CN101504416A - Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence - Google Patents
Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence Download PDFInfo
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- CN101504416A CN101504416A CNA2008100313509A CN200810031350A CN101504416A CN 101504416 A CN101504416 A CN 101504416A CN A2008100313509 A CNA2008100313509 A CN A2008100313509A CN 200810031350 A CN200810031350 A CN 200810031350A CN 101504416 A CN101504416 A CN 101504416A
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Abstract
The invention discloses a method for detecting Escherichia coli with the advantages of high sensitivity, quick speed, low cost and easy realization of automation. The basic principle is that: firstly, a first antibody is loaded by a gold coating magnetic particle, and an antigen to be detected and a second antibody are captured by the immune sandwich mode; under the action of external magnetic fields, a compound formed by the antigen-antibody and the gold magnetic particle can be separated; in solution containing (HCL-NaCl-Br2) strong oxidizer, simple substance Au on the surface of the gold magnetic particle is oxidized into Au<3+>, and because the antigen-antibody sandwich structure is strongly negatively charged, the free Au<3+> can be quantitatively adsorbed around the antigen-antibody sandwich structure; and a certain amount of luminol is added to cause chemical luminescence in situ around the magnetic particle, and the strength of the chemical luminescence is quickly detected so as to quantitatively detect the Escherichia coli. The biological sensor system supplies a gold ion source for improving sensitivity and efficiency based on in-situ oxidization of the gold coating magnetic particle, and generates a biological molecular identification system with chemical luminescence by utilizing the magnetic particle to capture and enrich the biological molecule Escherichia coli O157:H7 antigen in specific complementary pairing of a sample to be analyzed. The method avoids the complexity and non-specific interference in a second antibody nanometer gold label, and can be used for quickly and highly sensitively detecting the Escherichia coli. Therefore, the method is a novel method for detecting the Escherichia coli, which has the advantages of quickness, direct viewing, simple operation, and high efficiency.
Description
Affiliated technical field
The present invention relates to a kind of gold-coating magnetic granule in-situ initiation high-sensibility chemical luminescence and detect colibacillary new method.
Background technology
Escherichia coli O 157: the H7 infectious diarrhea is the serious enteric infectious disease of newfound in recent years harm, it is except that causing diarrhoea, hemorrhagic enteritis, also can take place hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP) etc. serious and freeze, latter's state of an illness is dangerous, the case fatality rate height.As long as discovering 10 Escherichia coli O 157: H7 just is enough to cause a disease.At present, relevant colibacillary detection method has had many reports.Escherichia coli O 157 commonly used: the H7 detection method mainly contains three classes: bacteriology separation, immunology detection and molecular Biological Detection.
In the method that relevant both at home and abroad research biology sensor detects Escherichia coli, wherein comparatively be typically a kind of chemiluminescence immunoassay based on gold nano particulate.This method has been inquired into the top condition of gold nano particulate dissolving and chemical luminescent detecting based on the chemiluminescence reaction system of gold nano particulate dissolving.It is that original antibody is fixed on the magnetic bead, and secondary antibody is modified on the collaurum, catches formation such as target biological molecules then by the mode of sandwich method and contains many anti-compounds, and the chemical treatment oxidation makes the Au of magnetic bead surfaces be oxidized to Au again
3+, add luminol (Luminol, luminous amine) and cause chemiluminescence, thereby applied chemistry luminescent immunoassay method is measured human immunity hyperglobulinemia G, utilize this method can produce the biology sensor of measuring human immunity hyperglobulinemia G.
Inspired by this, the present invention seeks in line with set up a kind of hypersensitive, speed fast, low-cost, realize the colibacillary method of detection of robotization easily.Propose a kind of new mentality of designing, promptly utilize golden magnetic granule in-situ oxidation to cause high-sensibility chemical luminescence and carry out the Escherichia coli detection.
