阅读说明：本技术 基于抗菌肽-磷酸盐纳米复合物的致病菌免疫分析生物传感器及其制备方法和应用 (Based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor and its preparation method and application ) 是由 万家余 柏华松 韩烨 于 2019-10-16 设计创作，主要内容包括：基于抗菌肽-磷酸盐纳米复合物的致病菌免疫分析生物传感器及其制备方法和应用,涉及食源性致病菌检测领域。该生物传感器包括Fe<Sub>3</Sub>O<Sub>4</Sub>-Magainins I磷酸铜纳米复合物和Cecropin P1-转化酶磷酸钙纳米复合物。通过合成两种抗菌肽-磷酸盐纳米复合物,以Fe<Sub>3</Sub>O<Sub>4</Sub>-Magainins I磷酸铜纳米复合物为捕获元件进行细菌磁性分离,以Cecropin P1-转化酶磷酸钙纳米复合物为信号转导元件；加入食源性致病菌目标物与两个纳米复合物形成夹心免疫结构传感器；磁性分离后信号转导元件中的蔗糖转化酶水解蔗糖为葡萄糖,血糖仪直接定量。本发明灵敏度高、特异性强、精确性高、操作简单、检测快速、成本低。(Based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor and its preparation method and application, it is related to food-borne pathogens detection field.The biosensor includes Fe 3 O 4 - Magainins I cupric phosphate nano-complex and Cecropin P1- invertase calcium phosphate nano compound.By synthesizing two kinds of antibacterial peptides-phosphate nano compound, with Fe 3 O 4 - Magainins I cupric phosphate nano-complex is that capture element carries out bacterium Magnetic Isolation, using Cecropin P1- invertase calcium phosphate nano compound as Signal Transduction Components；Food-borne pathogens object is added and two nano-complexes form sandwich immunoassay structure sensor；Invertase sucrose hydrolysis after Magnetic Isolation in Signal Transduction Components is glucose, blood glucose meter direct quantitative.High sensitivity of the present invention, high specificity, accuracy are high, easy to operate, detection is quick, at low cost.)

1. based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor characterized by comprising

Fe₃O₄- Magainins I cupric phosphate nano-complex；

Cecropin P1- invertase calcium phosphate nano compound.

2. as described in claim 1 based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor Preparation method, which comprises the following steps:

Step 1: synthesis Fe₃O₄- Magainins I cupric phosphate nano-complex；

Step 2: synthesis CecropinP1- invertase calcium phosphate nano compound.

3. preparation method according to claim 2, which is characterized in that in step 1, Fe₃O₄- Magainins I cupric phosphate The synthetic method of nano-complex specifically includes the following steps:

(1) 10mg Fe is taken₃O₄It is added in the PBS of 8mL, 10mM, pH7.4 with 0.6mg antibacterial peptide Magainins I；

(2) copper-bath of 400 μ L, 120mM is added, is settled to 10mL using ultrapure water, concussion is mixed, is incubated at 25 DEG C 16 hours；

(3) 12000 revs/min of centrifugation 5min generate black precipitate, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, gained freezes Dry powder is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH7.4, gained Fe₃O₄- Magainins I cupric phosphate is nano combined The final concentration of 5mg/ml of object.

4. preparation method according to claim 3, which is characterized in that the amino acid sequence of the antibacterial peptide Magainin I For GIGKFLHSAGKFGKAFVGEIMKS.

5. preparation method according to claim 2, which is characterized in that in step 2, Cecropin P1- invertase phosphoric acid The synthetic method of calcium nano-complex specifically includes the following steps:

(1) 0.4mg antibacterial peptide CecropinP1 and 3.6mg invertase is taken to be added in the PBS of 8mL, 4mM, pH7.0；

(2) CaCl of 160 μ L, 200mM is added₂Solution is settled to 10mL using ultrapure water, is uniformly mixed, by mixed liquor at 25 DEG C It is lower to stand 12 hours；

(3) 12000 revs/min of centrifugation 5min generate white depositions, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, gained freezes Dry powder is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH 7.0, gained Cecropin P1- invertase calcium phosphate nano is multiple Close the final concentration of 2.5mg/ml of object.

