of bovine serum albumin (Bovine Serum Albumin, BSA) was 2%.

Still further, the redox probe is a mixed aqueous solution containing potassium ferricyanide, potassium ferrocyanide and potassium chloride; wherein, in the mixed aqueous solution, the molar concentration of the potassium ferricyanide and the potassium ferrocyanide is 0.5mmol/L, and the molar concentration of the potassium chloride is 0.1mol/L.

The application also provides a method for detecting salmonella by the electrochemical impedance spectrum biosensor, which comprises the following steps:

1) Firstly, immersing a working electrode into a solution to be tested, slightly disturbing the solution to enable salmonella enteritidis phage SEP37 to capture salmonella in the solution to be tested;

2) The working electrode after salmonella is captured is gently washed by sterile distilled water, the working electrode is installed in a three-electrode measuring device, high-purity nitrogen is firstly introduced into a reactor containing a redox probe before measurement starts, and the measuring process is always maintained in a nitrogen atmosphere;

3) EIS measurements were made using an electrochemical analyzer and data analysis was performed using software VSimpVin 3.60 to fit electrode surface charge transfer impedance (Charge Transfer Resistance, rct) values.

Preferably, the operation parameters of the electrochemical analyzer are as follows: the amplitude of the ac disturbance was 10mV.

Preferably, the electrochemical analyzer detects a frequency of 0.1 to 10 ⁵ Hz。

The application has the beneficial effects that:

1. the phage-based electrochemical impedance spectroscopy sensor for detecting salmonella is free of non-specific recognition and cross-response due to the highly specific effect of phage.

2. The introduction of AuNPs changes the biocompatibility of the electrode, enhances the current effect, and thus improves the sensitivity of the sensor.

3. The phage is covalently immobilized by means of the mercaptoethylamine, so that the phage is combined on the composite electrode more stably, and the repeatability and stability of the sensor are improved.

4. For the concentration of 1X 10 to 1X 10 ⁶ The salmonella bacteria liquid with the CFU/mL has good linear response, the detection limit is 17CFU/mL, and the detection time is 25min.

In conclusion, the salmonella enteritidis phage SEP37 is obtained by screening, the phage SEP37 has the characteristic of high specificity, an electrochemical impedance spectrum sensor obtained by utilizing the phage SEP37 is a potential tool capable of rapidly and accurately detecting salmonella in various samples, and the detection method of the sensor has the characteristics of low detection limit, high specificity, good stability and high detection speed; the sensor is used for detecting the marked lake water, lettuce and chicken samples, and the detection result is verified by using the traditional microorganism culture method, so that the result shows that the sensor can rapidly and accurately detect and quantify the bacterial concentration in the samples.

Drawings

Fig. 1: phage SEP37 plaque picture with salmonella enteritidis ATCC13076 as host bacteria;

fig. 2: transmission electron microscope photograph of phage SEP37;

fig. 3: phage SEP37 biological properties;

in the figure, a: the optimal multiplicity of infection of phage SEP37,

b: phage SEP37 adsorption rate profile,

c: phage SEP37 one-step growth curve, in the figure 'L' refers to latency, 'B' refers to burst,

d: temperature stability of phage SEP37,

e: pH stability of phage SEP37;

fig. 4: a biosensor preparation flow chart;

fig. 5: SEM of AuNPs is loaded on the surface of the bare electrode;

in the figure, a: SEM of bare electrode surface;

b: 2.5. Mu.L of SEM of the colloidal gold solution was added dropwise,

c: add dropwise 5 μl of SEM of colloidal gold solution,

d: 7.5. Mu.L of SEM of the colloidal gold solution was added dropwise,

fig. 6: the preparation condition of the GDE-AuNPs-Cys-PhageSEP37-BSA composite electrode is optimized;

in the figure, a: the optimization result of the self-assembly time of the mercaptoethylamine,

b: results of optimization of phage incubation time,

c: as a result of the optimization of the BSA blocking time,

d: the result of the optimization of the incubation time of the bacteria,

fig. 7: response results of EIS during preparation of GDE-AuNPs-Cys-PhageSEP37-BSA composite electrode;

