CN102384974A - Application of transition metal oxide - Google Patents
Application of transition metal oxide Download PDFInfo
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- CN102384974A CN102384974A CN2011102111344A CN201110211134A CN102384974A CN 102384974 A CN102384974 A CN 102384974A CN 2011102111344 A CN2011102111344 A CN 2011102111344A CN 201110211134 A CN201110211134 A CN 201110211134A CN 102384974 A CN102384974 A CN 102384974A
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Abstract
The invention relates to detection and analysis of biochemical materials, in particular to an application of a transition metal oxide. The transition metal oxide is used as a signal label and used for detecting the content of specific identification biological molecules. According to the invention, the biological molecules, such as a protein factor, nucleic acid, microbe, virus and the like, are rapidly detected and analyzed by using the transition metal oxide as the signal label, the change of microbial populations is detected and controlled by combining the specific identification of the microbe and the antibody and the characteristics of transition metal oxide nano material similar to a catalytic enzyme, and microbes harmful to the human body and the environment can be rapidly detected. Compared with the traditional measurement of visible light density, the application has remarkable specificity to the detected microbes by using the characteristic of the transition metal oxide, and has high accuracy. By using the immunoreactions of the label of the transition metal oxide nano material, the application has the advantages of good stability, difficulty in inactivation, low price and the like.
Description
Technical field
The present invention relates to the detection and the analysis of biochemical, specifically a kind of application of transition metal oxide.
Background technology
At present, many methods have been used to the detection and the analysis of biochemical.Traditional method is controlled the variation of microbial population with the most probable number MPN method, and this method needs long preenrichment, and then through relevant biochemical test, the time that the process need of these a series of complicacies is general 15 days.In addition, enzyme linked immunoassay, fluorescence immunoassay hybridization technique and improved most probable number MPN method also are used to the detection of SRB.Although these technology have good Application Prospect, when using, also can there be some problems with the online detection of original position.Such as, because the long rise period, SRB needs several days time to obtain enough metabolic products, thereby makes improved MPN method need considerable time; In addition, for the immune response of enzyme chain, although some closed reagents hinder non-specific adsorption, this is not very effective.Although the molecular biotechnology accuracy is high, its operative technique is complicated, and cost is high.Recently, utilize the enzyme linked immunoassay method to detect microorganism and also obtained research.This method mainly is based on the antibody that passes through and discerns microorganism, detects microorganism through the chromogenic reaction through enzyme more then.
For sulphate reducing bacteria, at present, that the most frequently used is Rizk [the ABD-EL-Malek Y. of Britain in 1958; Rizk S.G..Counting of Sulphate-reducing Bacteria in Mixed Bacterial Populations.Nature; 1958; 182; 538.] on Nature, deliver improved MPN cultivation, utilize gradient dilution to cultivate to detect and control the quantity of sulphate reducing bacteria population, but this method needs considerable time (20 days).Adopted the method that directly detects the characterization compound of sulphate reducing bacteria afterwards, such as detection method [Tatnal R.E. based on the distinctive adenosine of sulphate reducing bacteria-5-phosphoric acid sulfuric anhydride reductase; Stanton K.M.; Ebersole R.C..Methods of Testing for the Presence of Sulfate-reducing Bacteria.Corrosion, NACE, Houston; 1988; 88,1-34.], this method can detect the whether existence of sulphate reducing bacteria fast; But the sensitivity that detects is not high, and needs the destroy microorganisms cell.Recently; States such as the U.S., Germany and Japan begin to utilize the nucleic acid molecules technology such as adopting polymerase chain reaction, fluorescence in situ hybridization technique and the polymorphic length of restriction fragment to detect sulphate reducing bacteria [Stubner S..Quantification of Gram-negative Sulphate-reducing Bacteria in Rice Field Soil by 16S rRNA Gene-targeted Real-time PCR.J.Microbiol.Meth. based on the distinctive 16S rRNA of this microorganism; 2004; 57,219230; L ü cker S.; Steger D.; Kjeldsen K.U.; MacGregor B.J.; Wagner M.; Loy A.Improved 16S rRNA-targe ted Probe Set for Ana lysis of Sulfate-reducing Bacteriaby Fluorescence in situ Hybridization.J.Microbiol.Meth., 2007,69,523528; Gaylarde C.; Cook P..New Rapid Methods for the Identification of Sulphate-reducing Bacteria.Int.Biodeter.Biodegr., 1990,26,337-345.].The main cause that this method can not be generalizable is that the cost that detects is high, and needs the technical professional.Therefore, a kind of method that effectively detects sulphate reducing bacteria fast of research has very important use value.