6. preparation method according to claim 5, which is characterized in that the amino acid sequence of the antibacterial peptide Cecropin P1 It is classified as SWLSKTAKKLENSAKKRISEGIAIAIQGGPR.

7. as described in claim 1 based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor Detection method, which comprises the following steps:

Step 1: synthesis Fe₃O₄- Magainins I cupric phosphate nano-complex；

Step 2: synthesis CecropinP1- invertase calcium phosphate nano compound；

Step 3: preparing Escherichia coli O 157: H7 sample；

Step 4: utilizing Fe₃O₄- Magainins I cupric phosphate nano-complex, Cecropin P1- invertase calcium phosphate nano Compound is to Escherichia coli O 157: H7 sample detects.

8. detection method according to claim 7, which is characterized in that in step 1, Fe₃O₄- Magainins I cupric phosphate The synthetic method of nano-complex specifically includes the following steps:

(1) 10mg Fe is taken₃O₄It is added in the PBS of 8mL, 10mM, pH7.4 with 0.6mg antibacterial peptide Magainins I；

(2) copper-bath of 400 μ L, 120mM is added, is settled to 10mL using ultrapure water, concussion is mixed, is incubated at 25 DEG C 16 hours；

(3) 12000 revs/min of centrifugation 5min generate black precipitate, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, gained freezes Dry powder is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH7.4, gained Fe₃O₄- Magainins I cupric phosphate is nano combined The final concentration of 5mg/ml of object.

9. detection method according to claim 7, which is characterized in that in step 2, Cecropin P1- invertase phosphoric acid The synthetic method of calcium nano-complex specifically includes the following steps:

(1) 0.4mg antibacterial peptide CecropinP1 and 3.6mg invertase is taken to be added in the PBS of 8mL, 4mM, pH7.0；

(2) CaCl of 160 μ L, 200mM is added₂Solution is settled to 10mL using ultrapure water, is uniformly mixed, by mixed liquor at 25 DEG C It is lower to stand 12 hours；

(3) 12000 revs/min of centrifugation 5min generate white depositions, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, gained freezes Dry powder is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH 7.0, gained Cecropin P1- invertase calcium phosphate nano is multiple Close the final concentration of 2.5mg/ml of object.

10. detection method according to claim 7, which is characterized in that step 3 and step 4 specifically includes the following steps:

(1) by Escherichia coli O 157: H7 bacterial strain is incubated overnight in Luria-Bertani cultured solution of broth in 37 DEG C, 4500rpm Be centrifuged 10min, PBS is washed twice, and is resuspended in the PBS of 1mM, pH 7.4, by bacterial suspension in PBS serial dilution 10 Times；

(2) by the Escherichia coli O 157 of 100 μ L various concentrations: H7 suspension is added in centrifuge tube, and PBS buffer solution is made negative right According to the Fe of 6 μ L of addition, 5mg/mL₃O₄The Cecropin of-Magainins I cupric phosphate nano-complex and 10 μ L, 2.5mg/ml P1- invertase calcium phosphate nano compound is uniformly mixed, 90min is cultivated at 37 DEG C, to form Fe₃O₄- Magainins I phosphorus Sour copper nano-complex-Escherichia coli O 157: H7-CecropinP1- invertase calcium phosphate nano compound is conjugated sandwich immunoassay Compound；

(3) it after Magneto separate, is washed twice with the PBS of 1mM, pH 8.5 to remove unbonded bacterial cell and non-specific binding Part obtains sandwich style immune complex；

(4) sucrose solution of 10 μ L, 500mM, pH 4.5 are added in sandwich style immune complex, are incubated at 55 DEG C 20min takes out 5 μ L supernatants, and carry out glucose assays, PGM signal and Escherichia coli O 157 using blood glucose meter PGM: H7 is generated Quantitative relationship.

