CN102384974B - Application of transition metal oxide - Google Patents
Application of transition metal oxide Download PDFInfo
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- CN102384974B CN102384974B CN201110211134.4A CN201110211134A CN102384974B CN 102384974 B CN102384974 B CN 102384974B CN 201110211134 A CN201110211134 A CN 201110211134A CN 102384974 B CN102384974 B CN 102384974B
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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 determination and analysis of biochemical, specifically a kind of application of transition metal oxide.
Background technology
At present, many methods have been used to the determination and analysis of biochemical.Traditional method is controlled the variation of microbial population with Maximum probable number method, this method needs long preenrichment, and then by relevant biochemical test, the process need time of general 15 days of this series of complex.In addition, enzyme linked immunoassay, fluorescence immunoassay hybridization technique and improved Maximum probable number method are also used to the detection of SRB.Although these technology have the prospect of good application, when applying the detection online with original position, also can there are some problems.Such as, because the long rise period, SRB needs the time of several days 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 molecular biotechnology accuracy is high, its operative technique is complicated, and cost is high.Recently, utilize enzyme linked immunoassay method to detect microorganism and also obtained research.This method is mainly based on identifying microorganism at the antibody passing through, and then by the chromogenic reaction by enzyme again, detects microorganism.
For sulphate reducing bacteria, at present, that the most frequently used is the Rizk[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 need considerable time (20 days).Adopted afterwards the method for the characterization compound of direct-detection sulphate reducing bacteria, such as detection method [the Tatnal R.E. based on the distinctive adenosine-5-of sulphate reducing bacteria 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 is the whether existence of detecting the contet of sulphate reducing bacteria fast, but the sensitivity detecting is not high, and needs destroy microorganisms cell.Recently, the U.S., Germany and the state such as Japanese start to utilize nucleic acid molecules technology such as adopting polymerase chain reaction, fluorescence in situ hybridization technique and the polymorphic length of restriction fragment to carry out detecting the contet of 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 detecting is high, and needs technical professional.Therefore, study a kind of effective method of detecting the contet of sulphate reducing bacteria fast and there is very important using 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 nearly 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, as far back as the analytical chemistry Shi Xiehui of Britain official in 1992, [the Gibson D.M. such as Gibson have been adopted; 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.] research the electrochemical method of leading based on electricity detect 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 by the enzyme chain immune response in biological chemistry detects salmonella.[the 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, by timing coulometry, 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.[the 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.] delivered by 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 is also applied in the detection of microorganism; 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, the also application extension application microorganism detection to the immune response based on different materials in microorganism detection from different biology sensors of research contents.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.].[the 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.] by the covering one deck fluorescent polymer at nanogold particle, then on its surface, add one deck fluorescence quenching.After microorganism is combined with nano particle, will make fluorescence quenching discharge, the fluorescence intensity of system will obtain increasing and detect microorganism.[the 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, finally by magnetic separation, detect coagulase negative staphylococcus, staphylococcus aureus and Staphylococcus epidermidis.[the 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.] propose with single wall nano carbon tube as conductive channel, at the specific antibody of nano carbon tube finishing one deck, by field effect transistor, detect the gemma of salmonella simultaneously.But the research being applied to for nano particle in the microorganism detection of electrochemica biological sensor is just at the early-stage.Recently, only had a small amount of bibliographical information, [the Lin Y.-H. such as Lin; 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 the method for the electrochemica biological sensor based on nano gold mark zymotechnic catalyzing hydrogen peroxide to detect Escherichia coli.
Summary of the invention
The object of the invention is to provide a kind of application of transition metal oxide.
For achieving the above object, the technical solution used in the present invention is:
A kind of application of transition metal oxide: transition metal oxide is the content size for detection of specific recognition biomolecule as signal mark.
Described transition metal oxide micro/nano level, size is 1-1000nm.Described transition metal oxide is manganese oxide, cobalt oxide, nickel oxide, vanadium oxide or vanadium oxide.The substrate of described transition metal oxide catalysis is dopamine, o-phenylenediamine, 2, and 2 '-azine-(3-acetyl phenyl thiazole sulfonic acid-6), tetramethyl benzidine, 4-AA or phenol are coupled substrate pair.
