CN105300963A - Preparing method and application of sandwich type electrochemical luminescence immunosensor for detecting marine pathogenic bacteria - Google Patents
Preparing method and application of sandwich type electrochemical luminescence immunosensor for detecting marine pathogenic bacteria Download PDFInfo
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Abstract
The invention discloses a preparing method and application of a sandwich type electrochemical luminescence immunosensor for detecting marine pathogenic bacteria. The method is characterized by comprising the steps of obtaining an immunomagnetic bead solution the surface of which is covered with marine pathogenic bacterium antibodies by immobilizing the marine pathogenic bacterium antibodies onto the surfaces of aminated magnetic beads; synthesizing multifunctional oxidized graphene through chemical bonding of the marine pathogenic bacterium antibodies and an electrochemical illuminant; conducting electrode pretreatment and immobilizing immunomagnetic beads onto the surface of a magnetic glassy carbon electrode; finally adsorbing the marine pathogenic bacteria to be detected and the multifunctional oxidized graphene onto the surface of the magnetic glassy carbon electrode in sequence. The obtained sandwich type electrochemical luminescence immunosensor serves as a working electrode, a platinum electrode is taken as a counter electrode and an Ag/AgCl electrode or saturated calomel electrode is taken as a reference electrode to calculate the accurate concentration of the marine pathogenic bacteria in a sample solution to be detected. Detection speed is high, the sensitivity and accuracy of detection results are high, and specificity is high.
Description
Technical field
The present invention relates to electrochemiluminescence immunosensor, especially relating to preparation method and the application thereof of the sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria.
Background technology
Marine Pathogenic Bacteria is a kind of protokaryon unicellular organism, in spherical, shaft-like, spiral fashion etc.Marine Pathogenic Bacteria can cause marine fishery animals and plants ill, such as: it is crushing dead that vibrio parahaemolytious can cause Juvenile Litopenaeus vannamei to occur.Some Marine Pathogenic Bacterias are important pathogens of zoonosis, and the mankind, by the skin contact seawater infection pathogen of take food contaminated marine product or breakage, can cause the diseases such as tympanitis, urinary system infection contamination, septicemia even dead.Therefore detecting Marine Pathogenic Bacteria is rapidly and accurately protection human health and the important channel of reducing economic loss.
At present for the mensuration of Marine Pathogenic Bacteria, conventional culture methods comprises the process such as Zengjing Granule, selectivity cultivation, and length consuming time, complex operation, recall rate are low.Detection method based on immunology utilizes the specificity of antigen-antibody identification, comprises Enzyme-linked Immunosorbent Assay (ELISA) method etc., but sensitivity needs to improve further.Detection method such as PCR (PCR) based on nucleic acid is a kind of sensitive detection method, but more easily occurs false positive results.Therefore, Marine Pathogenic Bacteria detection technique quick, sensitive, accurate, easy and simple to handle is developed extremely important.
Electrochemiluminescence (Electrochemiluminescence, ECL) is a kind of chemiluminescence reaction caused by galvanochemistry at electrode surface, is the product that chemiluminescence combines with galvanochemistry.Electrochemiluminescence immunosensor is the technology that current collection chemiluminescence and immunosensor are integrated, and has the advantages such as highly sensitive, easy and simple to handle.Graphene oxide is a kind of new carbon of excellent performance, there is higher specific surface area and abundant functional group, based on functional graphene oxide prepared by graphene oxide, can effectively to extend the Helmholtz face of electrode surface, remarkable enhancing electrochemiluminescence, improving sensitivity, is the ideal material building electrochemical luminous sensor label.Immunomagnetic beads is the immunological technique that development in recent years is got up, the high degree of specificity of distinctive for solidified reagents advantage and immunological response is incorporated into one by it, use the synthetic method synthesis superparamagnetism functionalization tri-iron tetroxide bead of core-shell structure copolymer, its surface group and antibody is utilized to carry out coupling, can in conjunction with corresponding antigen.At present with immunomagnetic ca pture bacterium, with multifunction graphene oxide for label, have no report for the sandwich electrochemiluminescence immunosensor detecting Marine Pathogenic Bacteria.