fig. 8: schematic representation of a phage-based electrochemical impedance spectroscopy biosensor for detecting salmonella;

in the figure, a measuring device 1 comprising three electrodes, a reactor 1.1 containing a redox probe, a working electrode 1.2, a platinum wire counter electrode 1.3, an Ag/AgCl reference electrode 1.4, a nitrogen supply device 2, and a P4000A-PARSTAT 4000A electrochemical analyzer 3;

fig. 9: a phage-based electrochemical impedance spectroscopy sensor for detecting salmonella in PBS buffer to detect a measurement of salmonella ATCC 13076;

in the figure, a: the concentration range detected by the sensor is 1 multiplied by 10 ¹ ～1×10 ⁸ At CFU/mL, the response of the EIS,

b: the Rct data corresponding to the EIS measurement results are further processed,

c: composite electrode and concentration of 1X 10 ³ SEM images after incubation of CFU/mL salmonella ATCC13076 for 30min,

d: composite electrode and concentration of 1X 10 ⁵ SEM images after incubation of CFU/mL salmonella ATCC13076 for 30min and magnified images thereof,

fig. 10: a phage-based electrochemical impedance spectroscopy sensor-specific result map for detecting salmonella;

fig. 11: a graph of electrochemical impedance spectroscopy sensor stability results for phage-based detection of salmonella;

fig. 12: EIS response results of phage-based electrochemical impedance spectroscopy sensors for salmonella detection in different samples (lake water, lettuce and chicken breast);

in the figure, a: the EIS response of salmonella ATCC13076 in the lake water was labeled,

b: EIS response results of Salmonella ATCC13076 in the bearded lettuce,

c: EIS response results of Salmonella ATCC13076 in the chicken breast,

d: and (3) further processing results of Rct data corresponding to EIS measurement results of lake water, lettuce and chicken breast samples.

Detailed Description

The present application is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art.

Example 1: phage isolation and screening, purification proliferation and morphological analysis

1. Phage SEP37 isolation and screening

Collecting market sewage samples of white sand Africa farm and sideline products in Wuhan, filtering with filter paper, taking 5mL of filtrate and 5mL of salmonella enteritidis ATCC13076 cultured to logarithmic phase, culturing overnight in 20mL of LB for 12h, filtering with a 0.22 μm microporous filter membrane, and repeating the filtrate once according to the method to obtain phage stock solution. Separating and identifying whether phage exist or not by adopting a double-layer flat plate method, gently mixing 100 mu L of salmonella enteritidis ATCC13076 100 mu L cultured for 6-8 h and 3.8mL of semisolid culture medium at 45-50 ℃ with the phage stock solution, pouring the mixture onto the LA lower layer culture medium, standing the mixture after solidification, and culturing the mixture in an incubator at 37 ℃ for about 6 h.

As shown in fig. 1: the presence of transparent circles in the figure demonstrates the presence of phage.

2. Phage purification and proliferation

Phage SEP37 was isolated by coupling to OD ₆₀₀ Culture of salmonella enteritidis ATCC13076 =0.6 was co-cultured, filtered, centrifuged. After salmonella enteritidis ATCC13076 had been grown for 8 hours at 37℃with shaking (180 rpm), the cultures were inoculated in 2 250mL Erlenmeyer flasks at a ratio of 1:20, each flask containing 50mL LB. When the OD of the bacterial culture ₆₀₀ When=0.2, about 100 μl of 10 is added per bottle ⁶ PFU/mL phage SEP37, about 3-4 hours after phage addition, phage complete lysis and release from the host bacteria, the lysate at 8000r/min,4 ℃ centrifugation for 20min, discarding the precipitate, the phage-containing solution obtained through 0.22 μm filter membrane filtration to remove residual bacteria, then at 40,000 r/min,4 ℃ centrifugation for 1h, discarding the supernatant, phage-containing precipitate re-suspended in PBS solution for standby.