Technical field

The present invention relates to food-borne pathogens detection technique fields, and in particular to one kind is based on antibacterial peptide-phosphate nano Pathogenic bacteria immunoassay biosensor of compound and its preparation method and application.

Background technique

Pathogenic bacteria infection constitutes a serious threat to global public health, and high incidence and high mortality give social band Huge financial burden is carried out.Effectively to prevent pathogenic bacterial infection, carry out point-of care (POC) pathogeny detection to clinical diagnosis, Food Inspection and environmental monitoring are of great significance, and especially in world low developed area, meaning is huger.It is traditional based on The method of culture is always the goldstandard of Analysis of pathogenic bacteria.But these methods often time and effort consuming does not meet pathogen POC detection Requirement.Polymerase chain reaction (PCR) program is also widely used for qualitatively and quantitatively analysis of pathogenic bacteria.However, external source pollution can It can lead to false positive or negative findings.Equally, nucleic acid needs to extract, and the portability of technology is also restrained.

In order to establish a kind of simple, quick, sensitive detection of pathogens method, based on Molecular recognition of medicine (such as antibody and Aptamer) POC immunoassays method for biosensor development, cause various circles of society and more and more pay close attention to.Based on antibody Sandwich immunoassays due to its high degree of specificity antigen binding ability and become currently used platform.However, antibody Preparation process is troublesome, and production cost is also very high.Aptamer is also proposed as substitution molecular recognition elements at present.With antibody It is compared with aptamers, peptide has higher antitypy and stability.Peptide also has short and clearly structure, they are to many analyses Object (such as protein, nucleic acid and metal ion) has specificity and strong affinity.For its property, peptide as develop it is sensitive, Effective recognition component of convenient biosensor receives significant attention.

A kind of antibacterial defence system of the antibacterial peptide (AMPs) as organism has height to high antigenic bacterial cell surface The affinity and specificity of degree.So far, many technologies have been used for (such as fluorogen, enzyme and receiving peptide and signal tracer Rice grain) it combines or peptide is fixed on carrier surface (such as magnetic bead, glass slide, graphene and gold electrode).However, these methods need The chemical group derivatization of AMPs is wanted to combine carrier or chemical modification with marking signal marker.The a large amount of examinations of these methods consumption Agent, it is expensive, and preparation process is excessively complicated.If using inappropriate immobilization scheme, AMP activity be may be subjected to Damage, for example, due to AMPs N- terminal derivative and cause to decline bacterium affinity, or due to AMPs excess marker Amino specific biological element causes its microorganism to combine active forfeiture.The bottleneck that in order to solve, should design one kind simply has The method of effect to realize that peptide is coupled large biological molecule (such as protein, enzyme), and is integrated to carrier surface (such as magnetic bead, glass slide Deng), while keeping the activity of peptide.

Summary of the invention

The object of the present invention is to provide a kind of high sensitivity, high specific, high precision, it is easy to operate, detection quickly, at This is low based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor and preparation method thereof and answering With.

Used technical solution is as follows in order to solve the technical problem by the present invention:

Of the invention is a kind of based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor, main Include:

Fe₃O₄- Magainins I cupric phosphate nano-complex；

Cecropin P1- invertase calcium phosphate nano compound.

A kind of system based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor of the invention Preparation Method mainly comprises the steps that

Step 1: synthesis Fe₃O₄- Magainins I cupric phosphate nano-complex；

Step 2: synthesis CecropinP1- invertase calcium phosphate nano compound.

As preferred embodiment, in step 1, Fe₃O₄The synthesis side of-Magainins I cupric phosphate nano-complex Method specifically includes the following steps:

(1) 10mg Fe is taken₃O₄It is added in the PBS of 8mL, 10mM, pH7.4 with 0.6mg antibacterial peptide Magainins I；

(2) copper-bath of 400 μ L, 120mM is added, is settled to 10mL using ultrapure water, concussion mixes, at 25 DEG C It is incubated for 16 hours；

(3) 12000 revs/min of centrifugation 5min generate black precipitate, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, institute It obtains freeze-dried powder and is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH7.4, gained Fe₃O₄- Magainins I cupric phosphate nanometer The final concentration of 5mg/ml of compound.