Summary of the invention
Technical matters to be solved by this invention is to provide that a kind of detection speed is fast, testing result sensitivity and accuracy is high, the preparation method of the sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria of high specificity and application thereof.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of preparation method of the sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria, comprises the following steps:
(1) synthesis of immunomagnetic beads
The amination magnetic bead of 5 ~ 10mg is added in clean flask, add the phosphate buffered solution that 2 ~ 5mL contains the 0.1mol/LpH7.4 of 3wt% glutaraldehyde again, 37 DEG C hatch 3h after, wash 3 ~ 5 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 2 ~ 5mL; Add 50 ~ 150 μ L0.5mg/mL Marine Pathogenic Bacteria primary antibodie solution, hatch 1h for 37 DEG C, wash 3 ~ 5 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, be settled to 2 ~ 5mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add bovine serum albumin solution 50 ~ 150 μ L of 2wt% with closed nonspecific activity site, hatch 1h for 37 DEG C, wash 3 ~ 5 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, 2 ~ 5mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, the immunomagnetic beads solution that surface coverage has Marine Pathogenic Bacteria antibody can be obtained, store for future use at 4 DEG C;
(2) synthesis of multifunction graphene oxide
The 1mg/mL graphene oxide (GO) of 150 ~ 250 μ L is added in clean test tube, ultrasonic 1h, then 100 ~ 300 μ L coupling reagents are added, mix, drip 0.1mol/L watery hydrochloric acid to pH value of solution=4 ~ 6 simultaneously, after oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 0.5 ~ 1mL; Add 40 ~ 60 μ L10
-5~ 0.001mol/L electrochemiluminescence liquid solution and the anti-solution of 40 ~ 60 μ L0.01 ~ 0.1mg/mL Marine Pathogenic Bacteria two, mix, drip 0.1mol/L sodium hydroxide solution to pH value of solution=8 ~ 10 simultaneously, normal temperature oscillation incubation 4h, the centrifugal 15min of 8000r, after removing supernatant, be settled to 0.5 ~ 1mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add bovine serum albumin solution 80 ~ 120 μ L of 2wt% again with closed nonspecific activity site, normal temperature oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, 0.5 ~ 1mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, the multifunction graphene oxide solution of chemical bonding while of Marine Pathogenic Bacteria antibody and electrochemiluminescent can be obtained, store for future use at 4 DEG C;
(3) electrode modification
Be the alundum (Al2O3) slurries polishing that the magnetic glassy carbon electrode of 3 ~ 5mm uses 1.0 μm, 0.3 μm, 0.05 μm successively by diameter, with ethanol, water successively ultrasonic 2min, clean, nitrogen dries up; Get the immunomagnetic beads solution that 5 ~ 10 μ L steps (1) obtain, drip and be applied to the magnetic glassy carbon electrode center processed, namely immunomagnetic beads is firmly adsorbed on electrode surface equably; Namely the immobilized magnetic glassy carbon electrode having immunomagnetic beads is obtained after washing;
(4) assembling of sandwich electrochemiluminescence immunosensor
By in the immobilized solution having the magnetic glassy carbon electrode of immunomagnetic beads to be immersed in containing Marine Pathogenic Bacteria to be measured of step (3) gained, at 37 DEG C, hatch 1h; After cleaning, then drip in multifunction graphene oxide solution that painting 5 ~ 10 μ L step (2) obtains, at 37 DEG C, hatch 1h; After cleaning, be namely assembled into the sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria.
Described electrochemiluminescent is the terpyridyl ruthenium derivative containing amido functional group, and solvent is the Tris-HCl damping fluid of 0.1mol/LpH=7.4.
Described coupling reagent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and 10:1 is soluble in water after mixing in molar ratio obtains for N-hydroxy-succinamide (NHS), in described coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 10 ~ 100mmol/L, and described N-hydroxy-succinamide (NHS) volumetric molar concentration is 1 ~ 10mmol/L.
Described phosphate solution is Na
2hPO
412H
2o-NaH
2pO
42H
2o system buffer solution.
Described Marine Pathogenic Bacteria is vibrio parahaemolytious (VP), Vibrio vulnificus (VV), Vibrio harveyi (VH), enterobacter cloacae (EC) or Huanghai Sea Shewanella (SM).
Utilize above-mentioned sandwich electrochemiluminescence immunosensor to detect the method for Marine Pathogenic Bacteria, concrete steps are as follows: using above-mentioned sandwich electrochemiluminescence immunosensor as working electrode; Adopt platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Above-mentioned three-electrode system is put into the buffer solution containing coreaction reagent, starts electrochemical reaction, measure electrochemiluminescence intensity, obtain the electrochemiluminescence intensity level that Marine Pathogenic Bacteria solution to be measured is corresponding; The actual concentrations of Marine Pathogenic Bacteria in testing sample solution can be calculated according to the quantitative relationship between electrochemiluminescence intensity level and Marine Pathogenic Bacteria solution concentration logarithm.
The described buffer solution containing coreaction reagent is the Na of 0.1mol/LpH7 ~ 8 of the tripropyl amine (TPA) containing 20 ~ 40mmol/L
2hPO
412H
2o-NaH
2pO
42H
2o system buffer solution.
The condition of described electrochemical reaction is as follows: potential step chronoamperometry, pulse width: 0.25 second; Recurrent interval: 30 seconds; Initial voltage: 1V; Pulse voltage: 1.5V.
Principle: Marine Pathogenic Bacteria first antibody is marked on amination magnetic bead surfaces and has prepared immunomagnetic beads, for catching Marine Pathogenic Bacteria by the present invention; Electrochemiluminescent is chemically bonded to together with Marine Pathogenic Bacteria second antibody surface of graphene oxide and has prepared functional graphene oxide; Then sandwich method is adopted to construct the electrochemical immunosensor detecting Marine Pathogenic Bacteria.Functional graphene oxide has huge surface area and good electric conductivity, can effectively to extend the Helmholtz face of electrode, make loaded electrochemiluminescent be all within the Helmholtz face of electrode, do not affect the energy between electrochemiluminescent and electrode, electron transmission, also can not affect second antibody activity, final realization increases substantially the object of detection sensitivity.Sandwich electrochemiluminescence immunosensor employing " signal-on " of detection Marine Pathogenic Bacteria prepared by the present invention (open by signal, when just referring to that detected object exists, detection signal strengthens) pattern, namely after antibody and Marine Pathogenic Bacteria react, electrochemiluminescence intensity strengthens along with the concentration increase of Marine Pathogenic Bacteria, compared to electrochemiluminescence intensity along with the concentration increase of Marine Pathogenic Bacteria weakens " signal-off " (signal pass, when just referring to that detected object exists, detection signal weakens) pattern, there is good stability, background current is little, detection limit is low, sensitivity advantages of higher.
