CN113999820A

CN113999820A - Salmonella enteritidis phage SEP37 and electrochemical impedance spectroscopy sensor and detection method thereof

Info

Publication number: CN113999820A
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: 2022-02-01
Anticipated expiration: 2041-10-14
Also published as: CN113999820B

Abstract

The invention discloses a salmonella enteritidis phage SEP37, an electrochemical impedance spectroscopy sensor and a detection method thereof, wherein the preservation number is as follows: CCTCC NO: m20211127; the sensor takes the phage as a biological recognition element, and consists of a measuring device comprising three electrodes, a nitrogen supply device and an electrochemical analyzer, wherein the electrochemical analyzer is communicated with an upper computer. The phage SEP37 has the characteristic of high specificity, the electrochemical impedance spectroscopy sensor obtained by using the phage SEP37 is a potential tool capable of quickly 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 lake water, the lettuce and the chicken samples with the labels, and the result shows that the sensor can quickly and accurately detect and quantify the bacterial concentration in the samples.

Description

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

Technical Field

The invention relates to the technical field of biosensors, in particular to a salmonella enteritidis phage SEP37, an electrochemical impedance spectroscopy 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, but the existing detection methods have more or less defects, such as time and labor consumption in microbial culture and physiological and biochemical identification, the need of obtaining high-affinity antibodies by an immunological method, the inability of distinguishing living and dead cells by a molecular biological method, the easy occurrence of false positive results and the like, so that the rapid and accurate detection of the salmonella is always a hotspot of research. In recent years, a biosensor based on a phage has been considered as an efficient and simple means for detecting a food-borne pathogen, and has attracted attention from researchers, and is expected to be a detection means having good selectivity, high sensitivity, a high analysis speed, and a low cost.

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

firstly, they are ubiquitous in nature and have a high tolerance to harsh environments;

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

third, they are easier to genetically and chemically modify and thus possess more stable and controllable properties, while their production costs are lower and their survival time is longer.

In recent years, on one hand, the electrochemical biosensor based on the phage becomes a research hotspot due to the inherent advantages thereof, such as strong specificity, low detection limit, good stability and the like; on the other hand, Electrochemical Impedance Spectroscopy (EIS) detection methods are widely used in Electrochemical biosensors because they are extremely sensitive to modification of an electrode surface and a biological recognition process, and can accurately detect a change in a fine Charge Transfer Impedance (Rct) caused by an analyte bound to a biological recognition element.

Disclosure of Invention

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

In order to achieve the purpose, the invention relates to a Salmonella enteritidis bacteriophages SEP37 with the preservation number: CCTCC NO: m20211127.

The Salmonella enteritidis bacteriophage (Salmonella enteritidis bacteriophage) SEP37 is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2021127, date of deposit 2021, 9/1, address: wuhan university in Wuhan, China.

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

The invention also provides an electrochemical impedance spectroscopy biosensor for detecting salmonella based on phage, which takes the phage as a biological identification element, and consists of a measuring device comprising three electrodes, a nitrogen supply device and a P4000A-PARSTAT4000A electrochemical analyzer, wherein the electrochemical analyzer is communicated with a superior computer.

Furthermore, 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 respectively a working electrode, a platinum wire counter electrode and an Ag/AgCl reference electrode (the working electrode has the double functions of specifically recognizing and capturing salmonella and conducting an electric signal; the platinum wire counter electrode is used for forming a closed loop, and Ag/AgCl is used as a reference of standard potential).

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

1) mechanically grinding, polishing, cleaning and activating the gold disc electrode;

2) physically depositing the gold nanoparticles to the working surface of the gold disc electrode to obtain GDE-AuNPs;

3) then, GDE-AuNPs are immersed in mercaptoethylamine solution at4 ℃ for 12 hours; obtaining GDE-AuNPs-Cys

4) Then, GDE-AuNPs-Cys is immersed into the activated phage SEP37 suspension for 4h to obtain GDE-AuNPs-Cys-PhageSEP37

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

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

in the step 2) of the said step,

the diameter of the gold nanoparticles (AuNPs) is 25-30 nm,

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

the titer of salmonella enteritidis phage SEP37 used was: 5X 10¹⁰PFU/mL，

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

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

The invention also provides a method for detecting salmonella by using the electrochemical impedance spectroscopy biosensor, which comprises the following steps:

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

2) the working electrode after capturing salmonella is gently flushed 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 begins, and the measurement process is always maintained in the nitrogen atmosphere;

3) 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 (Rct) values.

