CN105203756A - Method for preparing quick magnetic separation electrochemistry immunosensor and method for detecting staphylococcus aureus - Google Patents
Method for preparing quick magnetic separation electrochemistry immunosensor and method for detecting staphylococcus aureus Download PDFInfo
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- CN105203756A CN105203756A CN201510603728.8A CN201510603728A CN105203756A CN 105203756 A CN105203756 A CN 105203756A CN 201510603728 A CN201510603728 A CN 201510603728A CN 105203756 A CN105203756 A CN 105203756A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
Abstract
The invention discloses a method for preparing a quick magnetic separation electrochemistry immunosensor and a method for detecting staphylococcus aureus and belongs to the technical field of biosensing. Supernatant liquid is removed through separation and concentration under the effect of a magnetic field, sediment is dissolved through 0.1 M HNO3 and then subjected to ultrasounds, and the dissolved matter is then mixed with HAC/NaAC. A polished and cleaned electrode is steeped into the mixed solution, and staphylococcus aureus is detected through an SWV electrochemical method. Test results show that the concentration range of staphylococcus aureus detected by the sensor is 2.21*10<2>-2.21*10<7> CFU mL-1, and the detection limit is 83 CFU mL-1. In addition, the immunosensor successfully conducts quantitative detection on staphylococcus aureus in milk, compared with a traditional microorganism method, by means of the technology, the analyzing time is greatly shortened, operation is simple, and the requirement for quickly detecting staphylococcus aureus in the fields of food, environments and the like can be better met.
Description
Technical field
The invention provides a kind of Magneto separate electrochemistry immuno-sensing fast and to staphylococcus aureus high-sensitivity detecting method, belong to biosensor technique field.
Background technology
Staphylococcus aureus (
staphylococcusaureus) be a kind of gram-positive cocci being extensively present in the Nature.Staphylococcus aureus enterotoxin (
staphylococcusaureusenterotoxins, SES) be the extracellular toxin that a kind of staphylococcus aureus secretes, can cause food poisoning, can cause the symptoms such as chest pain, vomiting, diarrhoea, toxic shock, its median lethal dose is 0.02 μ g/kg.Food containing higher protein content, as milk and milk products, has often been detected SEB (SEB) and has existed.SEB is more stable, after several minutes through minute heating of high temperature number, still has remaining, should attach great importance to.
The classic method of current detection staphylococcus aureus: colony counting method, MTF method and filter membrane method.These three kinds of methods all some common shortcomings of tool as: detecting step is comparatively numerous and diverse, and sense cycle is longer, and labour intensity is larger.For adapting to the needs of food industry, the method that this several years developments have gone out several fast detecting Staphylococcus aureus is as: small-sized biological test chemical, immunology test, method, polymerase chain reaction etc. based on nucleic acid probe.The advantages such as these methods have conveniently, quick.But also exist some shortcomings as: testing cost is higher, more high to the instrument dependency degree of robotization.And in recent years, in environmental monitoring and field of food safety, biology sensor plays more and more important role in pathogen, organic detection.In view of its high sensitive and selectivity, immune sensing is considered to one of the most effective testing tool.
CdTe quantum is generally comparatively stable, water-soluble, can accept exciting light and produce fluorescence.Under crosslinking chemical effect, the quantum dot of Dispersal risk functionalization, can form stable chemical bond between antibody and quantum dot, and the connection product obtained combines firmly, comparatively stable, can specific detection antigen.Magnetic nano-balls is the magnetic material of nanoscale, and except the optics with general nano particle and character of surface, magnetic nano-balls also has catalytic activity, good magnetic steering, the character such as biology mixes, biodegradation, can be combined with many biomolecule, make it surface-functionalized.Fe
3o
4the maximum magnetic nano-balls being.Fe
3o
4particle diameter is little, toxicity is low, highly sensitive, stable performance, starting material are easy to get, and its surface area is comparatively large in addition, and surfactivity is also higher.Therefore suitable crosslinking agents can be selected specific antibodies to be attached on magnetic nano-balls, thus to make magnetic nano-balls with certain specific function.Under cross-linking reagent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) effect, functional magnetic nanosphere can combine with specific antigen, under outside magnetic field acting force, the magnetic functionalization biomolecule of tool can move to magnetic direction, thus concentrates on specific position.
Immuno magnetic cell separation technology, is the magnetic bead and antibody (or antigen) specific binding that utilize antigen (or antibody) to wrap quilt, then under the effect of externally-applied magnetic field, sample can be separated from complex matrices, reaches the object that example enrichment is concentrated.Immuno magnetic cell separation technology fast and high specificity, possesses the advantage of immobilization reagent, does not need to treat bacterial detection and carry out increasing bacterium, contribute to the detectability reducing original detection means.Thus in food, medical science etc., have its huge using value.