As preferred embodiment, the amino acid sequence of the antibacterial peptide Magainin I is GIGKFLHSAGKFGKAFVGEIMKS。

As preferred embodiment, in step 2, the synthesis of Cecropin P1- invertase calcium phosphate nano compound Method specifically includes the following steps:

(1) 0.4mg antibacterial peptide CecropinP1 and 3.6mg invertase is taken to be added to the PBS of 8mL, 4mM, pH7.0 In；

(2) CaCl of 160 μ L, 200mM is added₂Solution is settled to 10mL using ultrapure water, is uniformly mixed, by mixed liquor 12 hours are stood at 25 DEG C；

(3) 12000 revs/min of centrifugation 5min generate white depositions, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, institute It obtains freeze-dried powder and is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH 7.0, gained Cecropin P1- invertase calcium phosphate is received The final concentration of 2.5mg/ml of rice compound.

As preferred embodiment, the amino acid sequence of the antibacterial peptide Cecropin P1 is SWLSKTAKKLENSAKKRISEGIAIAIQGGPR。

A kind of inspection based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor of the invention Survey method, comprising the following steps:

Step 1: synthesis Fe₃O₄- Magainins I cupric phosphate nano-complex；

Step 2: synthesis CecropinP1- invertase calcium phosphate nano compound；

Step 3: preparing Escherichia coli O 157: H7 sample；

Step 4: utilizing Fe₃O₄- Magainins I cupric phosphate nano-complex, Cecropin P1- invertase calcium phosphate Nano-complex is to Escherichia coli O 157: H7 sample detects.

As preferred embodiment, in step 1, Fe₃O₄The synthesis side of-Magainins I cupric phosphate nano-complex Method specifically includes the following steps:

(1) 10mg Fe is taken₃O₄It is added in the PBS of 8mL, 10mM, pH7.4 with 0.6mg antibacterial peptide Magainins I；

(2) copper-bath of 400 μ L, 120mM is added, is settled to 10mL using ultrapure water, concussion mixes, at 25 DEG C It is incubated for 16 hours；

(3) 12000 revs/min of centrifugation 5min generate black precipitate, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, institute It obtains freeze-dried powder and is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH7.4, gained Fe₃O₄- Magainins I cupric phosphate nanometer The final concentration of 5mg/ml of compound.

As preferred embodiment, in step 2, the synthesis of Cecropin P1- invertase calcium phosphate nano compound Method specifically includes the following steps:

(1) 0.4mg antibacterial peptide CecropinP1 and 3.6mg invertase is taken to be added to the PBS of 8mL, 4mM, pH7.0 In；

(2) CaCl of 160 μ L, 200mM is added₂Solution is settled to 10mL using ultrapure water, is uniformly mixed, by mixed liquor 12 hours are stood at 25 DEG C；

(3) 12000 revs/min of centrifugation 5min generate white depositions, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, institute It obtains freeze-dried powder and is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH 7.0, gained Cecropin P1- invertase calcium phosphate is received The final concentration of 2.5mg/ml of rice compound.

As preferred embodiment, step 3 and step 4 specifically includes the following steps:

(1) by Escherichia coli O 157: H7 bacterial strain is incubated overnight in Luria-Bertani cultured solution of broth in 37 DEG C, 4500rpm is centrifuged 10min, and PBS is washed twice, and is resuspended in the PBS of 1mM, pH 7.4, and bacterial suspension is connected in PBS Continuous 10 times of dilution；

(2) by the Escherichia coli O 157 of 100 μ L various concentrations: H7 suspension is added in centrifuge tube, and PBS buffer is made The Fe of 6 μ L, 5mg/mL is added in negative control₃O₄- Magainins I cupric phosphate nano-complex and 10 μ L, 2.5mg/ml Cecropin P1- invertase calcium phosphate nano compound is uniformly mixed, 90min is cultivated at 37 DEG C, to form Fe₃O₄- Magainins I cupric phosphate nano-complex-Escherichia coli O 157: H7-CecropinP1- invertase calcium phosphate nano is compound Object is conjugated sandwich immunoassay compound；