At present, the preparation method of magnetic nanoparticle and functionalization aspect have had the exploratory development of the system of going deep into, and can adopt the multiple technologies approach to prepare magnetic nanoparticle and the functional magnetic nano particle with nucleocapsid structure.Technological process is simple, and step is simple; Good stability can standing storage (more than half a year); Shape and size can be controlled as required; On these nano magnetic particles, biomacromolecules such as fluorophor, bind molecule and protein (as antibody) and nucleic acid be can effectively modify, and good stability, but long preservation kept by the specificity of modified biological molecule.On this technical foundation, be expected to set up a kind of overdelicate chemiluminescence detection system, break through colibacillary detectability.
Goal of the invention
The objective of the invention is for provide a kind of hypersensitive, speed fast, low-cost, realize the colibacillary new method of detection of robotization easily, so that the chemiluminescence biosensor of preparation hypersensitive and good stability, and advantages such as it is simple to make it have experimental procedure, easy and simple to handle.
Summary of the invention
Purpose of the present invention realizes by following proposal:
The preparation of gold-coating magnetic composite nanoparticle comprises the steps:
1. the golden magnetic nanoparticle (Fe that coats
3O
4Or γ-Fe
2O
3) preparation:
(a) prepare Fe with coprecipitation
3O
4Utilize the mixed solution of ferric ion and ferrous ion, under the effect of alkali (NaOH), make Fe by coprecipitation
3O
4Nano particle; Then, with the magnetic particle amination of amino silane with gained; Again at gold ion (Au
3+) under the condition that exists, the Fe that utilizes the synthetic gold of sonochemical method to coat
3O
4Nano particle.
(b) with Fe
3O
4Nano particle is the forerunner, and by direct oxidation method, roasting obtains γ-Fe under the about 400 ℃ of conditions of high temperature
2O
3Nano particle; Subsequently, with amino silane with γ-Fe
2O
3The magnetic particle amination; Again at gold ion (Au
3+) under the condition that exists, the γ-Fe that utilizes the synthetic gold of sonochemical method to coat
2O
3Nano particle.
2. gold-coating magnetic granule surface biological functionalization combines with antigen-antibody and forms immune sandwich structure:
(c) utilize mercaptoethylmaine to make coloured glaze base in the finishing of golden magnetic grain, form on its surface sulfydryl key (-SH); Then, under the effect of glutaraldehyde, make because aldehyde radical is easy to bonding absorption monoclonal antibody, so can form monoclonal antibodyization gold magnetic grain by aldehyde radical in the finishing of golden magnetic grain.
(d) the golden magnetic grain with monoclonal antibodyization carries out specific adsorption to Escherichia coli; Then, carry out second antibody again and modify, both are in conjunction with forming special immune sandwich structure; Add strong oxidizer (HCl-NaCl-Br
2) after the system, make that the Au simple substance on the magnetic grain is oxidized to Au
3+Ion is because above-mentioned sandwich structure is strong electronegativity, free Au
3+Can be around it by specific adsorption; After the luminol processing, can set up chemical luminous system.
The present invention has following advantage compared to existing technology:
(1) magnetic nano-particle (Fe of gold coating
3O
4Or γ-Fe
2O
3) preparation be based on chemosynthesis variation route under the ultrasound condition; And, catch the basis for luminescence system provides gold ion source and bio-identification simultaneously by biological functional acquisition monoclonal antibody Au coating magnetic granule, avoided using the loaded down with trivial details of colloid gold label second antibody.
(2) the biological functional modification by magnetic nano-particle prepares the antigen-antibody articulated system, and the in-situ oxidation initiation chemiluminescence effect of utilizing gold to coat, realize novel colon bacillus 0157: the super sensitivity detection of the preparation of H7 chemiluminescence biosensor and colon bacillus 0157: H7.Than the method for the detection of biological molecule of original use, through the magnetic grain specific adsorption that gold coats, produce chemiluminescence after, operate more succinctly, sensitivity is higher, the Detection of antigen that is expected to break through present colon bacillus 0157: H7 is limit.
Description of drawings
The preparation of Fig. 1 gold magnetic grain is embodiment I process flow diagram.
The golden magnetic grain of Fig. 2 monoclonal antibodyization is embodiment II process flow diagram.
Fig. 3 is embodiment III process flow diagram in conjunction with the golden magnetic grain sandwich structure of second antibody.