With respect to traditional method; Recent two decades comes the method for flourish nano biological sensor to have the ability that high sensitivity, low detectability and real-time online detect, and this makes this method become one of focus reason of microbial rapid detection research field over past ten years.Along with the progress of research method, the content of research and result are also more deep.According to the result for retrieval from SciFinder Scholar, [Gibson D.M. such as Gibson have been adopted as far back as the analytical chemistry Shi Xiehui of Britain official in 1992; Coombs P.; Pimbley D.W..Automated conductance method for the detect ion of Salmonella in foods-collaborative study.J.AOAC Int.; 1992; 75,293-302.] electrochemical method of leading based on electricity of research detects the salmonella in the foods such as egg, fish and milk.[the Brewster J.D. such as Brewster of the U.S. in 1996; Gehring A.G.; Mazenko R.S.; Van Houten L.J.; Crawford C.J..Immunoelectrochemical Assays for Bacteria:Use of Epifluorescence Microscopy And Rapid-Scan Electrochemical Techniques in Development of an Assay for Salmonella.Anal.Chem.; 1996; 68,4153-4159.] biology sensor set up based on square wave voltammetry through the enzyme chain immune response in the biological chemistry detects salmonella.[Ertl P. such as Canadian Mikkelsen of calendar year 2001; Mikkelsen S.R..Electrochemical Biosensor Array for the Ident ification of Microorganisms Based on Lectin-Lipopolysaccharide Recognition.Anal.Chem.; 2001; 73; 4241-4248.] utilize the specific identification of agglutinin microorganism, distinguish and detect six kinds of microorganisms such as wax-shaped bacillus, golden yellow wine moon bright grape ball, proteus vulgaris, Escherichia coli, clostridium perfringen and saccharomyces cerevisiae through the timing coulometry.[Ruan C. such as Ruan in 2002; Yang L.; Li Y.; Immunobiosensor Chips for Detection of Escherichia coli O157:H7 Using Electrochemical Impedance Spectroscopy.Anal.Chem.; 2002; 74,4814-4820.] deliver the use AC impedance electrochemical technology and detected Escherichia coli.In recent years, electric potential type and anodic stripping voltammetry type biology sensor [the Bohem D.A. that also in the detection of microorganism, is applied; Gottlieb P.A.; Hua S.Z..On-chip Microfluidic Biosensor for Bacterial Detect and Identification.Sensor.Actuat.B-Chem., 2007,126,508-514; Dungchaia W.; Siangprohb W.; Chaicumpac W.; Tongtawed P.; Chailapakula O..Salmonella typhi Determination using Voltammetric Amplification of Nanoparticles:A Highly Sensitive Strategy for Metalloimmunoassay Based on a Copper-enhanced Gold Label.Talanta; 2008; 77,727 732.].