Compared with prior art, the invention has the advantages that:
(1) high sensitivity.Detectability of the present invention reaches 1CFU/mL, and detection method detection traditional is at present limited to 10
4~ 10CFU/mL.Reason is: adopt the electrochemiluminescence immunization method of " signal-on " pattern, use multifunction graphene oxide.
(2) high selectivity.The present invention is based on specific recognition between Marine Pathogenic Bacteria antibody and Marine Pathogenic Bacteria and combine the sandwich electrochemiluminescence immunosensor built, disturb bacterial classification not catch by Marine Pathogenic Bacteria antibody, therefore noiseless to this detection system.
(3) pin-point accuracy.Sandwich electrochemical luminous sensor prepared by the present invention adopts " signal-on " pattern, there is good stability, background current is little, detection limit is low, highly sensitive advantage compared with " signal-off " pattern, the equal >92% of the recovery.
In sum, the present invention has prepared with immunomagnetic ca pture bacterium, with multifunction graphene oxide for label, for detecting the sandwich electrochemiluminescence immunosensor of Marine Pathogenic Bacteria.This sensor have employed " signal-on " sandwich electrochemiluminescence immunization method, have high sensitivity, high specific, fast, stable, high repeatability and other advantages, can realize, to the detection of super low concentration Marine Pathogenic Bacteria, having a good application prospect.
Accompanying drawing explanation
Fig. 1 is the schematic diagram that sandwich electrochemiluminescence immunosensor detects Marine Pathogenic Bacteria;
Fig. 2 is ECL intensity (y)-concentration (x) the logarithmic relationship figure of variable concentrations vibrio parahaemolytious (VP);
Fig. 3 is ECL intensity (y)-concentration (x) the logarithmic relationship figure of variable concentrations Vibrio vulnificus (VV);
Fig. 4 is ECL intensity (y)-concentration (x) the logarithmic relationship figure of variable concentrations Vibrio harveyi (VH);
Fig. 5 is ECL intensity (y)-concentration (x) the logarithmic relationship figure of variable concentrations enterobacter cloacae (EC);
Fig. 6 is ECL intensity (y)-concentration (x) the logarithmic relationship figure of variable concentrations Huanghai Sea Shewanella (SM).
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment one
For detecting a preparation method for the sandwich electrochemiluminescence immunosensor of Marine Pathogenic Bacteria, comprise the following steps:
(1) synthesis of immunomagnetic beads
The amination magnetic bead of 5 ~ 10mg is added in clean flask, add the phosphate buffered solution that 2 ~ 5mL contains the 0.1mol/LpH7.4 of 3wt% glutaraldehyde again, 37 DEG C hatch 3h after, wash 3 ~ 5 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 2 ~ 5mL; Add 50 ~ 150 μ L0.5mg/mL Marine Pathogenic Bacteria primary antibodie solution, hatch 1h for 37 DEG C, wash 3 ~ 5 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, be settled to 2 ~ 5mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add bovine serum albumin solution 50 ~ 150 μ L of 2wt% with closed nonspecific activity site, hatch 1h for 37 DEG C, wash 3 ~ 5 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, 2 ~ 5mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, the immunomagnetic beads solution that surface coverage has Marine Pathogenic Bacteria antibody can be obtained, store for future use at 4 DEG C;
(2) synthesis of multifunction graphene oxide
The 1mg/mL graphene oxide (GO) of 150 ~ 250 μ L is added in clean test tube, ultrasonic 1h, then 100 ~ 300 μ L coupling reagents are added, mix, drip 0.1mol/L watery hydrochloric acid to pH value of solution=4 ~ 6 simultaneously, after oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 0.5 ~ 1mL; Add 40 ~ 60 μ L10
-5~ 0.001mol/L electrochemiluminescence liquid solution and the anti-solution of 40 ~ 60 μ L0.01 ~ 0.1mg/mL Marine Pathogenic Bacteria two, mix, drip 0.1mol/L sodium hydroxide solution to pH value of solution=8 ~ 10 simultaneously, normal temperature oscillation incubation 4h, the centrifugal 15min of 8000r, after removing supernatant, be settled to 0.5 ~ 1mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add bovine serum albumin solution 80 ~ 120 μ L of 2wt% again with closed nonspecific activity site, normal temperature oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, 0.5 ~ 1mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, the multifunction graphene oxide solution of chemical bonding while of Marine Pathogenic Bacteria antibody and electrochemiluminescent can be obtained, store for future use at 4 DEG C;
(3) electrode modification
Be the alundum (Al2O3) slurries polishing that the magnetic glassy carbon electrode of 3 ~ 5mm uses 1.0 μm, 0.3 μm, 0.05 μm successively by diameter, with ethanol, water successively ultrasonic 2min, clean, nitrogen dries up; Get the immunomagnetic beads solution that 5 ~ 10 μ L steps (1) obtain, drip and be applied to the magnetic glassy carbon electrode center processed, namely immunomagnetic beads is firmly adsorbed on electrode surface equably; Namely the immobilized magnetic glassy carbon electrode having immunomagnetic beads is obtained after washing;
(4) assembling of sandwich electrochemiluminescence immunosensor
By in the immobilized solution having the magnetic glassy carbon electrode of immunomagnetic beads to be immersed in containing Marine Pathogenic Bacteria to be measured of step (3) gained, at 37 DEG C, hatch 1h; After cleaning, then drip in multifunction graphene oxide solution that painting 5 ~ 10 μ L step (2) obtains, at 37 DEG C, hatch 1h; After cleaning, be namely assembled into the sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria.