Embodiment
Embodiment I:
1) gets 2g FeCl respectively
24H
2O and 5.2g FeCl
36H
2The concentrated hydrochloric acid 0.85mL of O and 12.1mol/L is dissolved in 200mL H
2Among the O, ultrasonic deoxidation; Then above drips of solution is added to 250mL, in the 0.75mol/L NaOH solution, all are reflected at temperature is 80 ℃, stirs N
2Carry out under the protection.Along with the carrying out of reaction, the precipitation of black appears in the reactant liquor.After reaction finishes, utilize externally-applied magnetic field that the gained precipitation is separated from reaction medium, can obtain Fe
3O
4Magnetic nano-particle.2) drip 0.4mLAPTES then in above mixed solution, stir 7h under the room temperature; Carry out magnetic and separate, obtain the Fe that APTES modifies
3O
4Magnetic nano-particle.3) with the solution (1g/L, the Fe that prepare more than the 15mL
3O
4), 0.6mmol/LAuCl
3HCl4H
2O aqueous solution 14mL is distributed in the 100mL deionized water, stirs; Drip the 0.2mol/L citric acid and receive solution 0.3mL, under certain frequency, under the room temperature ultrasonic up to the reaction solution color by the light yellow black that becomes gradually.Obtain the Fe that gold coats
3O
4Magnetic nano-particle.
1) gets the above-mentioned Fe for preparing
3O
4Magnetic nano-particle 2g puts into muffle furnace, is heated to 400 ℃, the about 2-3 of roasting hour, can make γ-Fe
2O
3Magnetic nano-particle.2) drip 0.4mLAPTES then in above mixed solution, stir 7h under the room temperature; Carry out magnetic and separate, obtain γ-Fe that APTES modifies
2O
3Magnetic nano-particle.3) again with the solution (1g/L, the Fe that prepare more than the 15mL
3O
4), 0.6mmol/LAuCl
3.HCl4H
2O aqueous solution 14mL is distributed in the 100mL deionized water, stirs; Drip the 0.2mol/L citric acid and receive solution 0.3mL, under certain frequency, under the room temperature ultrasonic up to the reaction solution color by the light yellow black that becomes gradually.Obtain γ-Fe that gold coats
2O
3Magnetic nano-particle.
Experimental program II:
1) get the above-mentioned golden magnetic particle that has prepared of 20mg, place a test tube, add the cysteamine solution of 20mg/mL, at ambient temperature, logical argon shield, reaction 48h.2) 10mL, 12% glutaraldehyde water solution are added in the above-mentioned mixed liquor, at room temperature, vibration evenly hatch after 12 hours again, and the golden magnetic grain of aldehyde radical is separated by magnetic in the modification, use PB buffer solution for cleaning 3 times.3) the golden magnetic grain of getting the aldehyde radicalization of 100 μ g join 2.5mL, 10% first antibody phosphate buffer (Phosphate Buffer, PB, pH=7.4) in, 40 ℃ of hatchings 2 hours down, and with this solution mixing that constantly vibrates.By the reaction of the amidine functional group in aldehyde radical and the monoclonal antibody molecule latter is connected to particle surface.4) after reaction finishes, utilize externally-applied magnetic field that particle is isolated from reaction medium, and with PB solution wash products repeatedly, last concentration with 4.0mg/mL is dispersed in the PB solution, obtains the golden magnetic grain of monoclonal antibodyization, and is stand-by.