Along with the development of biological chemistry and material science, research contents also from different biology sensors in the application extension of microorganism detection to the application of immune response microorganism detection based on different materials.The development of special nanometer material science and technology; Common nano particle is widely applied to detection [the Alivisatos P..The Use of Nanocrystals in Biological Detect ion.Nat.Biotechnol. of microorganism; 2004,22 (1), 47-52; Batt C.A..Food Pathogen Detection, Science, 2007,316,1579-1580.].[Phillips R.L. such as Bunz; Miranda O.R.; You C.-C.; Rotello V.M.; Bunz U.H.F..Rapid and Eifficient Identification of Bacteria Using Gold-Nanoparticle-Poly (para-phenyleneethynylene) Construct s.AngeW.Chem.Int.Ed.; 2008; 47; 2590-2594.] through covering one deck fluorescent polymer, add one deck fluorescent quenching agent then on its surface at nanogold particle.Microorganism will make the fluorescent quenching agent discharge with after nano particle combines, and the fluorescence intensity of system will obtain increasing and detect microorganism.[Gu H. such as Xu; Ho P.-L.; Tsang K.W.T.; Wang L.; Xu B..Using Biofunctional Magnetic Nanoparticles to Capture Vancomycin-Resistant Enterococci and other Gram-Positive Bacteria at Uitralow Concentration.J.Am.Chem.Soc.; 2003; 125; 15702-15703.] be reported in magnetic nanoparticle surface coverage one deck specific antibody identification microorganism, separate through magnetic at last and detect CN-S, staphylococcus aureus and MRSE.[Villamizar R.A. such as Maroto; Maroto A.; Rius F.X.; Inza I.; Figueras M.J..Fast detection of Salmonella Infantis with carbon nanotube field effect transistors.Biosens.Bioelectron.; 2008; 24; 279-283.] as conductive channel, the while is detected the gemma of salmonella at the specific antibody of nano carbon tube finishing one deck through field effect transistor with single wall nano carbon tube in proposition.But the research that is applied to for nano particle in the microorganism detection of electrochemica biological sensor is just at the early-stage.Had only a spot of bibliographical information, [Lin Y.-H. such as Lin recently; Chen S.-H.; Chuang Y.-C.; Lu Y.-C.; Shen T.Y.; Chang C.A.; Lin C.-S..Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modified screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7.Biosens.Bioelectron.; 2008; 23,1832-1837.] utilize method to detect Escherichia coli based on the electrochemica biological sensor of nano gold mark zymotechnic catalyzing hydrogen peroxide.
Summary of the invention
The object of the invention is to provide a kind of application of transition metal oxide.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
A kind of application of transition metal oxide: transition metal oxide is used for the content size of detection specificity identification biomolecule as the signal mark.
Said transition metal oxide micro/nano level, size is 1-1000nm.Said transition metal oxide is manganese oxide, cobalt oxide, nickel oxide, vanadium oxide or vanadium oxide.The substrate of said transition metal oxide catalysis is a dopamine, o-phenylenediamine, 2, and it is right that 2 '-azine-(3-acetyl phenyl thiazole sulfonic acid-6), tetramethyl benzidine, 4-amino-antipyrine or phenol coupling join substrate.
The advantage that the present invention had: the present invention utilizes transiting state metal oxide (manganese oxide, cobalt oxide, nickel oxide, vanadium oxide and vanadium oxide) to come fast detecting and molecular biosciences molecule as the signal mark; Such as protein factor, nucleic acid, microorganism, virus etc.; It is the characteristic that combines the specific recognition of microorganism and antibody and the class catalyzing enzyme that the transiting state metal oxide-based nanomaterial has; Detect the variation of control microbial population with it, can fast detecting to human body and environment detrimental microorganisms.With respect to the measurement of traditional visible light optical density, utilize this specific character of transiting state metal oxide that the microorganism that is detected is had significant specificity, accuracy is high simultaneously.Utilize the immune response of transiting state metal oxide-based nanomaterial mark, have good stability, be not easy inactivation, advantages such as low price.
Description of drawings
Fig. 1 for the embodiment of the invention provide based on peroxidase enzyme linked immunoassay experiment flow figure with based on the immune response experiment flow figure of transiting state metal oxide (is example with the manganese oxide nano material) mark.
The variation diagram of the electron scanning micrograph of the manganese oxide nanometer sheet (a) that Fig. 2 provides for the embodiment of the invention, manganese oxide nanosphere (b), manganese oxide nano wire (c), manganese oxide nano-complex (d), manganese oxide nanometer group (e) and manganese oxide nano wire catalytic substrate colour developing in not having peroxide systems.
Catalytic kinetics lab diagram of the manganese oxide nanometer sheet (a) that Fig. 3 provides for the embodiment of the invention, manganese oxide nanosphere (b), manganese oxide nano wire (c), manganese oxide nano-complex (d) and manganese oxide nanometer group (e) (figure A) and double reciprocal plot (figure B).