Summary of the invention
The object of invention is to provide a kind of simple to operate, and the response time is short, cheap, highly sensitive with the biology sensor detecting staphylococcus aureus.The present invention solves the problems of the technologies described above adopted technical scheme: a kind of electrochemica biological sensor for detecting staphylococcus aureus, comprises auxiliary electrode, contrast electrode and working electrode.It is characterized in that: synthesized antibody functionalized magnetic nano-particle (Fe first respectively
3o
4-Ab) and antibody functionalized quantum dot (QDs-Ab).Two kinds of functionalized nano probes are attached to staphylococcus aureus surface simultaneously, and are separated rapidly by magnetic fields, and combined with electrochemical Stripping Voltammetry method can realize Staphylococcus aureus in food and detect fast.
A kind of Magneto separate electrochemistry immuno-sensing fast and to the highly sensitive detection of staphylococcus aureus, carries out according to following step:
(1) preparation of CdTe quantum:
6mM mercaptopropionic acid (MPA) joins 2mMCdCl
2in solution, by the NaOH solution of 1M, PH is adjusted to 9.0, pass into N
2mixing 30min, more slowly add 0.0625MNaHTe solution, form dark yellow CdTe quantum.The quantum dot solution backflow 10h obtained, can reach particle diameter is 2.6nm, concentration 4.7 μMs.By the quantum dot solution made by 15000g ultrafiltration 10min under 4 DEG C of conditions to remove excessive mercaptopropionic acid, and rinse twice repeatedly, finally use the PBS constant volume of pH7.4, and the refrigerator being placed in 4 DEG C is preserved.
Wherein in step (1) each reactant according to Cd
2+/ MPA/HTe
-thing mass ratio is that 1:3:0.5 adds.
(2) CdTe quantum functionalization:
Get synthetic CdTe quantum solution (4.7 μMs), concentration is the Staphylococcus aureus antibody of 5mg/ml, and cross-linking reagent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) solid particle, 1h is stirred under mixed room temperature, after centrifuging, place the Refrigerator store of 4 DEG C.
Wherein in step (2), the volume ratio of CdTe quantum solution/Staphylococcus aureus antibody is 15:1, and the mass ratio of Staphylococcus aureus antibody/EDC/NHS is 1:6:6.
(3) magnetic nano-balls functionalization:
Be the PBS buffer solution (pH7.4) that 15:1 gets laboratory and prepares according to volume ratio, concentration is 5mg/mL Staphylococcus aureus antibody, Fe
3o
4pressed powder and crosslinking chemical 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC), at room temperature hybrid reaction 1h, places the Refrigerator store of 4 DEG C after centrifuging.
Wherein in step (3), the volume ratio of PBS buffer solution (pH7.4)/Staphylococcus aureus antibody is 15:1, Staphylococcus aureus antibody/Fe
3o
4the mass ratio of pressed powder/EDC is 1:6:6.
(4) working electrode pre-service:
By bare electrode successively with deionized water, absolute ethyl alcohol and deionized water ultrasonic 10min respectively, repeatedly polish to minute surface with the alumina powder that particle diameter is 1 μm, 0.3 μm, 0.05 μm successively again, then be placed in ethanol, deionized water for ultrasonic cleaning 10min respectively, finally dry up with nitrogen for subsequent use.
(5) modification of working electrode and the preparation of sensor:
Cultivate under getting 37 DEG C of conditions, the staphylococcus aureus after gradient dilution, add the antibody functionalized magnetic bead (Fe of step (3)
3o
4-Ab), after adding rotor stirring reaction 10min under room temperature, add the quantum dot (QDs-Ab) that step (2) is antibody functionalized, under room temperature, react 30min.After reaction terminates, take out rotor, standing adsorption 20min on magnet.Carefully take out supernatant liquor with liquid-transfering gun, and add testing laboratory self-control HNO
3, and ultrasonic 40min (0.1M).
Wherein staphylococcus aureus, Fe
3o
4-Ab, QDs-Ab and HNO
3each volume ratio is 2:1:1:2.5, behind PBS cleaning electrode surface, is immersed 1 ~ 2mLHAC/NaAC(pH4.6) in solution, rotor stirs.