Wherein electrochemiluminescent is the terpyridyl ruthenium derivative containing amido functional group, and solvent is the Tris-HCl damping fluid of 0.1mol/LpH=7.4; Coupling reagent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and 10:1 is soluble in water after mixing in molar ratio obtains for N-hydroxy-succinamide (NHS), in described coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 10 ~ 100mmol/L, and described N-hydroxy-succinamide (NHS) volumetric molar concentration is 1 ~ 10mmol/L; Phosphate solution is Na
2hPO
412H
2o-NaH
2pO
42H
2o system buffer solution.
Above-mentioned Marine Pathogenic Bacteria is vibrio parahaemolytious (VP), Vibrio vulnificus (VV), Vibrio harveyi (VH), enterobacter cloacae (EC) or Huanghai Sea Shewanella (SM).
Specific embodiment two
Sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria detects the method for Marine Pathogenic Bacteria, and concrete steps are as follows:
In the immobilized Marine Pathogenic Bacteria solution having the magnetic glassy carbon electrode of immunomagnetic beads to be immersed in respectively containing variable concentrations in the surface that above-mentioned specific embodiment one step (3) is prepared, incubation 1h at 37 DEG C, after intermediate water cleaning, be immersed in the multifunction graphene oxide solution that above-mentioned specific embodiment one step (2) prepares, take out after hatching 1h at 37 DEG C, as working electrode after intermediate water cleaning; Adopt platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Above-mentioned three-electrode system is put into the buffer solution containing coreaction reagent, start electrochemical reaction, measure electrochemiluminescence intensity, obtain the electrochemiluminescence intensity level that the Marine Pathogenic Bacteria solution of a series of variable concentrations is corresponding, set up the quantitative relationship between electrochemiluminescence intensity level and Marine Pathogenic Bacteria solution concentration logarithm; The actual concentrations of Marine Pathogenic Bacteria in testing sample solution can be calculated according to quantitative relationship.
Above-mentioned buffer solution is the Na2HPO of 0.1mol/LpH7 ~ 8 of the tripropyl amine (TPA) containing 20 ~ 40mmol/L
412H
2o-NaH
2pO
42H
2o system buffer solution.
The condition of above-mentioned electrochemical reaction is as follows: potential step chronoamperometry, pulse width: 0.25 second; Recurrent interval: 30 seconds; Initial voltage: 1V; Pulse voltage: 1.5V.
Above-mentioned Marine Pathogenic Bacteria is vibrio parahaemolytious (VP), Vibrio vulnificus (VV), Vibrio harveyi (VH), enterobacter cloacae (EC) or Huanghai Sea Shewanella (SM).
Specific embodiment three
A kind of for detecting the preparation method of the sandwich electrochemiluminescence immunosensor of vibrio parahaemolytious (VP), comprise the following steps:
(1) synthesis of immunomagnetic beads
The amination magnetic bead of 8mg is added in clean flask, add the phosphate buffered solution that 3.5mL contains the 0.1mol/LpH7.4 of 3wt% glutaraldehyde again, 37 DEG C hatch 3h after, 4 times are washed by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 2 ~ 5mL; Add 100 μ L0.5mg/mL vibrio parahaemolytious primary antibodie solution, hatch 1h for 37 DEG C, wash 4 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, be settled to 3mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add the bovine serum albumin solution 100 μ L of 2wt% with closed nonspecific activity site, hatch 1h for 37 DEG C, 4 times are washed by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, 3mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, the immunomagnetic beads solution that surface coverage has vibrio parahaemolytious primary antibodie can be obtained, store for future use at 4 DEG C;
(2) synthesis of multifunction graphene oxide
The 1mg/mL graphene oxide (GO) of 200 μ L is added in clean test tube, ultrasonic 1h, then 200 μ L coupling reagents are added, mix, drip 0.1mol/L watery hydrochloric acid to pH value of solution=5 simultaneously, after oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 0.8mL; Add 50 μ L10
-4mol/L electrochemiluminescence liquid solution and the anti-solution of 50 μ L0.05mg/mL vibrio parahaemolytious two, mix, drip 0.1mol/L sodium hydroxide solution to pH value of solution=9 simultaneously, normal temperature oscillation incubation 4h, the centrifugal 15min of 8000r, after removing supernatant, be settled to 0.8mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add the bovine serum albumin solution 100 μ L of 2wt% again with closed nonspecific activity site, normal temperature oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, 0.8mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, vibrio parahaemolytious two can be obtained and resist the multifunction graphene oxide solution with chemical bonding while of electrochemiluminescent, store for future use at 4 DEG C; Wherein electrochemiluminescent is the terpyridyl ruthenium derivative containing amido functional group, and solvent is the Tris-HCl damping fluid of 0.1mol/LpH=7.4; Coupling reagent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and 10:1 is soluble in water after mixing in molar ratio obtains for N-hydroxy-succinamide (NHS), in coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 50mmol/L, and described N-hydroxy-succinamide (NHS) volumetric molar concentration is 5mmol/L; Phosphate solution is Na
2hPO
412H
2o-NaH
2pO
42H
2o system buffer solution;
(3) electrode modification
Be the alundum (Al2O3) slurries polishing that the magnetic glassy carbon electrode of 4mm uses 1.0 μm, 0.3 μm, 0.05 μm successively by diameter, with ethanol, water successively ultrasonic 2min, clean, nitrogen dries up; Get the immunomagnetic beads solution that 8 μ L steps (1) obtain, drip and be applied to the magnetic glassy carbon electrode center processed, namely immunomagnetic beads is firmly adsorbed on electrode surface equably; Namely the immobilized magnetic glassy carbon electrode having immunomagnetic beads is obtained after washing;
(4) assembling of sandwich electrochemiluminescence immunosensor
By in the immobilized solution having the magnetic glassy carbon electrode of immunomagnetic beads to be immersed in containing vibrio parahaemolytious to be measured of step (3) gained, at 37 DEG C, hatch 1h; After cleaning, then drip in multifunction graphene oxide solution that painting 8 μ L step (2) obtains, at 37 DEG C, hatch 1h; After cleaning, be namely assembled into the sandwich electrochemiluminescence immunosensor for detecting vibrio parahaemolytious.