The manganese oxide nano wire (a) that Fig. 4 provides for the embodiment of the invention and the catalytic activity of HRP enzyme (b) along with the variation of pH (figure A), temperature (figure B) and concentration of hydrogen peroxide (scheming C) for the effect figure of manganese oxide (wherein; PH is 5.1, temperature is 50 ℃, and the catalytic activity under the hydrogen peroxide condition is maximum having.P H is 3 for the HRP enzyme, temperature is 40 ℃, and hydrogen peroxide condition concentration is that the catalytic activity of 10mM is maximum.)。
The different pH that Fig. 5 provides for the embodiment of the invention influence figure (scheming A) for manganese oxide nanometer wire rod (a) material and peroxidase (b) stability; (manganese oxide nano wire wherein: the pH that deposits that adapts to most is pH5.0 to different temperatures, and depositing its activity influence of temperature is little for the figure (figure B) that influences of material and enzyme stability.The HRP enzyme: be most that storage temperature is lower than 40 ℃, optimal pH is 5-7.)。
Nano material activity analysis figure behind four kinds of antibody modifications of the manganese oxide (c) of the manganese oxide (b) that Fig. 6 provides for the embodiment of the invention, alginic acid modification and chitosan-modified manganese oxide material (d) (figure A) based on manganese oxide (a), the sweet modification of dextrose; The component analysis figure (scheming B) of the qualitative analysis antibody modification behind four kinds of antibody modifications of manganese oxide of modifying based on the manganese oxide (b) of manganese oxide (a), the sweet modification of dextrose, alginic acid (c) and chitosan-modified manganese oxide material (d).
The employing HRP labelling technique that Fig. 7 provides for the embodiment of the invention detects the design sketch (figure A) of microorganism; Adopt manganese oxide nanowire labels technology to detect the design sketch (figure B) of microorganism.
Embodiment
Through embodiment the present invention is further specified below.The manganese dioxide nano line 10 μ g mL that provide in the embodiment of the invention simultaneously
-1Or 3ng mL
-1Horseradish peroxidase (HRP) is at the 0.2M sodium-acetate buffer
Embodiment 1:
The preparation of manganese dioxide nano-plates, referring to J.Am.Chem.Soc.2008,130,15938-15943 pertinent literature report.Be specially: 20mL is loaded with 0.6M TMA and 3%H
2O
2Solution adds the manganese chloride solution of 10 milliliters of 0.3M.Consequent suspending liquid is at room temperature to stir 12 hours, and is centrifugal then, with Milli-Q water and absolute ethanol washing, freeze drying then.Meanwhile, other manganese dioxide nano particle, i.e. nanospheres.
Manganese dioxide nano pin and nanometer rods, referring to J.Cry.Grow.2008,310, the hydro-thermal method of 716-722 pertinent literature report is come synthetic method.Be specially: the manganese sulfate of 2mM potassium permanganate manganese sulfate and 2mM is dissolved in Milli-Q water (80 milliliters).Be transferred to agitated reactor then, sealing, and be 0.5～8 160 ℃ heating hydro-thermal reaction time, centrifugal then to 72 hours these products, clean last freeze drying (see figure 2) with Mili-Q water.
Through glucosan (DT), shitosan (CS) or crosslinked manganese dioxide of alginic acid (AA) and antibody, thereby obtain the manganese dioxide composites of antibody modification.
Manganese dioxide is fixed with antibody through polymer-modified:
The glucosan (DT) of manganese dioxide nano line (50mg) and 25mg, shitosan (CS) or alginic acid (AA) be mixed in 50mL Milli-Q water.This potpourri at room temperature stirs 72h, changes into yellowly to sepia up to suspending liquid, and (explain and stablize glucosan, shitosan, the formation of alginic acid or plating manganese dioxide nano particle) is then centrifugal and wash with Milli-Q and to remove unnecessary polymkeric substance, freeze drying at last.