The present invention has advantages of: 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 signal mark, such as protein factor, nucleic acid, microorganism, virus etc., it is the characteristic of the class catalyzing enzyme that has in conjunction with the specific recognition of microorganism and antibody and transiting state metal oxide-based nanomaterial, with it, detect to detect control the variation of microbial population, can fast detecting to human body and the harmful microorganism of environment.With respect to the measurement of traditional visible ray optical density, utilize this specific character of transiting state metal oxide to there is significant specificity to detected microorganism, accuracy is high simultaneously.Utilize the immune response of transiting state metal oxide-based nanomaterial mark, there is good stability, be not easy inactivation, the advantages such as low price.
Accompanying drawing explanation
Fig. 1 for the embodiment of the present invention provide based on peroxidase enzyme linked immunoassay experiment flow figure and the immune response experiment flow figure based on transiting state metal oxide (take manganese oxide nano material as example) 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 present invention, manganese oxide nanosphere (b), manganese oxide nano wire (c), manganese oxide nano-complex (d), manganese oxide nanometer side (e) and manganese oxide nano wire catalytic substrate colour developing in there is no peroxide systems.
The catalytic kinetics lab diagram of the manganese oxide nanometer sheet (a) that Fig. 3 provides for the embodiment of the present invention, manganese oxide nanosphere (b), manganese oxide nano wire (c), manganese oxide nano-complex (d) and manganese oxide nanometer side (e) (figure A) and double reciprocal plot (figure B).
The manganese oxide nano wire (a) that Fig. 4 provides for the embodiment of the present 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 having, the catalytic activity under Hydrogen Peroxide is maximum.For HRP enzyme, p H is 3, temperature is 40 ℃, and the catalytic activity that Hydrogen Peroxide concentration is 10mM is maximum.)。
The different pH that Fig. 5 provides for the embodiment of the present invention affect figure (scheming A) for manganese oxide nanometer wire rod (a) material and peroxidase (b) stability; Different temperatures affects figure (figure B) (manganese oxide nano wire wherein: the pH that deposits adapting to is most pH5.0, and depositing its activity influence of temperature is little for material and enzyme stability.HRP enzyme: be most storage temperature lower than 40 ℃, optimal pH is 5-7.)。
Nano material activity analysis figure after four kinds of antibody modifications of the manganese oxide (c) of the manganese oxide (b) based on manganese oxide (a), the sweet modification of dextrose that Fig. 6 provides for the embodiment of the present invention, alginic acid modification and chitosan-modified manganese oxide material (d) (figure A); The component analysis figure of the qualitative analysis antibody modification after four kinds of antibody modifications of the manganese oxide (c) of the manganese oxide (b) based on manganese oxide (a), the sweet modification of dextrose, alginic acid modification and chitosan-modified manganese oxide material (d) (figure B).
The employing HRP labelling technique that Fig. 7 provides for the embodiment of the present 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
Below by embodiment, the present invention will be further described.The manganese dioxide nano line 10 μ g mL that simultaneously provide in the embodiment of the present invention
-1or 3ng mL
-1horseradish peroxidase (HRP) is at 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, then centrifugal, with Milli-Q water and absolute ethanol washing, then freeze drying.Meanwhile, other manganese dioxide nano particle, i.e. nanospheres.
Manganese dioxide nano pin and nanometer rods, referring to J.Cry.Grow.2008, the hydro-thermal method of 310,716-722 pertinent literature report is carried out synthetic method.Be specially: the manganese sulfate of 2mM potassium permanganate manganese sulfate and 2mM is dissolved in Milli-Q water (80 milliliters).Then be transferred to reactor, sealing, and be 0.5～8 the heating water thermal response time of 160 ℃, to 72 hours these products, then centrifugal, clean last freeze drying (seeing Fig. 2) with Mili-Q water.
By glucosan (DT), shitosan (CS) or alginic acid (AA), be cross-linked manganese dioxide and antibody, thereby obtain the manganese dioxide composites of antibody modification.
Manganese dioxide is fixed by polymer-modified and antibody:
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, until suspending liquid changes yellow into sepia, (illustrate and stablize glucosan, shitosan, the formation of alginic acid or plating manganese dioxide nano particle) then centrifugal and with Milli-Q washing, remove unnecessary polymkeric substance, last freeze drying.