(3) it after Magneto separate, is washed twice with the PBS of 1mM, pH 8.5 to remove unbonded bacterial cell and non-specificity Bound fraction obtains sandwich style immune complex；

(4) sucrose solution of 10 μ L, 500mM, pH 4.5 are added in sandwich style immune complex, are incubated at 55 DEG C 20min is educated, 5 μ L supernatants are taken out, carry out glucose assays, PGM signal and Escherichia coli O 157 using blood glucose meter PGM: H7 is produced Raw quantitative relationship.

Inventive principle: the present invention uses Fe₃O₄The Magainins I-Cu of insertion₃(PO₄)₂Flower-like nanometer composite material (Fe₃O₄- Magainins I cupric phosphate nano-complex, abbreviation MMP) it is used as capture element, it is used for Escherichia coli O 157: H7's Magnetic Isolation；Cecropin P1- invertase-Ca is used simultaneously₃(PO₄)₂(the Cecropin P1- conversion of flower-like nanometer composite material Enzyme calcium phosphate nano compound, abbreviation PIP) as the Signal Transduction Components in sensor sandwich immunoassay composite construction；In large intestine In the presence of bacillus O157:H7, conjugation sandwich immunoassay is formd between two phosphate nano compounds based on antibacterial peptide Compound；Sucrose hydrolysis is glucose by the invertase being conjugated in sandwich immunoassay compound.Therefore, which reflects Escherichia coli O 157: the level of H7 can carry out reliably quantitative on blood glucose meter PGM.

A kind of exempted from based on antibacterial peptide-phosphate nano compound pathogenic bacteria the beneficial effects of the present invention are: of the invention Epidemic disease analyzes biosensor, two kinds of antibacterial peptides-phosphate nano compound is mainly utilized, wherein with Fe₃O₄-Magainins I Cupric phosphate nano-complex is that capture element carries out bacterium Magnetic Isolation, compound with Cecropin P1- invertase calcium phosphate nano Object is as Signal Transduction Components.Antibacterial peptide Magainins I and antibacterial peptide CecropinP1 all have excellent Escherichia coli O157:H7 binding ability, therefore it is chosen as the identification molecule of sandwich style immunoassay biosensor.

The present invention is prepared for AMPs- phosphate nano-composite material pair in mild one kettle way, and passes through AMPs- phosphorus Silicate nanometer composite material realizes signal-obtaining to interlayer structure and personal blood glucose meter (PGM).Compared with prior art, of the invention It has the advantage that

(1) highly sensitive.The present invention is established and is based on by the way that cation A MPs self assembly to be fixed on phosphate crystal The sandwich style immunoassay biosensor of nano-complex pair, detection limit are about 100 times or more of existing method.Sun Ion peptide is preferentially adsorbed on negatively charged bacterial cell membrane by the positive charge of side chain amino acid, cation A MPs from group Dress process will not reduce the net positive charge on side chain, and in this process, peptide molecule passes through amide groups in metal ion and peptide chain Coordination forms complex compound with phosphate.Therefore, still as the cation A MPs of the nanocomposite centering of bacterium identification molecule Outstanding electrostatic interaction is kept with anion bacterial membrane.Since nano-complex is deposited with the aggregate form of AMP abundant , therefore the present invention is made to become a kind of more effective molecular-recognition biosensors for immunoassays.In addition, mild load Body (phosphate) provides enough biocompatibility sites, and the immobilization requirement of AMP is met in a manner of being freely orientable, from And the identification activity of AMP is effectively kept.Equally, the high-specific surface area structure of nano-complex pair has given full play to AMPs peptide The accessibility that chain combines the targeting of bacterial cell.These factors ensure the sandwich immunoassays based on nano-complex pair Bio-sensor system is sensitive to target detection.