Using the above-mentioned sandwich electrochemiluminescence immunosensor for detecting vibrio parahaemolytious as working electrode; Adopt platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Three-electrode system is put into buffer solution, starts electrochemical reaction, measure electrochemiluminescence intensity; Obtain the electrochemiluminescence intensity level that the vibrio parahaemolytious solution of a series of variable concentrations is corresponding, set up the quantitative relationship between electrochemiluminescence intensity level and vibrio parahaemolytious solution concentration; ECL intensity (y)-concentration (x) the log-linear relation of variable concentrations vibrio parahaemolytious VP as shown in Figure 2.Linear equation is: y=960.7*logx+17.3, coefficient R
2=0.9823, the range of linearity is 4 – (1 × 10
8) CFU/mL, detect and be limited to 1CFU/mL.Linear good, can be used for the unknown concentration detecting vibrio parahaemolytious in sample.
Specific embodiment four
A kind of for detecting the preparation method of the sandwich electrochemiluminescence immunosensor of Vibrio vulnificus (VV), comprise the following steps:
(1) synthesis of immunomagnetic beads
The amination magnetic bead of 5mg is added in clean flask, add the phosphate buffered solution that 2mL contains the 0.1mol/LpH7.4 of 3wt% glutaraldehyde again, 37 DEG C hatch 3h after, 3 times are washed by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 2mL; Add 50 μ L0.5mg/mL Vibrio vulnificus primary antibodie solution, hatch 1h for 37 DEG C, after additional magnet magnetic resolution, wash 3 times by the phosphate buffered solution of 0.1mol/LpH7.4, after removing supernatant, be settled to 2mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add the bovine serum albumin solution 50 μ L of 2wt% with closed nonspecific activity site, hatch 1h for 37 DEG C, 3 times are washed by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, 2mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, the immunomagnetic beads solution that surface coverage has Vibrio vulnificus primary antibodie can be obtained, store for future use at 4 DEG C;
(2) synthesis of multifunction graphene oxide
The 1mg/mL graphene oxide (GO) of 150 μ L is added in clean test tube, ultrasonic 1h, then 100 μ L coupling reagents are added, mix, drip 0.1mol/L watery hydrochloric acid to pH value of solution=4 simultaneously, after oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 0.5mL; Add 40 μ L0.001mol/L electrochemiluminescence liquid solutions and the anti-solution of 40 μ L0.1mg/mL Marine Pathogenic Bacteria two, mix, drip 0.1mol/L sodium hydroxide solution to pH value of solution=8 simultaneously, normal temperature oscillation incubation 4h, the centrifugal 15min of 8000r, after removing supernatant, be settled to 0.5mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add the bovine serum albumin solution 80 μ L of 2wt% again with closed nonspecific activity site, normal temperature oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, 0.5mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, Vibrio vulnificus two can be obtained and resist the multifunction graphene oxide solution with chemical bonding while of electrochemiluminescent, store for future use at 4 DEG C; Wherein electrochemiluminescent is the terpyridyl ruthenium derivative containing amido functional group, and solvent is the Tris-HCl damping fluid of 0.1mol/LpH=7.4; Coupling reagent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and 10:1 is soluble in water after mixing in molar ratio obtains for N-hydroxy-succinamide (NHS), in described coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 10mmol/L, and described N-hydroxy-succinamide (NHS) volumetric molar concentration is 10mmol/L; Phosphate solution is Na2HPO
412H
2o-NaH
2pO
42H
2o system buffer solution;
(3) electrode modification
Be the alundum (Al2O3) slurries polishing that the magnetic glassy carbon electrode of 3mm uses 1.0 μm, 0.3 μm, 0.05 μm successively by diameter, with ethanol, water successively ultrasonic 2min, clean, nitrogen dries up; Get the immunomagnetic beads solution that 5 μ L steps (1) obtain, drip and be applied to the magnetic glassy carbon electrode center processed, namely immunomagnetic beads is firmly adsorbed on electrode surface equably; Namely the immobilized magnetic glassy carbon electrode having immunomagnetic beads is obtained after washing;
(4) assembling of sandwich electrochemiluminescence immunosensor
By in the immobilized solution having the magnetic glassy carbon electrode of immunomagnetic beads to be immersed in containing Vibrio vulnificus to be measured of step (3) gained, at 37 DEG C, hatch 1h; After cleaning, then drip in multifunction graphene oxide solution that painting 5 μ L step (2) obtains, at 37 DEG C, hatch 1h; After cleaning, be namely assembled into the sandwich electrochemiluminescence immunosensor for detecting Vibrio vulnificus.