With above-mentioned glucosan, shitosan or alginic acid coat the manganese dioxide nano line and are used as sulfate reducing bacteria resisting antibody immobilization carrier.Before antibody coupling, 1mg mL
-1Glucan-modified manganese oxide nano wire (10mL) and 2mg mL
-1Sodium metaperiodate (1mL) hybrid reaction.The glucosan of the manganese dioxide nano line finishing that activates is used 0.1mg mL then
-1Hatch sulfate reducing bacteria resisting antibody and fix, use 1mg mL at 4 ℃ of 12 hours antibody
-1Sodium borohydride (2mL) cessation reaction.The manganese dioxide nano line of antibody modification leaves 4 ℃ in
1mg mL
-1Chitosan-modified manganese oxide nano wire (10mL) and 2mg mL
-1Sodium metaperiodate (1mL) hybrid reaction.The shitosan of the manganese dioxide nano line finishing that activates is used 0.1mg mL then
-1Hatch sulfate reducing bacteria resisting antibody and fix, use 1mg mL at 4 ℃ of 12 hours antibody
-1Sodium borohydride (2mL) cessation reaction.The manganese dioxide nano line of antibody modification leaves 4 ℃ in
1mg mL
-1Manganese oxide nano wire (10mL) that alginic acid is modified and 2mg mL
-1Sodium metaperiodate (1mL) hybrid reaction.The alginic acid of the manganese dioxide nano line finishing that activates is used 0.1mg mL then
-1Hatch sulfate reducing bacteria resisting antibody and fix, use 1mg mL at 4 ℃ of 12 hours antibody
-1Sodium borohydride (2mL) cessation reaction.The manganese dioxide nano line of antibody modification leaves 4 ℃ in
One 100 μ L 0.1mg mL
-1The manganese dioxide nano particle of antibody modification and 100 μ L 0.1mg mL
-1The albumin A of FITC mark is confirmed the antibody of anti-SRB, and the concentration of the nanometer titanium dioxide manganese solution of this antibody modification is the cultivation of 0.2 μ L.The PBS damping fluid that consequent compound washes 0.1M contains 0.1% bovine serum albumin(BSA) (pH value 7.2), to remove unnecessary fluorescein-labeled albumin A and fluorescence analysis and measurement suspension (see figure 6).
Get the foregoing description gained manganese oxide nanometer sheet (a), manganese oxide nanosphere (b), manganese oxide nano wire (c), each 10 μ g of manganese oxide nano-complex (d) respectively, add 50 to 800 μ MTMB respectively, reaction 5min, assaying reaction product absorbance then.Draw the curve of TMB concentration and absorbance, during then through Michaelis-Menten equation, V=Vmax * [S]/(Km+ [S]) computational dynamics constant (see figure 3).
Detect manganese dioxide nano line and HRP simultaneously respectively to pH value (1-12), the dependence (see figure 4) of temperature (5-95 ℃) catalytic activity adopts concentration of hydrogen peroxide (0-400mM) to study.The manganese dioxide of nano wire and HRP have carried out a series of research (see figure 5)s at pH and temperature stability.
Above-mentioned through changing the dynamic experiment that carries out that carries out under the reaction conditions, do not having under the concentration of hydrogen peroxide condition, by the reaction rate Mechanism Study of 50 to 800 μ M TMB.All measurements are observed under the 652nm wavelength, use Beckman DU650 spectrophotometer wavelength absorbance.This kind of enzyme dynamics and kinetic parameter have carried out on the estimation basis, during Michaelis-Menten equation, and V=Vmax * [S]/(Km+ [S]), wherein V is current reaction velocity, and Vmax is a maximum response speed, and [s] is concentration of substrate, and Km is a Michaelis constant.At michaelis-Menton kinetics is a kind of enzyme kinetics, and wherein having introduced relevant reaction rate is the irreversible enzyme reaction Rate Models of concentration of substrate.
Then with sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation once more that add above-mentioned polystyrene porous plate, add in every hole then that 1%BSA seals non-specific site and with PBS give a baby a bath on the third day after its birth time (pH value 7.4); The last SRB that in every hole, drips different dilute concentrations again cultivates two hours (from 1.8 * 10 in 96 orifice plates
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1), add the anti-rat immune globulin of the rabbit with the HRP mark (Wuhan Boster Biological Technology Co., Ltd.) of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of manganese dioxide mark again
-1), hatched at last in 1 hour, the SA of horseradish peroxidase-labeled or the antibodies of manganese dioxide mark are measured in the bacterial cell surfactivity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).The foregoing description gained nano material of manganese dioxide sample is added on the porous plate to every hole 100 μ L (10 μ g mL respectively
-1), and at room temperature hatch.After each hatching, porous plate is carrying out four cleanings with the PBS solution that contains 0.05%Tween20.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.Through measuring SRB concentration from 1.8 * 10
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1Absorbance numerical value, draw the curve (as shown in Figure 7) of microorganism concn and absorbance then.