(2) high specific.Other bacteriums in the present invention other than object are noiseless to this detection architecture, this is because this The building of the sensor of invention is built upon on the basis of Ag-Ab specific recognition.Amphipathic cationic antibacterial peptide Magainins I keeps high specific to Gram-negative enteropathogenic E. Coli O157:H7.

(3) high precision.Testing result of the present invention is accurate, reliable, rate of recovery 86.5%-117%, relative standard deviation For 2.9%-6.9%.

(4) easy to operate, detection is quickly.After the present invention synthesizes double antibacterial peptide-phosphate nano compounds, food is only added Borne pathogen sample realizes that " step is incubated for method ", PGM Rapid reading are detected two steps and be can be completed.The present invention is almost not required to Other extra times are wanted, detection is rapidly completed, simple to operate.

(5) at low cost.Due to no longer adding other expensive reagents in the present invention, preparation and testing cost is greatly saved.

Detailed description of the invention

Fig. 1 is Fe prepared by embodiment 1₃O₄The SEM of-Magainins I cupric phosphate nano-complex (MMP) schemes and XRD Peak figure.

Fig. 2 be embodiment 2 prepare Cecropin P1- invertase calcium phosphate nano compound (PIP) SEM figure and XRD peak figure.

Fig. 3 is the catalytic activity point for the Cecropin P1- invertase calcium phosphate nano compound (PIP) that embodiment 2 synthesizes Analyse result and holding time activity analysis result.

Fig. 4 is that the present invention applies antibacterial peptide-phosphate nano compound pair sandwich immunoassay quickly to detect food source The schematic diagram of property pathogenic bacteria.

Fig. 5 various concentration Escherichia coli O 157: the PGM signal value and Escherichia coli O 157 of H7: the line of the log concentration of H7 Sexual intercourse figure.

Fig. 6 concentration is 10⁵The comparison diagram of the PGM signal value of the different strains of CFU/mL.

Specific embodiment

Of the invention is a kind of based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor, main Include:

Fe₃O₄- Magainins I cupric phosphate nano-complex (MMP)；

Cecropin P1- invertase calcium phosphate nano compound (PIP).

A kind of system based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor of the invention Preparation Method mainly comprises the steps that

Step 1: synthesis Fe₃O₄- Magainins I cupric phosphate nano-complex (MMP)

(1) 10mg Fe is taken₃O₄It is added in the PBS of 8mL, 10mM, pH7.4 with 0.6mg antibacterial peptide Magainins I；

(2) copper-bath of 400 μ L, 120mM is added, is settled to 10mL using ultrapure water, concussion mixes, at 25 DEG C It is incubated for 16 hours；

(3) 12000 revs/min of centrifugation 5min generate black precipitate, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, institute It obtains freeze-dried powder and is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH7.4, gained Fe₃O₄- Magainins I cupric phosphate nanometer The final concentration of 5mg/ml of compound.

Wherein, the amino acid sequence of antibacterial peptide Magainin I is GIGKFLHSAGKFGKAFVGEIMK S.

Step 2: synthesis CecropinP1- invertase calcium phosphate nano compound (PIP)

(1) 0.4mg antibacterial peptide CecropinP1 and 3.6mg invertase is taken to be added to the PBS of 8mL, 4mM, pH7.0 In；

(2) CaCl of 160 μ L, 200mM is added₂Solution is settled to 10mL using ultrapure water, is uniformly mixed, by mixed liquor 12 hours are stood at 25 DEG C；

(3) 12000 revs/min of centrifugation 5min generate white depositions, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, institute It obtains freeze-dried powder and is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH 7.0, gained Cecropin P1- invertase calcium phosphate is received The final concentration of 2.5mg/ml of rice compound.

Wherein, the amino acid sequence of antibacterial peptide Cecropin P1 is SWLSKTAKKLENSAKKRISEG IAIAIQGGPR.