3. Phage morphology analysis

After the phage is negatively stained with phosphotungstic acid, the phage is placed under a transmission electron microscope to observe the phage morphology, and the specific operation steps are as follows: after the copper mesh is immersed in phage suspension for 10min, the redundant liquid is sucked by filter paper, the copper mesh is placed in 0.5% phosphotungstic acid dye for dyeing, then naturally dried, the prepared copper mesh is observed in phage form under a transmission electron microscope, and the size of the copper mesh is measured by software Digital Micrograph Demo 3.9.1.

As shown in fig. 2: phage SEP37 belongs to the order Rheuviridae, the family myoviridae. The head part of the icosahedron is provided with a tail part which comprises a tail sheath and a tail pipe and is retractable; the diameter of the head is 108.7 plus or minus 2.7nm, and the total length of the tail is 101.8 plus or minus 3.8nm.

The salmonella enteritidis phage (Salmonella Enteritidis bacteriophage) SEP37 (hereinafter referred to as phage SEP 37) is preserved in China center for type culture Collection, and the preservation number is: cctccc NO: m20211127, storage date 2021, 9, 1, address: university of martial arts in chinese.

Example 2: phage SEP37 biological Property analysis

1. Phage SEP37 host profiling

Phage host profile was determined using the spotting method:

take 100. Mu.L OD ₆₀₀ Adding the bacterial liquid to be measured with the concentration of 0.6 into a warm semi-solid culture medium, uniformly mixing, pouring the mixture into a pre-prepared LA flat plate, and taking 5 mu L of the mixture with the titer of 1 multiplied by 10 after solidification ⁹ PFU/mL phage were added dropwise to the upper plate surface, dried and then incubated in an incubator at 37℃for 4-6 hours, and the cleavage was observed, with the results shown in Table 1.

TABLE 1 phage SEP37 host profile

Note that: ATCC (ATCC) ^a ，American Type Culture Collection；SJTU ^b ，Sha nghai Jiao Tong University；CMCC ^c ，Center for Medical Culture Collections；CVCC ^d ，China Veterinary Culture Collection Center；“++”strong intensity(Clear plaque)；“+”weak intensity(Opaque plaque)；“-”no lytic activity(No plaque).

2. Phage optimal infection complex (MOI)

The multiplicity of infection (Multiplicity of Infection, MOI) refers to the ratio of the number of phages to the number of host bacteria at the time of initial infection. Mixing phage with host bacteria according to certain MOI values (0.001, 0.01, 0.1, 1, 10, 100 and 1000), culturing at 37 ℃ for 3.5h, centrifuging at 9000r/min for 10min, and measuring phage titers in supernatants corresponding to different MOI values by a double-layer plate method. The test was set up in 3 replicates.

As shown in fig. 3A, phage titers were maximized at moi=10, i.e., the optimal multiplicity of infection of phage was 10, indicating that more phage could be propagated when phage were mixed with host bacteria in a 10:1 ratio.

3. Adsorption rate

Fresh phage solution and host bacterial suspension are mixed in a centrifuge tube according to the optimal MOI value, and shake cultivation is carried out at 37 ℃. The titers of phages in the supernatants were determined by double-layer plating every 5min, starting at 0min. The test was set up in 3 replicates. Adsorption rate = 1- (phage titer unadsorbed per time point/phage titer at 0 min) ×100%, and the results of phage adsorption rate to host bacteria are shown in figure 3B.

As can be seen from FIG. 3B, the optimal adsorption rate of phage SEP37 was 64.70% and the time to reach the optimal adsorption rate was 25min, at which time the phage was adsorbed to the host bacteria in the maximum amount.

4. One-step growth curve

The one-step growth curve of phage reflects its growth law. Phage and host bacteria were mixed at the optimal MOI values, incubated at 37℃for 25min to allow phage to adsorb onto bacteria, then centrifuged at 8000r/min at 4℃for 2min, the supernatant discarded, and resuspended twice with equal volumes of LB to remove unadsorbed phage. Adding the liquid into 9mL of LB culture medium, placing in a shaking table at 37 ℃ for shake culture, sampling 300 mu L every 10min from 0min, centrifuging at 8000r/min for 2min, and measuring the titer of phage in the supernatant by adopting a double-layer plate method. The test was set up in 3 replicates. The incubation period, lytic amount of phage (lytic amount = phage titer at end of lysis/host concentration at initial stage of infection) can be seen from the one-step growth plot (fig. 3C). The incubation period of phage SEP37 was 40min, the lysis period was 140min, and the lysis capacity was 13.7PFU/cell.