Then with sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation once more that add above-mentioned polystyrene porous plate, add in every hole then that 1%BSA seals non-specific site and with PBS give a baby a bath on the third day after its birth time (pH value 7.4); The last SRB that in every hole, drips different dilute concentrations again cultivates two hours (from 1.8 * 10 in 96 orifice plates
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1), add the anti-rat immune globulin of the rabbit with the HRP mark (Wuhan Boster Biological Technology Co., Ltd.) of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of cobalt oxide mark again
-1), hatched at last in 1 hour, the SA of horseradish peroxidase-labeled or the antibodies of cobalt oxide mark are measured in the bacterial cell surfactivity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).The foregoing description gained cobalt oxide nano material sample is added on the porous plate to every hole 100 μ L (10 μ g mL respectively
-1), and at room temperature hatch.After each hatching, porous plate is carrying out four cleanings with the PBS solution that contains 0.05%Tween20.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.Through measuring SRB concentration from 1.8 * 10
1Cfu mL
-1To 1.8 * 10
8CfumL
-1Absorbance numerical value, draw the curve (as shown in Figure 7) of microorganism concn and absorbance then.
Then with sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation once more that add above-mentioned polystyrene porous plate, add in every hole then that 1%BSA seals non-specific site and with PBS give a baby a bath on the third day after its birth time (pH value 7.4); The last SRB that in every hole, drips different dilute concentrations again cultivates two hours (from 1.8 * 10 in 96 orifice plates
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1), add the anti-rat immune globulin of the rabbit with the HRP mark (Wuhan Boster Biological Technology Co., Ltd.) of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of nickel oxide mark again
-1), hatched at last in 1 hour, the SA of horseradish peroxidase-labeled or the antibodies of nickel oxide mark are measured in the bacterial cell surfactivity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).The foregoing description gained nickel oxide nano material sample is added on the porous plate to every hole 100 μ L (10 μ g mL respectively
-1), and at room temperature hatch.After each hatching, porous plate is carrying out four cleanings with the PBS solution that contains 0.05%Tween20.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.Through measuring SRB concentration from 1.8 * 10
1Cfu mL
-1To 1.8 * 10
8CfumL
-1Absorbance numerical value, draw the curve of microorganism concn and absorbance then.
Then with sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation once more that add above-mentioned polystyrene porous plate, add in every hole then that 1%BSA seals non-specific site and with PBS give a baby a bath on the third day after its birth time (pH value 7.4); The last SRB that in every hole, drips different dilute concentrations again cultivates two hours (from 1.8 * 10 in 96 orifice plates
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1), add the anti-rat immune globulin of the rabbit with the HRP mark (Wuhan Boster Biological Technology Co., Ltd.) of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of nickel oxide mark again
-1), hatched at last in 1 hour, the SA of horseradish peroxidase-labeled or the antibodies of nickel oxide mark are measured in the bacterial cell surfactivity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).The foregoing description gained nickel oxide nano material sample is added on the porous plate to every hole 100 μ L (10 μ g mL respectively
-1), and at room temperature hatch.After each hatching, porous plate is carrying out four cleanings with the PBS solution that contains 0.05%Tween20.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.Through measuring SRB concentration from 1.8 * 10
1Cfu mL
-1To 1.8 * 10
8CfumL
-1Absorbance numerical value, draw the curve of microorganism concn and absorbance then.