Preferably, the operating parameters of the electrochemical analyzer are as follows: the amplitude of the AC perturbation was 10 mV.

Preferably, the detection frequency of the electrochemical analyzer is 0.1-10⁵Hz。

The invention has the beneficial effects that:

1. phage-based electrochemical impedance spectroscopy sensors for salmonella do not have non-specific recognition due to the highly specific action of the phage, and no cross-response.

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 bacteriophage is covalently fixed by means of mercaptoethylamine, so that the bacteriophage is more stably combined on the composite electrode, and the repeatability and stability of the sensor are improved.

4. For the concentration of 1 × 10 to 1 × 10⁶The CFU/mL salmonella bacterial liquid has good linear response, the detection limit is 17CFU/mL, and the detection time is 25 min.

In conclusion, the Salmonella enteritidis phage SEP37 is obtained by screening, the phage SEP37 has the characteristic of high specificity, the electrochemical impedance spectroscopy sensor obtained by utilizing the phage SEP37 is a potential tool capable of quickly 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 lake water, lettuce and chicken samples added with the labels, and the detection result is verified by using the traditional microbial culture method, and the result shows that the sensor can quickly and accurately detect and quantify the concentration of bacteria in the samples.

Drawings

FIG. 1: the phage SEP37 takes a plaque picture of salmonella enteritidis ATCC13076 as host bacteria;

FIG. 2: transmission electron micrograph of phage SEP 37;

FIG. 3: biological characteristics of bacteriophage SEP 37;

in the figure, A: the optimal multiplicity of infection for the bacteriophage SEP37,

b: the adsorption rate curve for phage SEP37,

c: the one-step growth curve of phage SEP37, wherein ` L ` indicates latency ` B ` indicates burst phase,

d: the temperature stability of the bacteriophage SEP37,

e: pH stability of phage SEP 37;

FIG. 4: a biosensor preparation flow chart;

FIG. 5: loading SEM of AuNPs on the surface of the bare electrode;

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

b: adding 2.5 μ L of colloidal gold solution SEM dropwise,

c: 5 mul of SEM of colloidal gold solution is dropped,

d: 7.5 mu L of colloidal gold solution SEM is added dropwise,

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

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

b: the result of the optimization of the incubation time of the phage,

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

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

FIG. 7: response results of EIS in the preparation process of the GDE-AuNPs-Cys-PhageSEP37-BSA composite electrode;

FIG. 8: a schematic diagram 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, a P4000A-PARSTAT4000A electrochemical analyzer 3;

FIG. 9: phage-based electrochemical impedance spectroscopy sensors for detecting salmonella measurement results of salmonella ATCC13076 in PBS buffer;

in the figure, A: the detection concentration range of the sensor is 1 multiplied by 10¹～1×10⁸CFU/mL, response results of EIS,

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

c: composite electrode and concentration of 1 × 10³SEM image after incubation of CFU/mL Salmonella ATCC13076 for 30min,

d: composite electrode and concentration of 1 × 10⁵SEM image and its enlarged view of CFU/mL Salmonella ATCC13076 after 30min incubation,

FIG. 10: a specific result chart of the electrochemical impedance spectroscopy sensor for detecting salmonella based on bacteriophage;

FIG. 11: a stability result graph of the electrochemical impedance spectroscopy sensor for detecting salmonella based on bacteriophage;

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

in the figure, A: the EIS response results of Salmonella ATCC13076 in lake water were normalized,

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

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

d: and (4) further processing results of the Rct data corresponding to the EIS measurement results of the lake water, the lettuce and the chicken breast meat samples.

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

Example 1: phage isolation and screening, purification value-added and morphological analysis

1. Phage SEP37 isolation and screening

Collecting a sample of agricultural byproduct market sewage of Baishazhou province in Wuhan city, filtering the sample by using filter paper, taking 5mL of filtrate and 5mL of salmonella enteritidis ATCC13076 cultured to logarithmic phase to be cultured in 20mL of LB overnight for 12h, filtering the culture solution by using a 0.22 mu m microporous membrane, and repeating the above steps on the filtrate to obtain a phage stock solution. Separating and identifying existence of phage by double-layer plate method, pouring 100 μ L of phage stock solution and 3.8mL of 45-50 deg.C semisolid culture medium on LA lower layer culture medium, pouring into 37 deg.C incubator for 6h after solidification.