5. Phage SEP37 thermal stability analysis

Diluting phage stock solution to titer of 1×10 ⁷ PFU/mL, and sub-packaging into 1mL sterile centrifuge tubes, placing the centrifuge tubes in constant temperature water baths at 30deg.C, 40deg.C, 50deg.C, 60deg.C, 70deg.C and 80deg.C for 0min,30min and 60min, respectively, and determining phage titer in each tube. The test was set up in 3 replicates. As can be seen from FIG. 3D, phage SEP37 has a substantially constant activity between 30 and 50℃and gradually decreases with time when the temperature is raised to 60℃and has already begun to decrease significantly in a short period of time when the temperature exceeds 70 ℃.

6. Phage SEP37 pH stability analysis

Phage suspension of known titer (1X 10) ⁷ PFU/mL) 100. Mu.L was added to 900. Mu.L of PBS buffer with different pH values (2-13), and the mixture was placed in a 37℃water bath for 2h, and the phage titer in each centrifuge tube was determined. The test was set up in 3 replicates. As can be seen from FIG. 3E, the phage remained at a relatively stable titer at pH 4-11, decreased at pH 3 and pH 12, and decreased to substantially 0 at pH 2 and pH 13, indicating that strong acids and bases directly destroyed the phage activity.

Example 3: electrochemical impedance spectrum biosensor for detecting salmonella based on bacteriophage

1. Preparation of working electrode (GDE-AuNPs-Cys-PhageSEP 37-BSA) composite electrode

1) Pretreatment of Gold Disk Electrode (GDE) to obtain amino-functionalized surface:

mechanically polishing the bare GDE to a mirror finish with 0.3 μm and 0.05 μm alumina/cement slurry on a polishing cloth, respectively, and then thoroughly ultrasonically cleaning with deionized water, acetone and ethanol, respectively; then at 0.5mol/L H ₂ SO ₄ The solution is circularly scanned for 100 times between-0.2 and 1.0V at a voltage of 100mV/s, the electrode is electrochemically cleaned, and then rinsed with deionized water and N ₂ Air flow drying;

2) Gold nanoparticles with the diameter of 25-30 nm are dripped on the working surface of the electrode to obtain GDE-AuNPs

3) After drying in a clean environment, immersing the gold disk electrode GDE-AuNPs carrying the AuNPs in a mercaptoethylamine solution with the concentration of 1mmol/L at the temperature of 4 ℃ for 12 hours, and forming a self-assembled monolayer structure under the interaction of thiol-gold, namely: GDE-AuNPs-Cys.

4) Preparing GDE-AuNPs-Cys-PhageSEP37 (phage SEP37 is fixed on the surface of GDE-AuNPs-Cys due to amide bond formation);

a. preparation of activated phage SEP37 suspension:

0.08mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (1-ethyl- (3-dimethylaminopropyl) carbodiimide, EDC) and 0.22mg of N-Hydroxysuccinimide (N-Hydroxysuccinimide, NHS) were added sequentially to the phage-containing pellet (5X 10) at intervals of 30min ¹⁰ PFU/mL) in 1mL PBS to give an activated phage SEP37 suspension;

b. immersing the GDE-AuNPs-Cys in the activated phage SEP37 suspension and incubating for 4 hours at 4 ℃, and then leaching with deionized water to remove the phage which is not firmly combined;

5) Finally, GDE-AuNPs-Cys-PhageSEP37 was incubated with 0.2% bovine serum albumin solution (BSA) for 30min (blocking unbound amino groups), and the electrode was rinsed with deionized water to obtain a working electrode, and a finished working electrode (i.e.:

GDE-AuNPs-Cys-PhageSEP 37-BSA) was stored in PBS solution at 4℃for subsequent experiments.