Above-mentioned quick Magneto separate electrochemistry immuno-sensing and to the highly sensitive detection of staphylococcus aureus, is characterized in that: fast Magneto separate electrochemical immunosensor working curve foundation and fast Magneto separate electrochemical immunosensor to the detection of actual milk sample.Prepared quick Magneto separate electrochemical immunosensor working curve is: when staphylococcus aureus concentration is 2.21 × 10
2~ 2.21 × 10
7cFUmL
-1time, its linear equation is: y=0.9934x-0.3526, and detection is limited to: 83CFUmL
-1.Prepared quick its performance of Magneto separate electrochemical immunosensor also comprises good specificity, repeatability and stability and the detection to actual milk sample.
Principle: the present invention successfully obtains CdTe quantum by Aqueous phase, carries out functionalization respectively again subsequently to quantum dot and magnetic nano-balls.The quantum dot of functionalization and magnetic nano-balls can specific bond staphylococcus aureuses, form the compound to be detected of sandwich structure.Externally-applied magnetic field bottom test tube, this compound is in bottom enrichment, and by removing supernatant and suitably washing, object thoroughly can be separated with impurity, carries out SWV Electrochemical Detection more subsequently.Typical curve and linear equation can be obtained according to the data obtained, the concentration of staphylococcus aureus in indirect determination testing sample, as shown in Figure 1, Figure 2 and Figure 3.
Compared with prior art, the invention has the advantages that:
(1) nano material specific surface area is large, and surfactivity site is more, and bioaffinity is strong, can be used to do carrier adsorption or support immune molecule, is conducive to improving the many performances of sensor.Immune nanometer magnetic bead and quantum dot, mainly through Immune discrimination, are caught thalline, quick separating under magnetic fields, do not destroy thalline biologic activity.Ag-Ab specific binding determines the high sensitivity of immunosensor in addition, high specificity, not by the interference of other materials.Magneto separate and electrochemical detection are also fast, simply.Whole operating process, fast easy, practicality is good, highly sensitive, for detecting in food whether provide another kind new method safely and effectively containing the staphylococcus aureus exceeded standard.
(2) nanometer technology combines with electro-chemistry immunity technology by this experiment, and by quick Magneto separate, quantitatively detects, have good specificity to the staphylococcus aureus in milk.Compared to traditional micro-biological process; this technology is used for the detection of Staphylococcus aureus in food and substantially reduces analysis time, simple to operate, can better meet the requirement of fast detecting Staphylococcus aureus in the fields such as food hygiene, safe drinking water, environmental protection and control infectious disease.
Accompanying drawing explanation
The preparation of the quick Magneto separate electrochemical immunosensor of Fig. 1 and the Cleaning Principle figure to staphylococcus aureus thereof;
Fig. 2 detects SWV curve map (a) 0 of variable concentrations staphylococcus aureus, (b) 2.2 × 10
2, (c) 2.2 × 10
3, (d) 2.2 × 10
4, (e) 2.2 × 10
5, (f) 2.2 × 10
6, (g) 2.2 × 10
7cFUmL
-1;
The canonical plotting of Fig. 3 variable concentrations staphylococcus aureus SWV peak current.
Embodiment
Be described in further detail the present invention below in conjunction with accompanying drawing embodiment, concrete operation method is as follows:
(1) preparation of CdTe quantum
26 μ L6mM mercaptopropionic acids (MPA) join 50mL2mMCdCl
2in solution, by the NaOH solution of 1M, pH is adjusted to 9.0, pass into N
2mixing 30min, more slowly add 0.8mL0.0625MNaHTe solution, form dark yellow CdTe quantum.Cd
2+/ MPA/The
-thing mass ratio is 1:3:0.5.The quantum dot solution backflow 10h obtained, can reach particle diameter is 2.6nm, concentration 4.7 μMs.By the quantum dot solution made by 15000g ultrafiltration 10min under 4 DEG C of conditions to remove excessive mercaptopropionic acid, and rinse twice repeatedly, finally become 700 μ L with the PBS constant volume of pH7.4, the refrigerator being placed in 4 DEG C is preserved.
(2) CdTe quantum functionalization
The cross-linking reagent that the present invention adopts has 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC), N-hydroxy-succinamide (NHS).Concrete operation method: get synthetic CdTe quantum solution 470 μ L, add antibody 30 μ L (5mg/ml), stir 1h under each 1mg mixed room temperature of EDC and NHS.After centrifuging, place the Refrigerator store of 4 DEG C.
(3) magnetic nano-balls functionalization
Get PBS buffer solution (pH7.4) the 470 μ L that testing laboratory prepares, Fe
3o
4pressed powder 1mg, crosslinking chemical 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) 1mg, joins in PBS buffer solution (pH7.4) respectively.Add antibody 30 μ L (5mg/mL).At room temperature react 1h, after centrifuging, place the Refrigerator store of 4 DEG C.