Then with sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation once more that add above-mentioned polystyrene porous plate, add in every hole then that 1%BSA seals non-specific site and with PBS give a baby a bath on the third day after its birth time (pH value 7.4); The last SRB that in every hole, drips different dilute concentrations again cultivates two hours (from 1.8 * 10 in 96 orifice plates
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1), add the anti-rat immune globulin of the rabbit with the HRP mark (Wuhan Boster Biological Technology Co., Ltd.) of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of vanadium oxide mark again
-1), hatched at last in 1 hour, the SA of horseradish peroxidase-labeled or the antibodies of vanadium oxide mark are measured in the bacterial cell surfactivity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).The foregoing description gained vanadium oxide nano material sample is added on the porous plate to every hole 100 μ L (10 μ g mL respectively
-1), and at room temperature hatch.After each hatching, porous plate is carrying out four cleanings with the PBS solution that contains 0.05%Tween20.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.Through measuring SRB concentration from 1.8 * 10
1Cfu mL
-1To 1.8 * 10
8CfumL
-1Absorbance numerical value, draw the curve of microorganism concn and absorbance then.
Then with sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation once more that add above-mentioned polystyrene porous plate, add in every hole then that 1%BSA seals non-specific site and with PBS give a baby a bath on the third day after its birth time (pH value 7.4); The last SRB that in every hole, drips different dilute concentrations again cultivates two hours (from 1.8 * 10 in 96 orifice plates
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1), add the anti-rat immune globulin of the rabbit with the HRP mark (Wuhan Boster Biological Technology Co., Ltd.) of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of vanadium oxide mark again
-1), hatched at last in 1 hour, the SA of horseradish peroxidase-labeled or the antibodies of vanadium oxide mark are measured in the bacterial cell surfactivity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).The foregoing description gained vanadium oxide nano material sample is added on the porous plate to every hole 100 μ L (10 μ g mL respectively
-1), and at room temperature hatch.After each hatching, porous plate is carrying out four cleanings with the PBS solution that contains 0.05%Tween20.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.Through measuring SRB concentration from 1.8 * 10
1Cfu mL
-1To 1.8 * 10
8CfumL
-1Absorbance numerical value, draw the curve of microorganism concn and absorbance then.
Then with sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation once more that add above-mentioned polystyrene porous plate, add in every hole then that 1%BSA seals non-specific site and with PBS give a baby a bath on the third day after its birth time (pH value 7.4); The last SRB that in every hole, drips different dilute concentrations again cultivates two hours (from 1.8 * 10 in 96 orifice plates
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1), add the anti-rat immune globulin of the rabbit with the HRP mark (Wuhan Boster Biological Technology Co., Ltd.) of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of cobalt oxide mark again
-1); Hatched in 1 hour at last, the SA of horseradish peroxidase-labeled or the antibodies of cobalt oxide mark are measured in the bacterial cell surfactivity of peroxidase, add substrate solution (2 of 50 μ M; 2 '-azine-(3-acetyl phenyl thiazole sulfonic acid-6), pH value 5.1).The foregoing description gained cobalt oxide nano material sample is added on the porous plate to every hole 100 μ L (10 μ g mL respectively
-1), and at room temperature hatch.After each hatching, porous plate is carrying out four cleanings with the PBS solution that contains 0.05%Tween20.After 10 minutes, reaction stops, and measures the absorbance at 425nm place.Through measuring SRB concentration from 1.8 * 10
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1Absorbance numerical value, draw the curve of microorganism concn and absorbance then.
Then with sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation once more that add above-mentioned polystyrene porous plate, add in every hole then that 1%BSA seals non-specific site and with PBS give a baby a bath on the third day after its birth time (pH value 7.4); The last SRB that in every hole, drips different dilute concentrations again cultivates two hours (from 1.8 * 10 in 96 orifice plates
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1), add the anti-rat immune globulin of the rabbit with the HRP mark (Wuhan Boster Biological Technology Co., Ltd.) of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of cobalt oxide mark again
-1), hatched at last in 1 hour, the SA of horseradish peroxidase-labeled or the antibodies of cobalt oxide mark are measured in the bacterial cell surfactivity of peroxidase, add substrate solution (dopamine of 50 μ M, pH value 5.1).The foregoing description gained cobalt oxide nano material sample is added on the porous plate to every hole 100 μ L (10 μ g mL respectively
-1), and at room temperature hatch.After each hatching, porous plate is carrying out four cleanings with the PBS solution that contains 0.05%Tween20.After 10 minutes, reaction stops, and measures the absorbance at 300nm place.Through measuring SRB concentration from 1.8 * 10
1Cfu mL
-1To 1.8 * 10
8Cfu mL
-1Absorbance numerical value, draw the curve of microorganism concn and absorbance then.