As shown in fig. 1: the presence of phage is evidenced by the appearance of a clear circle in the figure.

2. Multiplication of phage purification

Phage SEP37 was generated by interaction with OD₆₀₀Co-culturing 0.6 culture of salmonella enteritidis ATCC13076, filtering, and centrifuging. After culturing Salmonella enteritidis ATCC13076 at 37 ℃ for 8h with shaking (180rpm), the culture was inoculated into 2 250mL Erlenmeyer flasks each containing 50mL of LB at a ratio of 1: 20. When OD of bacterial culture₆₀₀When the concentration is 0.2, about 100. mu.L of 10 is added to each bottle⁶PFU/mL phage SEP37, after adding phage for 3-4 h, the phage is completely cracked and released from host bacteria, and the lysate is at 8000r/min, centrifuging at4 deg.C for 20min, discarding the precipitate, filtering the resultant phage-containing solution through a 0.22 μm filter to remove residual bacteria, centrifuging at 40000 r/min at4 deg.C for 1h, discarding the supernatant, and resuspending the phage-containing precipitate in PBS solution for use.

3. Phage morphology analysis

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

As shown in fig. 2: phage SEP37 belongs to the order uroviridae, the family myoviridae. The tail part comprises a tail sheath and a tail pipe and can be contracted; the diameter of the head part is 108.7 +/-2.7 nm, and the total length of the tail part is 101.8 +/-3.8 nm.

The Salmonella Enteritidis phage SEP37 (hereinafter referred to as phage SEP37) is deposited in the China center for type culture Collection with the following preservation numbers: CCTCC NO: m20211127, date of deposit 2021, 9/1, address: wuhan university in Wuhan, China.

Example 2: analysis of the biological Properties of phage SEP37

1. Analysis of phage SEP37 host spectra

The determination of the phage host spectrum adopts a spotting method:

100 μ L of OD was taken₆₀₀Adding the bacterial liquid to be measured of 0.6 into warm semisolid culture medium, mixing uniformly, pouring onto prepared LA plate, solidifying, and collecting 5 μ L of the liquid with titer of 1 × 10⁹And (3) dropwise adding the phage of PFU/mL onto the surface of the upper flat plate, drying, then inversely placing the flat plate in an incubator at 37 ℃ for culturing for 4-6 h, and observing the cracking condition, wherein the results are shown in Table 1.

TABLE 1 phage SEP37 host spectra

Note: ATCC (American type culture Collection)^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 multiplicity of infection (MOI)

Multiplicity of Infection (MOI) refers to the ratio of the number of phage to the number of host bacteria at the time of initial Infection. Mixing phage and host bacteria according to certain MOI value (0.001, 0.01, 0.1, 1, 10, 100, 1000), culturing at 37 deg.C for 3.5h, centrifuging at 9000r/min for 10min, and measuring phage titer in supernatant corresponding to different MOI values by double-layer plate method. The experiment was set up in 3 replicates.

As shown in fig. 3A, when the MOI of the phage is 10, the phage titer is maximized, i.e., the optimal multiplicity of infection of the phage is 10, which indicates that more phage can be proliferated when the number of phage and host bacteria is mixed in a ratio of 10: 1.

3. Adsorption rate

Mixing fresh phage liquid and host bacteria suspension in a centrifuge tube according to the optimal MOI value, and performing shake cultivation at 37 ℃. The titer of phage in the supernatant was determined by the double-plate method every 5min, starting from 0 min. The experiment was set up in 3 replicates. The adsorption rate was 1- (phage titer not adsorbed/phage titer at 0min per time point) × 100%, and the results of the adsorption rate of phage to host bacteria are shown in fig. 3B.