By above-mentioned glucosan, the coated manganese dioxide nano line of shitosan or alginic acid is 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 activating, then uses 0.1mg mL
-1hatch sulfate reducing bacteria resisting antibody and fix at 4 ℃ of 12 hours antibody, use 1mg mL
-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 activating, then uses 0.1mg mL
-1hatch sulfate reducing bacteria resisting antibody and fix at 4 ℃ of 12 hours antibody, use 1mg mL
-1sodium borohydride (2mL) cessation reaction.The manganese dioxide nano line of antibody modification leaves 4 ℃ in
1mg mL
-1the manganese 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 activating, then uses 0.1mg mL
-1hatch sulfate reducing bacteria resisting antibody and fix at 4 ℃ of 12 hours antibody, use 1mg mL
-1sodium borohydride (2mL) cessation reaction.The manganese dioxide nano line of antibody modification leaves 4 ℃ in
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 determined 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.Consequent compound rinses the PBS damping fluid of 0.1M containing 0.1% bovine serum albumin(BSA) (pH value 7.2), to remove unnecessary fluorescein-labeled albumin A, and fluorescence analysis and measurement suspension (seeing Fig. 6).
Get respectively above-described embodiment 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), add respectively 50 to 800 μ MTMB, reaction 5min, then assaying reaction product absorbance.Draw the curve of TMB concentration and absorbance, during then by Michaelis-Menten equation, V=Vmax * [S]/(Km+[S]) computational dynamics constant (seeing Fig. 3).
Detect respectively manganese dioxide nano line and HRP to pH value (1-12), the dependence (seeing Fig. 4) of temperature (5-95 ℃) catalytic activity, adopts concentration of hydrogen peroxide (0-400mM) to be studied simultaneously.The manganese dioxide of nano wire and HRP have carried out a series of research (seeing Fig. 5) at pH and temperature stability.
Above-mentioned by changing the dynamic experiment that carries out carrying out under reaction conditions, do not having under concentration of hydrogen peroxide condition, by the reaction rate Mechanism Study of 50 to 800 μ M TMB.All measurements, observe under 652nm wavelength, use Beckman DU650 spectrophotometer wavelength absorbance.This kind of enzyme dynamics and kinetic parameter have carried out estimating that basis is upper, during Michaelis-Menten equation, and V=Vmax * [S]/(Km+[S]), wherein V is current reaction velocity, and Vmax is maximum reaction rate, and [s] is concentration of substrate, and Km is Michaelis constant.At michaelis-Menton kinetics, be a kind of enzyme kinetics, wherein having introduced relevant reaction rate is the irreversible enzyme reaction Rate Models of concentration of substrate.
Then by sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation again that add above-mentioned polystyrene porous plate, then in every hole, add 1%BSA to seal non-specific site and wash (pH value 7.4) three times with PBS; The last SRB that drips again different dilute concentrations in every hole cultivates two hours (from 1.8 * 10 in 96 orifice plates
1cfu mL
-1to 1.8 * 10
8cfu mL
-1), then add the anti-rat immune globulin of rabbit (Wuhan Boster Biological Technology Co., Ltd.) with HRP mark of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of manganese dioxide mark
-1), within 1 hour, hatch finally, the antibody of the second antibody of horseradish peroxidase-labeled or manganese dioxide mark is combined in to the bacterial cell determination of surface activity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).Above-described embodiment gained nano material of manganese dioxide sample is added respectively on porous plate to every hole 100 μ L (10 μ g mL
-1), and at room temperature hatch.After each hatching, porous plate carries out four times at the PBS solution with containing 0.05%Tween20 and cleans.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.By measuring SRB concentration from 1.8 * 10
1cfu mL
-1to 1.8 * 10
8cfu mL
-1absorbance numerical value, then draw the curve (as shown in Figure 7) of microorganism concn and absorbance.