The results are shown in FIG. 4: the preparation process flow of the biosensor is shown.

Optimization of GDE-AuNPs-Cys-PhageSEP37-BSA preparation conditions

1) Determining the loading capacity of gold nano particles on a gold electrode; 2.5 mu L, 5 mu L and 7.5 mu L of colloidal gold with the diameter of 25-30 nm are respectively dripped on the surface of the electrode, and a scanning electron microscope is used for observing the loading condition of gold nanoparticles;

2) Determining self-assembly time of mercaptoethylamine; the time for immersing the electrode carrying AuNPs in the mercaptoethylamine solution was varied to 6h,12h,18h,24h,30h,36h, and the corresponding Rct was recorded, with the reduction Rct defined as Δrct=rct ₀ –Rct ₁ (Rct ₀ Is the Rct value of the composite electrode before the self-assembly of the mercaptoethylamine, and Rct ₁ Rct value of the composite electrode after self-assembly of mercaptoethylamine).

3) Phage for use in a humanDetermining incubation time; changing incubation time of phage to 1h,2h,3h,4h,5h,6h, recording corresponding Rct, increasing Rct can be defined as Δrct=rct ₂ –Rct ₁ (Rct ₂ Is the Rct value of the composite electrode after phage incubation).

4) Determination of BSA incubation time; incubating phage-carrying electrodes in 2% bsa for 10min,20min,30min,40min,50min,60min, recording the corresponding Rct, the increased Rct being defined as Δrct=rct ₃ –Rct ₂ (Rct ₃ Is the Rct value of the composite electrode after BSA incubation).

The results are shown in FIG. 5: SEM results showed that after 7.5. Mu.L (FIG. 5D) of colloidal gold was added dropwise, the gold nanoparticles adsorbed most uniformly and densely.

The results are shown in FIG. 6: (A) The result shows that when the self-assembly time of the mercaptoethylamine exceeds 12 hours, the delta Rct is basically unchanged; (B) It was shown that when phage SEP37 was incubated for 4h, Δrct was gradually stabilized; (C) It was shown that when BSA blocked for 30min, Δrct no longer increased and started to stabilize.

The results are shown in FIG. 7: the EIS response results of the overall process of preparing the composite electrode are shown.

Summarizing the optimal conditions for the preparation of GDE-AuNPs-Cys-PhageSEP37-BSA biosensors were: the GDE surface was then titrated with 7.5. Mu.L of colloidal gold solution, followed by self-assembly in 1mmol/L cysteamine solution for 12h, followed by incubation with phage SEP37 for 4h and finally blocking with 2% BSA for 30min.

3. Electrochemical impedance spectrum biosensor for detecting salmonella based on bacteriophage

As shown in fig. 8: the sensor takes phage SEP37 as a biological recognition element and consists of a measuring device 1 comprising three electrodes, a nitrogen supply device 2 and a P4000A-PARSTAT 4000A electrochemical analyzer 3; the electrochemical analyzer is communicated with an upper computer;

the measuring device comprises a reactor 1.1 containing a redox probe and three electrodes arranged in the redox probe, wherein the three electrodes are a working electrode 1.2, a platinum wire counter electrode 1.3 and an Ag/AgCl reference electrode 1.4 respectively.

The redox probe is a mixed aqueous solution containing potassium ferricyanide, potassium ferrocyanide and potassium chloride; wherein, in the mixed aqueous solution, the molar concentration of the potassium ferricyanide and the potassium ferrocyanide is 0.5mmol/L, and the molar concentration of the potassium chloride is 0.1mol/L.