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

4. One step growth curve

The one-step growth curve of the phage reflects the growth rule. The phage and host bacteria were mixed at the optimum MOI value, incubated at 37 ℃ for 25min to allow the phage to adsorb to the bacteria, then centrifuged at 8000r/min for 2min at4 ℃, the supernatant was discarded, and resuspended twice with an equal volume of LB to remove unadsorbed phage. Adding the liquid into 9mL LB culture medium, shaking and culturing at 37 ℃ in a shaking table, sampling 300 mu L every 10min from 0min, centrifuging at 8000r/min for 2min, and measuring the titer of the phage in the supernatant by adopting a double-layer plate method. The experiment was set up in 3 replicates. The incubation period, lysis amount of phage (lysis amount ═ end phase of lysis phage titer/initial host bacteria concentration at the time of infection) can be seen from the one-step growth plot (fig. 3C). The latency period of the phage SEP37 was 40min, the lysis period was 140min, and the lysis amount was 13.7PFU/cell.

5. Phage SEP37 thermostability assay

The phage stock was diluted to a titer of 1X 10⁷PFU/mL, and is divided into 1mL sterile centrifuge tubes, the centrifuge tubes are respectively placed in a constant temperature water bath kettle with the temperature of 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃, and the phage titer in each tube is respectively measured at the time of 0min, 30min and 60 min. The experiment was set up in 3 replicates. As can be seen from FIG. 3D, the activity of the phage SEP37 remained substantially unchanged between 30 ℃ and 50 ℃, gradually decreased with time when the temperature was increased to 60 ℃, and already started to decrease significantly in a short time when the temperature exceeded 70 ℃.

6. Phage SEP37 pH stability assay

Phage suspensions of known titer (1X 10)⁷PFU/mL) 100. mu.L of the phage suspension was added to 900. mu.L of PBS buffer solution with different pH values (2-13), and the phage titer in each centrifuge tube was measured after placing the buffer solution in a water bath at 37 ℃ for 2 hours. The experiment was set up in 3 replicates. As can be seen from FIG. 3E, the phage maintains a stable titer at pH 4-11, the titer decreases at pH 3 and pH 12, and the titer substantially decreases to 0 at pH 2 and pH 13, indicating that strong acid and strong base directly destroy the activity of the phage.

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

1. Preparation of working electrode (GDE-AuNPs-Cys-PhageSEP37-BSA) composite electrode

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

mechanically polishing the bare GDE to mirror finish by using 0.3 mu m and 0.05 mu m of alumina/cement slurry on polishing cloth respectively, and then performing thorough ultrasonic cleaning by using deionized water, acetone and ethanol respectively; then at 0.5mol/L H₂SO₄Circularly scanning the solution for 100 times at a voltage of 100mV/s between-0.2 and 1.0V, electrochemically cleaning the electrode, rinsing with deionized water, and using N₂Drying by airflow;

2) dripping gold nanoparticles with the diameter of 25-30 nm to the working surface of the electrode to obtain GDE-AuNPs

3) After drying in a clean environment, the gold disk electrode GDE-AuNPs carrying AuNPs is immersed in mercaptoethylamine solution at4 ℃ and 1mmol/L for 12h, and under the interaction of thiol-gold, a self-assembled monolayer structure is formed, namely: GDE-AuNPs-Cys.

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

a. preparation of activated phage SEP37 suspension:

0.08mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (1-ethyl- (3-dimethylamino) carbodiimide, EDC) and 0.22mg of N-Hydroxysuccinimide (N-Hydroxysuccinimide, NHS) were added to the phage-containing solution (5X 10) in this order at intervals of 30min¹⁰PFU/mL) in 1mL of PBS to obtain activated phage SEP37 suspension;

b. immersing the GDE-AuNPs-Cys into the activated phage SEP37 suspension, incubating for 4h at4 ℃, and then rinsing with deionized water to remove the phage with weak binding;

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

GDE-AuNPs-Cys-PhageSEP37-BSA), stored in PBS solution at4 ℃ for subsequent experiments.

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

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

1) Determining the loading capacity of the gold nanoparticles on the gold electrode; respectively dripping 2.5 mu L, 5 mu L and 7.5 mu L of colloidal gold with the diameter of 25-30 nm on the surface of the electrode, and observing the loading condition of the gold nanoparticles by using a scanning electron microscope;

2) determining self-assembly time of mercaptoethylamine; the immersion time of the electrode carrying the AuNPs in the mercaptoethylamine solution was changed to 6h, 12h, 18h, 24h, 30h and 36h, and the corresponding Rct was recorded, and the reduction of the Rct was defined as Δ Rct ═ Rct₀–Rct₁(Rct₀Is the Rct value of the composite electrode before self-assembly of mercaptoethylamine₁Rct value of the composite electrode after self-assembly of mercaptoethylamine).