Using the above-mentioned sandwich electrochemiluminescence immunosensor for detecting Vibrio vulnificus as working electrode; Adopt platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Three-electrode system is put into buffer solution, starts electrochemical reaction, measure electrochemiluminescence intensity; Obtain the electrochemiluminescence intensity level that the Vibrio vulnificus solution of a series of variable concentrations is corresponding, set up the quantitative relationship between electrochemiluminescence intensity level and Vibrio vulnificus solution concentration; ECL intensity (y)-concentration (x) the log-linear relation of variable concentrations Vibrio vulnificus VV as shown in Figure 3.Y=1141.8*logx+335.4, coefficient R
2=0.9873, the range of linearity is 4 – (1 × 10
8) CFU/mL, detect and be limited to 1CFU/mL.Linear good, can be used for the unknown concentration detecting Vibrio vulnificus in sample.
Specific embodiment five
A kind of for detecting the preparation method of the sandwich electrochemiluminescence immunosensor of Vibrio harveyi (VH), comprise the following steps:
(1) synthesis of immunomagnetic beads
The amination magnetic bead of 10mg is added in clean flask, add the phosphate buffered solution that 5mL contains the 0.1mol/LpH7.4 of 3wt% glutaraldehyde again, 37 DEG C hatch 3h after, 5 times are washed by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 5mL; Add 150 μ L0.5mg/mL Vibrio harveyi primary antibodie solution, hatch 1h for 37 DEG C, wash 5 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, be settled to 5mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add the bovine serum albumin solution 150 μ L of 2wt% with closed nonspecific activity site, hatch 1h for 37 DEG C, 5 times are washed by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, 5mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, the immunomagnetic beads solution that surface coverage has Vibrio harveyi primary antibodie can be obtained, store for future use at 4 DEG C;
(2) synthesis of multifunction graphene oxide
The 1mg/mL graphene oxide (GO) of 250 μ L is added in clean test tube, ultrasonic 1h, then 300 μ L coupling reagents are added, mix, drip 0.1mol/L watery hydrochloric acid to pH value of solution=6 simultaneously, after oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 1mL; Add 60 μ L10
-5mol/L electrochemiluminescence liquid solution and the anti-solution of 60 μ L0.01mg/mL Vibrio harveyi two, mix, drip 0.1mol/L sodium hydroxide solution to pH value of solution=8 simultaneously, normal temperature oscillation incubation 4h, the centrifugal 15min of 8000r, after removing supernatant, be settled to 0.5mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add bovine serum albumin solution 80 ~ 120 μ L of 2wt% again with closed nonspecific activity site, normal temperature oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, 1mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, Vibrio harveyi two can be obtained and resist the multifunction graphene oxide solution with chemical bonding while of electrochemiluminescent, store for future use at 4 DEG C; Wherein electrochemiluminescent is the terpyridyl ruthenium derivative containing amido functional group, and solvent is the Tris-HCl damping fluid of 0.1mol/LpH=7.4; Coupling reagent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and 10:1 is soluble in water after mixing in molar ratio obtains for N-hydroxy-succinamide (NHS), in described coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 100mmol/L, and N-hydroxy-succinamide (NHS) volumetric molar concentration is 1mmol/L; Phosphate solution is Na
2hPO
412H
2o-NaH
2pO
42H
2o system buffer solution;
(3) electrode modification
Be the alundum (Al2O3) slurries polishing that the magnetic glassy carbon electrode of 5mm uses 1.0 μm, 0.3 μm, 0.05 μm successively by diameter, with ethanol, water successively ultrasonic 2min, clean, nitrogen dries up; Get the immunomagnetic beads solution that 10 μ L steps (1) obtain, drip and be applied to the magnetic glassy carbon electrode center processed, namely immunomagnetic beads is firmly adsorbed on electrode surface equably; Namely the immobilized magnetic glassy carbon electrode having immunomagnetic beads is obtained after washing;
(4) assembling of sandwich electrochemiluminescence immunosensor
By in the immobilized solution having the magnetic glassy carbon electrode of immunomagnetic beads to be immersed in containing Vibrio harveyi to be measured of step (3) gained, at 37 DEG C, hatch 1h; After cleaning, then drip in multifunction graphene oxide solution that painting 10 μ L step (2) obtains, at 37 DEG C, hatch 1h; After cleaning, be namely assembled into the sandwich electrochemiluminescence immunosensor for detecting Vibrio harveyi.
Using the above-mentioned sandwich electrochemiluminescence immunosensor for detecting Vibrio harveyi as working electrode; Adopt platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Three-electrode system is put into buffer solution, starts electrochemical reaction, measure electrochemiluminescence intensity; Obtain the electrochemiluminescence intensity level that the Vibrio harveyi solution of a series of variable concentrations is corresponding, set up the quantitative relationship between electrochemiluminescence intensity level and Vibrio harveyi solution concentration; ECL intensity (y)-concentration (x) the log-linear relation of variable concentrations Vibrio harveyi VH as shown in Figure 4.Y=1091.4*logx+1265.1, coefficient R
2=0.9858, the range of linearity is 4 – (1 × 10
8) CFU/mL, detect and be limited to 1CFU/mL.Linear good, can be used for the unknown concentration detecting Vibrio harveyi in sample.
Specific embodiment six
A kind of for detecting the preparation method of the sandwich electrochemiluminescence immunosensor of enterobacter cloacae (EC) with above-mentioned specific embodiment three, its difference is that the Marine Pathogenic Bacteria adopted in preparation process is enterobacter cloacae.