Example 4: detection method of electrochemical impedance spectrum biosensor for detecting salmonella based on phage

1. Determination of bacterial incubation time

To determine the time for phage recognition and capture of host bacteria, the biosensor was associated with 5X 10 ⁵ CFU/mL Salmonella enteritidis ATCC13076 cells were incubated for 10min,15min,20min,25min,30min,35min, and then Rct was measured, the increase in Rct being defined as ΔRct=Rct ₄ –Ret ₃ (Rct ₄ To identify the Rct value after salmonella enteritidis ATCC13076 cells, ret ₃ Initial Rct value for composite electrode

The results are shown in FIG. 6: (D) It was shown that DeltaRct increases rapidly at 20-25 min, and then remains stable for 5-10 min, which is attributed to the fact that the phage SEP 37-based biosensor recognizes and immobilizes Salmonella at an early stage, but almost all phage recognition sites are occupied over time, and bacteria cannot be recognized and immobilized any more, so that the biosensor is incubated with the detection broth for 30min.

2. Detection of Salmonella enteritidis at different concentrations

The biosensor was immersed in a solution containing 1X 10 ¹ ～1×10 ⁸ Incubating in PBS (phosphate buffered saline) solution of CFU/mL salmonella enteritidis for 25min with slight disturbance, introducing high-purity nitrogen into the system for 15min before measurement starts, and maintaining the measurement process in nitrogen atmosphere all the time; when the solution to be detected is replaced, the combination of phage and host bacteria on the composite electrode in the previous step is destroyed by weak alkaline solution, and the electrode is slowly rinsed by deionized water, so that the response before bacteria detection is restored.

The results are shown in FIG. 9: (a) is the measurement of EIS; (B) Is the result of using the Rct values fitted by software VSimpVin 3.60 and further processed, when the bacterial concentration is 10 ¹ ～10 ⁶ In the CFU/mL range, delta Rct value and salmonella enteritidis concentration are measuredThe logarithm is linearly related, and the regression equation is y= 796.89x-484.77 (R ² = 0.9829), the detection limit is 17CFU/mL, and the detection time is 25min; (C) The capture concentration for the sensor was 1×10 ³ SEM pictures of CFU/mL of Salmonella enteritidis ATCC 13076; (D) The capture concentration for the sensor was 1×10 ⁵ SEM pictures of CFU/mL of Salmonella enteritidis ATCC13076 and magnified pictures thereof.

Example 5: phage-based detection specificity test of electrochemical impedance spectroscopy biosensor for detecting salmonella

By combining the biosensor with the same concentration (5X 10 ⁵ CFU/mL) and some non-salmonella strains were incubated together to evaluate the specificity of the sensor. Salmonella strains include Salmonella enteritidis 4 strains, salmonella typhimurium 4 strains, and other serotype 6 strains; non-salmonella strains include listeria monocytogenes strain 2, staphylococcus aureus strain 2, escherichia coli strain 2.

The results are shown in FIG. 10: the tested salmonella strains (14 strains, 100%) all caused a significant increase in impedance, while the 6 non-salmonella strains hardly caused an increase in impedance or were consistent with the blank, indicating that the sensor only can identify salmonella, but not other bacteria, with a high degree of specificity.

Example 6: phage-based detection stability test for salmonella-detecting electrochemical impedance spectroscopy biosensor

By comparing the same concentration (5X 10) at 4℃and 23℃for 6 weeks ⁵ CFU/mL) to evaluate the stability of the sensor.

The results are shown in FIG. 11: Δrct remained essentially unchanged for the first three weeks, and began to decrease at week 4 and decreased to 90% at week 6; there was no significant difference in Δrct at 4 ℃ and 23 ℃; the sensor maintains a higher stability than other types of biosensors.

Example 7: phage-based electrochemical impedance spectroscopy biosensor for detecting salmonella in different sample matrices

Respectively to do not containAnd (5) performing marking treatment on lake water, lettuce and chicken breast samples to be inspected. Firstly, carrying out simple filtering treatment on lake water, treating lettuce and chicken breast meat samples according to national standard, and then respectively adding salmonella enteritidis ATCC13076 into the lake water, lettuce and chicken breast meat to make the final concentration of the salmonella enteritidis ATCC13076 be 10 in sequence ¹ 、10 ² 、10 ³ 、10 ⁴ 、10 ⁵ And 10 ⁶ CFU/mL; taking one untagged sample as a control; measurements were made as in example 4.