3) Determining the incubation time of the phage; the incubation times of the phages were varied to 1h, 2h, 3h, 4h, 5h, 6h and the corresponding Rct was recorded, the increase in Rct being defined as Δ Rct ═ Rct₂–Rct₁(Rct₂Rct values for the composite electrode after phage incubation).

4) Determining the BSA incubation time; phage-bearing electrodes were incubated in 2% BSA for 10min, 20min, 30min, 40min, 50min, 60min and the corresponding Rct recorded, increasing Rct can be defined as Δ Rct ═ Rct₃–Rct₂(Rct₃Rct values for the composite electrodes after BSA incubation).

The results are shown in FIG. 5: SEM results show that 7.5. mu.L (FIG. 5C) of colloidal gold was added dropwise, and the gold nanoparticles were adsorbed most uniformly and densely.

The results are shown in FIG. 6: (A) the result shows that when the self-assembly time of mercaptoethylamine exceeds 12h, the delta Rct is basically kept unchanged; (B) shows that when the phage SEP37 is incubated for 4h, the Δ Rct is gradually stabilized; (C) it was shown that Δ Rct did not rise any more and began to stabilize when BSA blocked for 30 min.

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

Summary the optimal preparation conditions for the GDE-AuNPs-Cys-PhageSEP37-BSA biosensor were: the GDE surface was dripped with 7.5. mu.L of colloidal gold solution, followed by self-assembly in 1mmol/L cysteamine solution for 12h, co-incubation with phage SEP37 for 4h, and final blocking with 2% BSA for 30 min.

3. Electrochemical impedance spectroscopy 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-PARSTAT4000A electrochemical analyzer 3; the electrochemical analyzer is communicated with the upper computer;

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

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

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

1. Determination of incubation time of bacteria

To determine when the phage recognizes and captures the host bacteria, the biosensor is contacted with a 5X 10⁵CFU/mL salmonella enteritidis ATCC13076 cells were incubated for 10min, 15min, 20min, 25min, 30min, 35min, and then Rct was measured, the increment of Rct being defined as Δ Rct ═ Rct₄–Ret₃(Rct₄To identify Rct values, Ret, after Salmonella enteritidis ATCC13076 cells₃Initial Rct value for composite electrode)

The results are shown in FIG. 6: (D) it is shown that Δ Rct increases rapidly at 20-25 min and then remains stable for 5-10 min, which is attributed to the fact that the bacteriophage SEP 37-based biosensor recognizes and immobilizes Salmonella at an early stage, but almost all of the phage recognition sites are occupied over time and no longer recognize and immobilize bacteria, so the biosensor is incubated with the detection bacterial solution for 30 min.

2. Detection of Salmonella enteritidis at different concentrations

Sequentially immersing the biosensor in a solution containing 1 × 10¹～1×10⁸Incubating 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, and maintaining the measurement process in a nitrogen atmosphere; when the solution to be detected is replaced, the combination of the phage and the host bacteria on the composite electrode in the previous step is firstly destroyed by alkalescent solution, and the electrode is slowly washed by deionized water so as to recover the response before the bacteria detection.

The results are shown in FIG. 9: (A) is the measurement result of EIS; (B) is the result of Rct value fitted with software VSimpVin 3.60 and further processed when the bacterial concentration is 10¹～10⁶In the CFU/mL range, the value of the delta Rct is linearly related to the logarithm of the concentration of the salmonella enteritidis, and the regression equation is that y is 796.89x-484.77(R is²0.9829), the detection limit is 17CFU/mL, and the detection time is 25 min; (C) capture concentration for sensor 1 × 10³SEM picture of CFU/mL Salmonella enteritidis ATCC 13076; (D) capture concentration for sensor 1 × 10⁵SEM pictures and magnified pictures of CFU/mL Salmonella enteritidis ATCC 13076.