Using the above-mentioned sandwich electrochemiluminescence immunosensor for detecting enterobacter cloacae as working electrode; Adopt platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Three-electrode system is put into buffer solution, starts electrochemical reaction, measure electrochemiluminescence intensity; Obtain the electrochemiluminescence intensity level that the enterobacter cloacae solution of a series of variable concentrations is corresponding, set up the quantitative relationship between electrochemiluminescence intensity level and enterobacter cloacae solution concentration; ECL intensity (y)-concentration (x) the log-linear relation of variable concentrations enterobacter cloacae EC as shown in Figure 5.Y=1047.5*logx+522.4, coefficient R
2=0.9805, the range of linearity is 4 – 1 × 10
8cFU/mL, detects and is limited to 1CFU/mL.Linear good, can be used for the unknown concentration detecting enterobacter cloacae in sample.
Specific embodiment seven
A kind of for detecting the preparation method of the sandwich electrochemiluminescence immunosensor of Huanghai Sea Shewanella (SM) with above-mentioned specific embodiment three, its difference is that the Marine Pathogenic Bacteria adopted in preparation process is Huanghai Sea Shewanella.
Using the above-mentioned sandwich electrochemiluminescence immunosensor for detecting Huanghai Sea Shewanella as working electrode; Adopt platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Three-electrode system is put into buffer solution, starts electrochemical reaction, measure electrochemiluminescence intensity; Obtain the electrochemiluminescence intensity level that the Huanghai Sea Shewanella solution of a series of variable concentrations is corresponding, set up the quantitative relationship between electrochemiluminescence intensity level and Huanghai Sea Shewanella solution concentration; ECL intensity (y)-concentration (x) the log-linear relation of variable concentrations Huanghai Sea Shewanella SM as shown in Figure 6.Y=1237*logx+618.1, coefficient R
2=0.9807, the range of linearity is 4 – 1 × 10
8cFU/mL, detects and is limited to 1CFU/mL.Linear good, can be used for the unknown concentration detecting Huanghai Sea Shewanella in sample.
Specific embodiment eight
Specific test
Choose Vibrio vulnificus (VV), selectivity that Vibrio harveyi (VH), enterobacter cloacae (EC), Huanghai Sea Shewanella (SM), vibrio parahaemolytious (VP) 5 kinds of bacterium prove the sandwich electrochemiluminescence immunosensor for detecting vibrio parahaemolytious (VP) prepared by the method for above-mentioned specific embodiment three.The vibrio parahaemolytious electrochemiluminescence immunosensor of preparation is measured 10 respectively
8vV, VH, EC, SM of CFU/mL and blank group, corresponding electrochemiluminescence intensity all about 300, and measures the VP of 4000CFU/mL, and corresponding electrochemiluminescence intensity obviously strengthens, about 4500; Vibrio parahaemolytious electrochemiluminescence immunosensor is measured containing 10 simultaneously
8the VV, 10 of CFU/mL
8the VH, 10 of CFU/mL
8the EC, 10 of CFU/mL
8the mixed solution of the VP of SM and 4000CFU/mL of CFU/mL, also about 4500.Above result display: other bacterial classifications are dense, and signal is but roughly the same with blank, represents that other bacterial classifications detect noiseless to this sensor.Result shows: vibrio parahaemolytious electrochemiluminescence immunosensor selectivity is good, and common interference bacterial classification disturbs without conspicuousness.
In like manner, the method of above-mentioned specific embodiment four prepare for detecting the electrochemiluminescence immunosensor of Vibrio vulnificus (VV), the method for the electrochemiluminescence immunosensor and specific embodiment seven that detect enterobacter cloacae (EC) that prepared by the method for specific embodiment five for detecting the electrochemiluminescence immunosensor of Vibrio harveyi (VH), prepared by the method for specific embodiment six prepare good for the equal selectivity of electrochemiluminescence immunosensor detecting Huanghai Sea Shewanella (SM), common interference bacterial classification disturbs without conspicuousness.
Specific embodiment nine
The detection of Marine Pathogenic Bacteria in seawater
Accurately pipette seawater blank sample, carry out mark-on and reclaim detection, build sensor according to the specific experiment step in above-mentioned specific embodiment three-seven and detect by specific embodiment two method, testing result is in table 1.
The testing result of table 1 many kinds of Marine Pathogenic Bacterias
From table 1 testing result, the relative standard deviation (RSD) of result is less than 11%, and the recovery is 91.3 ~ 110.3%, and show that the present invention is high for the detection precision of Marine Pathogenic Bacteria in seawater, result accurately and reliably.
These results suggest that, the electrochemiluminescence immunosensor of detection Marine Pathogenic Bacteria that the present invention builds, highly sensitive, detectability is low, selectivity is high, simple to operate, result accurately and reliably.Only need change the antibody in this electrochemiluminescence immunosensor, the high sensitivity to different target Marine Pathogenic Bacteria, specificity can be realized, simply, fast detect.