Embodiment III:
1) gets the golden magnetic grain solution 25mL of above-mentioned monoclonal antibodyization, place a test tube, its artificial Escherichia coli bacteria liquid with 10% is fully mixed, under 40 ℃ of temperature, hatched 2 hours, carry out magnetic and separate, after PB buffer solution for cleaning 3 times, obtain first antibody-antigen-Jin magnetic grain complex.2) to wherein adding 2.5mL, 10% second antibody phosphate buffer is at room temperature hatched 1h, utilizes immune sandwich mode, catches second antibody, forms special antigen-antibody sandwich structure.Under the effect of externally-applied magnetic field, antigen-antibody is carried out magnetic with golden magnetic grain compound separate, cleaning fluid is removed.3) the 0.01M HCl-0.5M NaCl-0.5mM Br of adding 10mL
2Strong oxidant solution after about 10 minutes, places 60 ℃ baking oven with mixed liquor, is incubated 20 minutes, removes unnecessary bromine, and the Au simple substance on golden magnetic grain surface is oxidized to Au
3+, because antigen-antibody sandwich structure surface is strong electronegativity, free Au
3+Can quantitatively be adsorbed on around the sandwich structure.4) luminol of adding 0.01mM/mL, original position causes chemiluminescence around the magnetic grain, and the rapid test chemiluminescence intensity, and this luminous intensity and Escherichia coli amount are proportional, come quantitative with this.Detect and be limited to 10-10
6CFU/mL.
Claims (4)
1, a kind of gold-coating magnetic granule in-situ initiation high-sensibility chemical luminescence detects colibacillary new method, comprises the steps:
1.1 the magnetic nanoparticle (Fe that gold coats
3O
4Or γ-Fe
2O
3) preparation:
(a) prepare Fe with coprecipitation
3O
4Adopt the mixed solution of ferric ion and ferrous ion, under the effect of alkali (NaOH), make Fe by coprecipitation
3O
4Nano particle; Subsequently under the effect of amino silane, with the amination of gained magnetic particle; Again at gold ion (Au
3+) under the condition that exists, the Fe that utilizes the synthetic gold of sonochemical method to coat
3O
4Nano particle.
(b) with Fe
3O
4Nano particle is the forerunner, and by direct oxidation method, roasting obtains γ-Fe under the about 400 ℃ of conditions of high temperature
2O
3Nano particle; Subsequently, under the effect of amino silane, with γ-Fe
2O
3The magnetic particle amination; Again at gold ion (Au
3+) under the condition that exists, the γ-Fe that utilizes the synthetic gold of sonochemical method to coat
2O
3Nano particle.
1.2 combining with antigen-antibody, gold-coating magnetic granule surface biological functionalization forms immune sandwich structure:
(c) utilize mercaptoethylmaine to make golden magnetic grain (Fe
3O
4Or γ-Fe
2O
3) sulfydryl in the finishing, form on its surface sulfydryl key (-SH); Then, under the effect of glutaraldehyde, make and utilize aldehyde radical to be easy to adsorb this characteristic of bonding monoclonal antibody by aldehyde radical in the finishing of golden magnetic grain, form monoclonal antibodyization gold magnetic grain.
(d) with monoclonal antibodyization gold magnetic grain Escherichia coli are carried out specific adsorption, obtain first antibody antigen and golden magnetic grain compound; Subsequently, carry out second antibody and modify, second antibody combines with aforesaid compounds and forms special immune sandwich structure; Add strong oxidizer (HCl-NaCl-Br again
2) system, the Au simple substance on the magnetic grain is oxidized to Au
3+Ionic adsorption after the luminol processing, can obtain chemiluminescence sample system around Escherichia coli.
2, the method for this gold-coating magnetic granule preparation according to claim 1 is characterized in that the gold-coating magnetic granule of sonochemical method preparation can directly provide gold ion source and bio-identification to catch the basis through in-situ oxidation.
3, a kind of gold-coating magnetic granule in-situ initiation high-sensibility chemical luminescence according to claim 2 detects colibacillary new method, it is characterized in that in the described step (c), behind the last aldehyde radical of gold-coating magnetic granule modification, but specificity bonding adsorption antigen forms monoclonal antibodyization gold magnetic grain.
4, cause high-sensibility chemical luminescence and detect colibacillary new method according to claim 1,2 described a kind of gold-coating magnetic granule in-situs, it is characterized in that avoiding the loaded down with trivial details of second antibody nano gold mark.Can be easy set up a kind of special immune sandwich structure, obtain the highly sensitive detection architecture of a kind of chemiluminescence, be expected to improve colibacillary detectability.
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|---|---|---|---|
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|---|---|---|---|
| CNA2008100313509A CN101504416A (en) | 2008-05-20 | 2008-05-20 | Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence |
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Family
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