(4) modification of working electrode and the preparation of sensor
Cultivate under getting 37 DEG C of conditions, the staphylococcus aureus 20 μ L after gradient dilution, adds the antibody functionalized magnetic bead (Fe of step (3)
3o
4-Ab) 10 μ L, after adding rotor stirring reaction 10min under room temperature, add quantum dot (QDs-Ab) the 10 μ L that step (2) is antibody functionalized, under room temperature, react 30min.After reaction terminates, take out rotor, standing adsorption 20min on magnet.Carefully take out supernatant liquor with liquid-transfering gun, and add 25 μ L testing laboratory self-control HNO
3, and ultrasonic 40min (0.1M).Behind PBS cleaning electrode surface, being immersed 1mLHAC/NaAC(pH4.6) in solution, rotor stirs.
The present invention successfully obtains CdTe quantum by Aqueous phase, carries out functionalization respectively again subsequently to quantum dot and magnetic nano-balls.The quantum dot of functionalization and magnetic nano-balls can specific bond staphylococcus aureuses, form the compound to be detected of sandwich structure.Externally-applied magnetic field bottom test tube, this compound is in bottom enrichment, and by removing supernatant and suitably washing, object thoroughly can be separated with impurity, carries out SWV Electrochemical Detection more subsequently.Typical curve and linear equation can be obtained, the concentration of staphylococcus aureus in indirect determination testing sample according to the data obtained.
The detection of standard items
By the Fe utilizing specific embodiment two method to prepare
3o
4-Ab/
s.aureus/qDs-Ab sensor is placed in staphylococcus aureus standard solution and reacts, and standard solution concentration is followed successively by 2.21 × 10
2, 2.21 × 10
3, 2.21 × 10
4, 2.21 × 10
5, 2.21 × 10
6, 2.21 × 10
7cFUmL
-1, (standard items solvent is PBS buffer solution, pH7.4), working buffer system is HAC/NaAC(pH4.6), record square-wave pulse voltammetry (SWV) gained response current.Prepared electrochemical immunosensor working curve is: when staphylococcus aureus concentration is 2.21 × 10
2~ 2.21 × 10
7cFUmL
-1time, its linear equation is: y=0.9934x-0.3526, and detection is limited to: 83CFUmL
-1.Prepared quick Magneto separate electrochemical immunosensor is simple to operate, comprises good specificity in addition, repeatability and stability.
Specificity verification is tested
Choose other three kinds of bacterium: Escherichia coli, vibrio parahemolyticus and typhoid fever sramana (family name) bacterium, measure the specificity of this sensor as comparative, drip same concentration (1 × 10
4cFUmL
-1and 1 × 10
5cFUmL
-1) different strain is in electrode surface, when drip Escherichia coli, vibrio parahemolyticus and typhoid fever sramana (family name) bacterium time, the data value of peak current is all only less than 1 μ A, and there is no significant change between variable concentrations, the peak point current of corresponding staphylococcus aureus then has 4.07 μ A and 4.79 μ A, and numerical value has had obvious increase, and so huge change is mainly because of the specificity of antibody for antigen recognizing, again show, this immunosensor has high selectivity.
The detection of milk sample
In order to verify the actual application value of this immunosensor, the milk choosing interpolation staphylococcus aureus, as detecting sample (milk is the fresh products that local supermarket is bought), gets 1mL milk, with PBS(pH7.4) be diluted to 10mL sample.Sample is divided into the staphylococcus aureus (1.2 × 10 that three parts are added variable concentrations respectively
2, 1.2 × 10
3with 1.2 × 10
4cFUmL
-1), utilize this immunosensor to detect the sample of above-mentioned process respectively, the testing result recovery is 93.7%, 92.8% and 104.4%, and indicate this immunosensor high to staphylococcus aureus detection accuracy in actual sample, result is accurately and reliably.
Above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing, and those skilled in the art are in essential scope of the present invention, and the change made, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.