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing.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 (8)
1., for detecting a preparation method for the sandwich electrochemiluminescence immunosensor of Marine Pathogenic Bacteria, it is characterized in that comprising the following steps:
(1) synthesis of immunomagnetic beads
The amination magnetic bead of 5 ~ 10mg is added in clean flask, add the phosphate buffered solution that 2 ~ 5mL contains the 0.1mol/LpH7.4 of 3wt% glutaraldehyde again, 37 DEG C hatch 3h after, wash 3 ~ 5 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 2 ~ 5mL; Add 50 ~ 150 μ L0.5mg/mL Marine Pathogenic Bacteria primary antibodie solution, hatch 1h for 37 DEG C, wash 3 ~ 5 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, be settled to 2 ~ 5mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add bovine serum albumin solution 50 ~ 150 μ L of 2wt% with closed nonspecific activity site, hatch 1h for 37 DEG C, wash 3 ~ 5 times by the phosphate buffered solution of 0.1mol/LpH7.4 after additional magnet magnetic resolution, after removing supernatant, 2 ~ 5mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, the immunomagnetic beads solution that surface coverage has Marine Pathogenic Bacteria antibody can be obtained, store for future use at 4 DEG C;
(2) synthesis of multifunction graphene oxide
The 1mg/mL graphene oxide of 150 ~ 250 μ L is added in clean test tube, ultrasonic 1h, then 100 ~ 300 μ L coupling reagents are added, mix, drip 0.1mol/L watery hydrochloric acid to pH value of solution=4 ~ 6 simultaneously, after oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, the phosphate buffered solution of 0.1mol/LpH7.4 is settled to 0.5 ~ 1mL; Add 40 ~ 60 μ L10
-5~ 0.001mol/L electrochemiluminescence liquid solution and the anti-solution of 40 ~ 60 μ L0.01 ~ 0.1mg/mL Marine Pathogenic Bacteria two, mix, drip 0.1mol/L sodium hydroxide solution to pH value of solution=8 ~ 10 simultaneously, normal temperature oscillation incubation 4h, the centrifugal 15min of 8000r, after removing supernatant, be settled to 0.5 ~ 1mL by the phosphate buffered solution of 0.1mol/LpH7.4; Add bovine serum albumin solution 80 ~ 120 μ L of 2wt% again with closed nonspecific activity site, normal temperature oscillation incubation 1h, the centrifugal 15min of 8000r, after removing supernatant, 0.5 ~ 1mL is settled to by the phosphate buffered solution of 0.1mol/LpH7.4, the multifunction graphene oxide solution of chemical bonding while of Marine Pathogenic Bacteria antibody and electrochemiluminescent can be obtained, store for future use at 4 DEG C;
(3) electrode modification
Be the alundum (Al2O3) slurries polishing that the magnetic glassy carbon electrode of 3 ~ 5mm uses 1.0 μm, 0.3 μm, 0.05 μm successively by diameter, with ethanol, water successively ultrasonic 2min, clean, nitrogen dries up; Get the immunomagnetic beads solution that 5 ~ 10 μ L steps (1) obtain, drip and be applied to the magnetic glassy carbon electrode center processed, namely immunomagnetic beads is firmly adsorbed on electrode surface equably; Namely the immobilized magnetic glassy carbon electrode having immunomagnetic beads is obtained after washing;
(4) assembling of sandwich electrochemiluminescence immunosensor
By in the immobilized solution having the magnetic glassy carbon electrode of immunomagnetic beads to be immersed in containing Marine Pathogenic Bacteria to be measured of step (3) gained, at 37 DEG C, hatch 1h; After cleaning, then drip in multifunction graphene oxide solution that painting 5 ~ 10 μ L step (2) obtains, at 37 DEG C, hatch 1h; After cleaning, be namely assembled into the sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria.
2. the preparation method of the sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria according to claim 1, it is characterized in that: described electrochemiluminescent is the terpyridyl ruthenium derivative containing amido functional group, and solvent is the Tris-HCl damping fluid of 0.1mol/LpH=7.4.
3. the preparation method of the sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria according to claim 1, it is characterized in that: described coupling reagent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 10:1 is soluble in water after mixing in molar ratio obtains for N-hydroxy-succinamide, in described coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is 10 ~ 100mmol/L, and described N-hydroxy-succinamide volumetric molar concentration is 1 ~ 10mmol/L.
4. the preparation method of the sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria according to claim 1, is characterized in that: described phosphate solution is Na
2hPO
412H
2o-NaH
2pO
42H
2o system buffer solution.
5. the preparation method of the sandwich electrochemiluminescence immunosensor for detecting Marine Pathogenic Bacteria according to any one of claim 1-4, is characterized in that: described Marine Pathogenic Bacteria is vibrio parahaemolytious, Vibrio vulnificus, Vibrio harveyi, enterobacter cloacae or Huanghai Sea Shewanella.
6. utilize the sandwich electrochemiluminescence immunosensor according to any one of claim 1-5 to detect a method for Marine Pathogenic Bacteria, it is characterized in that concrete steps are as follows:
Using sandwich electrochemiluminescence immunosensor according to claim 1 as working electrode; Adopt platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Above-mentioned three-electrode system is put into the buffer solution containing coreaction reagent, starts electrochemical reaction, measure electrochemiluminescence intensity, obtain the electrochemiluminescence intensity level that Marine Pathogenic Bacteria solution to be measured is corresponding; The actual concentrations of Marine Pathogenic Bacteria in testing sample solution can be calculated according to the quantitative relationship between electrochemiluminescence intensity level and Marine Pathogenic Bacteria solution concentration logarithm.
7. sandwich electrochemiluminescence immunosensor according to claim 6 detects the method for Marine Pathogenic Bacteria, it is characterized in that: the described buffer solution containing coreaction reagent is the Na of 0.1mol/LpH7 ~ 8 of the tripropyl amine (TPA) containing 20 ~ 40mmol/L
2hPO
412H
2o-NaH
2pO
42H
2o system buffer solution.
8. sandwich electrochemiluminescence immunosensor according to claim 6 detects the method for Marine Pathogenic Bacteria, it is characterized in that: the condition of described electrochemical reaction is as follows: potential step chronoamperometry, pulse width: 0.25 second; Recurrent interval: 30 seconds; Initial voltage: 1V; Pulse voltage: 1.5V.
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