A kind of inspection based on antibacterial peptide-phosphate nano compound pathogenic bacteria immunoassay biosensor of the invention Survey method, comprising the following steps:

Step 1: synthesis Fe₃O₄- Magainins I cupric phosphate nano-complex (MMP)

(1) 10mg Fe is taken₃O₄It is added in the PBS of 8mL, 10mM, pH7.4 with 0.6mg antibacterial peptide Magainins I；

(2) copper-bath of 400 μ L, 120mM is added, is settled to 10mL using ultrapure water, concussion mixes, at 25 DEG C It is incubated for 16 hours；

(3) 12000 revs/min of centrifugation 5min generate black precipitate, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, institute It obtains freeze-dried powder and is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH7.4, gained Fe₃O₄- Magainins I cupric phosphate nanometer The final concentration of 5mg/ml of compound.

Step 2: synthesis CecropinP1- invertase calcium phosphate nano compound (PIP)

(1) 0.4mg antibacterial peptide CecropinP1 and 3.6mg invertase is taken to be added to the PBS of 8mL, 4mM, pH7.0 In；

(2) CaCl of 160 μ L, 200mM is added₂Solution is settled to 10mL using ultrapure water, is uniformly mixed, by mixed liquor 12 hours are stood at 25 DEG C；

(3) 12000 revs/min of centrifugation 5min generate white depositions, and twice, centrifugation removes supernatant to pure water；

(4) addition 5mL pure water, which is placed at -20 DEG C, freezes, and is subsequently placed in vacuum freeze drier and is freeze-dried, institute It obtains freeze-dried powder and is placed in -20 DEG C of preservations；

(5) freeze-dried powder is resuspended in the PBS of 1mM, pH 7.0, gained Cecropin P1- invertase calcium phosphate is received The final concentration of 2.5mg/ml of rice compound.

Step 3: preparing Escherichia coli O 157: H7 sample

By Escherichia coli O 157: H7 bacterial strain is incubated overnight in Luria-Bertani cultured solution of broth in 37 DEG C, 4500rpm is centrifuged 10min, and PBS is washed twice, and is resuspended in the PBS of 1mM, pH 7.4, and bacterial suspension is connected in PBS Continuous 10 times of dilution, it is spare.

Step 4: utilizing Fe₃O₄- Magainins I cupric phosphate nano-complex, Cecropin P1- invertase calcium phosphate Nano-complex is to Escherichia coli O 157: H7 sample detects:

(1) by the Escherichia coli O 157 of 100 μ L various concentrations: H7 suspension is added in centrifuge tube, and PBS buffer is made The Fe of 6 μ L, 5mg/mL is added in negative control₃O₄- Magainins I cupric phosphate nano-complex and 10 μ L, 2.5mg/ml Cecropin P1- invertase calcium phosphate nano compound is uniformly mixed, 90min is cultivated at 37 DEG C, to form Fe₃O₄- Magainins I cupric phosphate nano-complex-Escherichia coli O 157: H7-CecropinP1- invertase calcium phosphate nano is compound Object is conjugated sandwich immunoassay compound；

(2) it after Magneto separate, is washed twice with the PBS of 1mM, pH 8.5 to remove unbonded bacterial cell and non-specificity Bound fraction obtains sandwich style immune complex；

(3) sucrose solution of 10 μ L, 500mM, pH 4.5 are added in sandwich style immune complex, are incubated at 55 DEG C 20min is educated, 5 μ L supernatants are taken out, carry out glucose assays, PGM signal and Escherichia coli O 157 using blood glucose meter PGM: H7 is produced Raw quantitative relationship.The size for measuring a series of corresponding glucose concentration value of food-borne pathogens of various concentrations, establishes grape Quantitative relationship between sugared concentration value and food-borne pathogenic bacteria concentration (CFU/mL)；It can be measured according to the quantitative relationship unknown Food-borne pathogenic bacteria concentration in sample.It is an advantage of the invention that detection process realizes one-step method detection, reach accurate detection food source Property pathogenic bacteria purpose, have high sensitivity, high specific, it is easy to operate, detect it is quick, easily operated low with experimental cost etc. Advantage.

Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described, Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all Belong to the scope of protection of the invention.