Claims (6)
1. the preparation method of quick Magneto separate electrochemistry immuno-sensing, is characterized in that carrying out according to following step:
(1) preparation of CdTe quantum:
6mM mercaptopropionic acid (MPA) joins 2mMCdCl
2in solution, by the NaOH solution of 1M, PH is adjusted to 9.0, pass into N
2mixing 30min, more slowly add 0.0625MNaHTe solution, form dark yellow CdTe quantum; The quantum dot solution backflow 10h obtained, can reach particle diameter is 2.6nm, concentration 4.7 μMs; By the quantum dot solution made by 15000g ultrafiltration 10min under 4 DEG C of conditions to remove excessive mercaptopropionic acid, and rinse twice repeatedly, finally use the PBS constant volume of pH7.4, and the refrigerator being placed in 4 DEG C is preserved;
Wherein in step (1) each reactant according to Cd
2+/ MPA/HTe
-thing mass ratio is that 1:3:0.5 adds;
(2) CdTe quantum functionalization:
Get synthetic CdTe quantum solution (4.7 μMs), concentration is the Staphylococcus aureus antibody of 5mg/ml, and cross-linking reagent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) solid particle, 1h is stirred under mixed room temperature, after centrifuging, place the Refrigerator store of 4 DEG C;
Wherein in step (2), the volume ratio of CdTe quantum solution/Staphylococcus aureus antibody is 15:1, and the mass ratio of Staphylococcus aureus antibody/EDC/NHS is 1:6:6;
(3) magnetic nano-balls functionalization:
Be the PBS buffer solution (pH7.4) that 15:1 gets laboratory and prepares according to volume ratio, concentration is 5mg/mL Staphylococcus aureus antibody, Fe
3o
4pressed powder and crosslinking chemical 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC), at room temperature hybrid reaction 1h, places the Refrigerator store of 4 DEG C after centrifuging;
Wherein in step (3), the volume ratio of PBS buffer solution (pH7.4)/Staphylococcus aureus antibody is 15:1, Staphylococcus aureus antibody/Fe
3o
4the mass ratio of pressed powder/EDC is 1:6:6;
(4) working electrode pre-service:
By bare electrode successively with deionized water, absolute ethyl alcohol and deionized water ultrasonic 10min respectively, repeatedly polish to minute surface with the alumina powder that particle diameter is 1 μm, 0.3 μm, 0.05 μm successively again, then be placed in ethanol, deionized water for ultrasonic cleaning 10min respectively, finally dry up with nitrogen for subsequent use;
(5) modification of working electrode and the preparation of sensor:
Cultivate under getting 37 DEG C of conditions, the staphylococcus aureus after gradient dilution, add the antibody functionalized magnetic bead (Fe of step (3)
3o
4-Ab), after adding rotor stirring reaction 10min under room temperature, add the quantum dot (QDs-Ab) that step (2) is antibody functionalized, under room temperature, react 30min; After reaction terminates, take out rotor, standing adsorption 20min on magnet; Carefully take out supernatant liquor with liquid-transfering gun, and add testing laboratory self-control HNO
3, and ultrasonic 40min (0.1M);
Wherein staphylococcus aureus, Fe
3o
4-Ab, QDs-Ab and HNO
3each volume ratio is 2:1:1:2.5, behind PBS cleaning electrode surface, is immersed 1 ~ 2mLHAC/NaAC(pH4.6) in solution, rotor stirs.
2. the preparation method of quick Magneto separate electrochemistry immuno-sensing according to claim 1, is characterized in that, it is characterized in that wherein in step (1) each reactant according to Cd
2+/ MPA/HTe
-thing mass ratio is that 1:3:0.5 adds.
3. the preparation method of quick Magneto separate electrochemistry immuno-sensing according to claim 1, it is characterized in that, the volume ratio that it is characterized in that CdTe quantum solution/Staphylococcus aureus antibody in wherein step (2) is 15:1, and the mass ratio of Staphylococcus aureus antibody/EDC/NHS is 1:6:6.
4. the preparation method of quick Magneto separate electrochemistry immuno-sensing according to claim 1, it is characterized in that, the volume ratio that it is characterized in that PBS buffer solution (pH7.4)/Staphylococcus aureus antibody in wherein step (3) is 15:1, Staphylococcus aureus antibody/Fe
3o
4the mass ratio of pressed powder/EDC is 1:6:6.
5. the preparation method of quick Magneto separate electrochemistry immuno-sensing according to claim 1, is characterized in that, it is characterized in that wherein step (5) staphylococcus aureus, Fe
3o
4-Ab, QDs-Ab and HNO
3each volume ratio is 2:1:1:2.5, behind PBS cleaning electrode surface, is immersed 1 ~ 2mLHAC/NaAC(pH4.6) in solution, rotor stirs.
6. quick Magneto separate electrochemistry immuno-sensing according to claim 1 is to staphylococcus aureus high-sensitivity detecting method, it is characterized in that: the foundation of quick Magneto separate electrochemical immunosensor working curve and quick Magneto separate electrochemical immunosensor are to the detection of actual milk sample; Prepared quick Magneto separate electrochemical immunosensor working curve is: when staphylococcus aureus concentration is 2.21 × 10
2~ 2.21 × 10
7cFUmL
-1time, its linear equation is: y=0.9934x-0.3526, and detection is limited to: 83CFUmL
-1.
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Cited By (4)
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