The results are shown in FIG. 12: and (A), (B) and (C) are EIS measurement results, and EIS responses of the three samples are increased along with the increase of the bacterial concentration, so that the biosensor can detect salmonella in the samples. (D) The result of the oct values fitted using software VSimpVin 3.60 and further processed, the EIS response of lake water and lettuce was almost identical to that of the PBS group; the fitting curve of lake water is y= 931.75x-608.58, r2= 0.9899; lettuce has a fitted curve y= 1073.3x-359.09, r2= 0.9931; both samples had a linear range of 1X 10 ¹ ～1×10 ⁶ CFU/mL; the fitted curve of chicken breast is y= 1190.8x-159.97, r2= 0.9877, and the linear range is 1×10 ² ～1×10 ⁵ CFU/mL. The standard curve is used for quantifying three types of samples containing a certain number of target bacteria, and the results are verified by using a traditional microorganism culture method, so that the detection results of the biosensor are not significantly different from the detection results of the microorganism culture method. The results prove that the sensor can accurately detect and quantify salmonella in different sample matrixes, so the sensor provided by the application is a detection method which is short in time consumption, simple and convenient to operate, stable in property and low in cost, and has potential of being applied to actual sample detection.

Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the application, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the application.

Claims

1. Salmonella enteritidis phageSalmonellaenteritidisbacteriophage) SEP37, accession number: cctccc NO: m20211127.

2. An electrochemical impedance spectroscopy biosensor for detecting salmonella based on bacteriophage, characterized in that: the sensor takes the bacteriophage of claim 1 as a biological recognition element, and consists of a measuring device comprising three electrodes, a nitrogen supply device, and a P4000A-PARSTAT 4000A electrochemical analyzer, wherein the electrochemical analyzer is in communication with an upper computer.

3. The electrochemical impedance spectroscopy biosensor of claim 2, wherein: the measuring device comprises a reactor containing a redox probe and three electrodes arranged in the redox probe, wherein the three electrodes are respectively a working electrode, a platinum wire counter electrode and an Ag/AgCl reference electrode; the redox probe is a mixed aqueous solution containing potassium ferricyanide, potassium ferrocyanide and potassium chloride; wherein, in the mixed aqueous solution, the molar concentration of the potassium ferricyanide and the potassium ferrocyanide is 0.5mmol/L, and the molar concentration of the potassium chloride is 0.1mol/L.

4. The electrochemical impedance spectroscopy biosensor of claim 3, wherein: the working electrode is manufactured by the following steps:

1) Mechanically polishing, cleaning and activating the gold plate electrode;

3) Then immersing GDE-AuNPs in a mercaptoethylamine solution at 4 ℃ for 12h to obtain GDE-AuNPs-Cys;

4) Immersing GDE-AuNPs-Cys into activated phage SEP37 suspension for 4h to obtain GDE-AuNPs-Cys-PhageSEP37;

5) And finally, incubating GDE-AuNPs-Cys-PhageSEP37 with a bovine serum albumin solution for 30min, and washing to obtain the working electrode.

5. The electrochemical impedance spectroscopy biosensor of claim 4, wherein: the diameter of the gold disk electrode is 2mm,

in the step 2), the diameter of the gold nano-particles is 25-30 nm,

the molar concentration of the mercaptoethylamine used is 1mmol/L,

the use titer of salmonella enteritidis phage SEP37 was: 5X 10 ¹⁰ PFU/mL，

The mass fraction of bovine serum albumin is 2%.

6. A method for detecting salmonella by using the electrochemical impedance spectroscopy biosensor of any one of claims 3-5, wherein: the method is a non-diagnostic method comprising the steps of:

3) Electrochemical impedance spectroscopy EIS measurements were performed using an electrochemical analyzer and data analysis was performed using software VSimpVin 3.60 to fit electrode surface charge transfer impedance values.

7. The method according to claim 6, wherein: the operation parameters of the electrochemical analyzer are as follows: the ac disturbance amplitude was 10mV.

8. The method according to claim 6, wherein: the detection frequency of the electrochemical analyzer is 0.1-10 ⁵ Hz。