Claims (4)
1. the application of a transition metal oxide is characterized in that: transition metal oxide is used for the content size of detection specificity identification biomolecule as the signal mark.
2. by the application of the described transition metal oxide of claim 1, it is characterized in that: said transition metal oxide micro/nano level, comprise nano wire, nanosphere, porous nanometer material, size is 1-1000nm.
3. by the application of claim 1 or 2 described transition metal oxides, it is characterized in that: said transition metal oxide is manganese oxide, cobalt oxide, nickel oxide or vanadium oxide.
4. press the application of the described transition metal oxide of claim 1; It is characterized in that: the substrate of said transition metal oxide catalysis is a dopamine; O-phenylenediamine, 2, it is right that 2 '-azine-(3-acetyl phenyl thiazole sulfonic acid-6), tetramethyl benzidine, 4-amino-antipyrine or phenol coupling join substrate.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104076153A (en) * | 2014-05-14 | 2014-10-01 | 中国科学院海洋研究所 | Signal system for marking sucrase on basis of active functional molecules and application of signal system |
| CN106248930A (en) * | 2016-07-22 | 2016-12-21 | 厦门大学 | A kind of without enzymophathy substance colorimetric analysis detection sensor |
| CN106940314A (en) * | 2017-03-09 | 2017-07-11 | 首都师范大学 | A kind of developer of ascorbic acid detection and its application |
| CN108226473A (en) * | 2018-04-27 | 2018-06-29 | 中国科学院青岛生物能源与过程研究所 | A kind of application based on transition metal-catalyzed redox fluorescence regulation and control system in bioprotein marks analyte detection |
| CN110987833A (en) * | 2019-11-04 | 2020-04-10 | 河北农业大学 | Glucose biosensor material, preparation method and application |
| CN111077124A (en) * | 2019-12-30 | 2020-04-28 | 中国科学院烟台海岸带研究所 | Red-yellow-blue three-fluorescence emission sensor and preparation and application thereof |
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2011
- 2011-07-22 CN CN201110211134.4A patent/CN102384974B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| AFAF EL-ANSARY: "Nanoparticles as biochemical sensors", 《NANOTECHNOLOGY, SCIENCE AND APPLICATIONS》 * |
| ATUL ASATI: "Oxidase-Like Activity of Polymer-Coated Cerium Oxide Nanoparticles", 《ANGEWANDTE CHEMIE INTERNATION EDITION》 * |
| LIZENG GAO: "Intrinsic peroxidase-like activity of ferromagnetic nanoparticles", 《NATURE NANOTECHNOLOGY》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104076153A (en) * | 2014-05-14 | 2014-10-01 | 中国科学院海洋研究所 | Signal system for marking sucrase on basis of active functional molecules and application of signal system |
| CN106248930A (en) * | 2016-07-22 | 2016-12-21 | 厦门大学 | A kind of without enzymophathy substance colorimetric analysis detection sensor |
| CN106248930B (en) * | 2016-07-22 | 2017-12-08 | 厦门大学 | One kind is without enzymophathy substance colorimetric analysis detection sensor |
| CN106940314A (en) * | 2017-03-09 | 2017-07-11 | 首都师范大学 | A kind of developer of ascorbic acid detection and its application |
| CN108226473A (en) * | 2018-04-27 | 2018-06-29 | 中国科学院青岛生物能源与过程研究所 | A kind of application based on transition metal-catalyzed redox fluorescence regulation and control system in bioprotein marks analyte detection |
| CN110987833A (en) * | 2019-11-04 | 2020-04-10 | 河北农业大学 | Glucose biosensor material, preparation method and application |
| CN111077124A (en) * | 2019-12-30 | 2020-04-28 | 中国科学院烟台海岸带研究所 | Red-yellow-blue three-fluorescence emission sensor and preparation and application thereof |
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| CN102384974B (en) | 2014-03-05 |
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