Example 5: bacteriophage-based electrochemical impedance spectroscopy biosensor detection specificity test for salmonella detection

By mixing the biosensors with the same concentration (5X 10)⁵CFU/mL) and some non-salmonella strains were incubated together to assess the specificity of the sensor. The salmonella strains comprise 4 strains of salmonella enteritidis, 4 strains of salmonella typhimurium and 6 strains of other serotypes; the non-salmonella strains include listeria monocytogenes strain 2, staphylococcus aureus strain 2, and 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 caused little or no increase in impedance, consistent with the blank, indicating that the sensor was highly specific in recognizing only salmonella and not other bacteria.

Example 6: electrochemical impedance spectroscopy biosensor detection stability test for detecting salmonella based on bacteriophage

By comparing the same concentrations (5X 10) at4 ℃ and 23 ℃ over 6 weeks⁵CFU/mL) to evaluate the stability of the sensor.

The results are shown in FIG. 11: Δ Rct remained essentially unchanged for the first three weeks, began to decline at week 4, and declined to 90% at week 6; at4 ℃ and 23 ℃, Δ Rct is not significantly different; the sensor maintains a higher stability than other types of biosensors.

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

Respectively carrying out labeling treatment on lake water, lettuce and chicken breast samples which do not contain the bacteria to be detected. Firstly, simply filtering lake water, processing lettuce and chicken breast samples according to national standard, and then adding salmonella enteritidis ATCC13076 into the lake water, the lettuce and the chicken breast respectively to make the final concentration of 10¹、10²、10³、10⁴、10⁵And 10⁶CFU/mL; another sample without a label was taken as a control; measurements were made as in example 3.

The results are shown in FIG. 12: (A) and (B) and (C) are EIS measurement results, the EIS response of the three samples is increased along with the increase of the bacterial concentration, and the biosensor can detect the salmonella in the samples. (D) Is the result of Rct value fitted with software VSimpVin 3.60 and further processed, the EIS response of lake water and lettuce is nearly identical to that of PBS group; the fit curve of lake water is y-931.75 x-608.58, R2-0.9899; the fitted curve of the lettuce is y-1073.3 x-359.09, and R2-0.9931; the linear range of both samples was 1X 10¹～1×10⁶CFU/mL; the fitted curve of the chicken breast is 1190.8x-159.97, R2 is 0.9877, and the linear range is 1 × 10²～1×10⁵CFU/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 microbial culture method, and the results show that the detection results of the biosensor are not obviously different from the detection results of the microbial culture method. The results prove that the sensor can accurately detect and quantify the salmonella in different sample matrixes, so that 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 the potential of being applied to actual sample detection.

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. A Salmonella enteritidis bacteriophage (Salmonella enteritidis bacteriophage) SEP37, accession number: CCTCC NO: m20211127.

2. A phage-based electrochemical impedance spectroscopy biosensor for detecting Salmonella, comprising: the sensor uses the bacteriophage of claim 1 as a biological recognition element, and the sensor is composed of a measuring device comprising three electrodes, a nitrogen supply device, and a P4000A-PARSTAT4000A electrochemical analyzer, which is in communication with a superior computer.

3. The electrochemical impedance spectroscopy biosensor of claim 2, wherein: 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 respectively a working electrode, a platinum wire counter electrode and an Ag/AgCl reference electrode.

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

5) And finally, co-incubating the GDE-AuNPs-Cys-PhageSEP37 and 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 disc electrode is 2mm,

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

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

the titer of salmonella enteritidis phage SEP37 used was: 5X 10¹⁰PFU/mL，

The mass fraction of bovine serum albumin was 2%.

6. The phage-based electrochemical impedance spectroscopy biosensor for detecting salmonella of claim 3, wherein: the redox probe is a mixed aqueous solution containing potassium ferricyanide, potassium ferrocyanide and potassium chloride; wherein, the molar concentrations of the potassium ferricyanide and the potassium ferrocyanide in the mixed aqueous solution are both 0.5mmol/L, and the molar concentration of the potassium chloride is 0.1 mol/L.

7. A method for detecting salmonella by an electrochemical impedance spectroscopy biosensor is characterized by comprising the following steps: the method comprises the following steps:

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

8. The method of claim 7, wherein: the operating parameters of the electrochemical analyzer are as follows: the amplitude of the AC perturbation was 10 mV.

9. The method of claim 7, wherein: the detection frequency of the electrochemical analyzer is 0.1-10⁵Hz。