Then by sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation again that add above-mentioned polystyrene porous plate, then in every hole, add 1%BSA to seal non-specific site and wash (pH value 7.4) three times with PBS; The last SRB that drips again different dilute concentrations in every hole cultivates two hours (from 1.8 * 10 in 96 orifice plates
1cfu mL
-1to 1.8 * 10
8cfu mL
-1), then add the anti-rat immune globulin of rabbit (Wuhan Boster Biological Technology Co., Ltd.) with HRP mark of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of cobalt oxide mark
-1), within 1 hour, hatch finally, the antibody of the second antibody of horseradish peroxidase-labeled or cobalt oxide mark is combined in to the bacterial cell determination of surface activity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).Above-described embodiment gained cobalt oxide nano material sample is added respectively on porous plate to every hole 100 μ L (10 μ g mL
-1), and at room temperature hatch.After each hatching, porous plate carries out four times at the PBS solution with containing 0.05%Tween20 and cleans.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.By measuring SRB concentration from 1.8 * 10
1cfu mL
-1to 1.8 * 10
8cfumL
-1absorbance numerical value, then draw the curve (as shown in Figure 7) of microorganism concn and absorbance.
Then by sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation again that add above-mentioned polystyrene porous plate, then in every hole, add 1%BSA to seal non-specific site and wash (pH value 7.4) three times with PBS; The last SRB that drips again different dilute concentrations in every hole cultivates two hours (from 1.8 * 10 in 96 orifice plates
1cfu mL
-1to 1.8 * 10
8cfu mL
-1), then add the anti-rat immune globulin of rabbit (Wuhan Boster Biological Technology Co., Ltd.) with HRP mark of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of nickel oxide mark
-1), within 1 hour, hatch finally, the antibody of the second antibody of horseradish peroxidase-labeled or nickel oxide mark is combined in to the bacterial cell determination of surface activity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).Above-described embodiment gained nickel oxide nano material sample is added respectively on porous plate to every hole 100 μ L (10 μ g mL
-1), and at room temperature hatch.After each hatching, porous plate carries out four times at the PBS solution with containing 0.05%Tween20 and cleans.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.By measuring SRB concentration from 1.8 * 10
1cfu mL
-1to 1.8 * 10
8cfumL
-1absorbance numerical value, then draw the curve of microorganism concn and absorbance.
Then by sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation again that add above-mentioned polystyrene porous plate, then in every hole, add 1%BSA to seal non-specific site and wash (pH value 7.4) three times with PBS; The last SRB that drips again different dilute concentrations in every hole cultivates two hours (from 1.8 * 10 in 96 orifice plates
1cfu mL
-1to 1.8 * 10
8cfu mL
-1), then add the anti-rat immune globulin of rabbit (Wuhan Boster Biological Technology Co., Ltd.) with HRP mark of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of nickel oxide mark
-1), within 1 hour, hatch finally, the antibody of the second antibody of horseradish peroxidase-labeled or nickel oxide mark is combined in to the bacterial cell determination of surface activity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).Above-described embodiment gained nickel oxide nano material sample is added respectively on porous plate to every hole 100 μ L (10 μ g mL
-1), and at room temperature hatch.After each hatching, porous plate carries out four times at the PBS solution with containing 0.05%Tween20 and cleans.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.By measuring SRB concentration from 1.8 * 10
1cfu mL
-1to 1.8 * 10
8cfumL
-1absorbance numerical value, then draw the curve of microorganism concn and absorbance.
Then by sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation again that add above-mentioned polystyrene porous plate, then in every hole, add 1%BSA to seal non-specific site and wash (pH value 7.4) three times with PBS; The last SRB that drips again different dilute concentrations in every hole cultivates two hours (from 1.8 * 10 in 96 orifice plates
1cfu mL
-1to 1.8 * 10
8cfu mL
-1), then add the anti-rat immune globulin of rabbit (Wuhan Boster Biological Technology Co., Ltd.) with HRP mark of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of vanadium oxide mark
-1), within 1 hour, hatch finally, the antibody of the second antibody of horseradish peroxidase-labeled or vanadium oxide mark is combined in to the bacterial cell determination of surface activity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).Above-described embodiment gained vanadium oxide nano material sample is added respectively on porous plate to every hole 100 μ L (10 μ g mL
-1), and at room temperature hatch.After each hatching, porous plate carries out four times at the PBS solution with containing 0.05%Tween20 and cleans.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.By measuring SRB concentration from 1.8 * 10
1cfu mL
-1to 1.8 * 10
8cfumL
-1absorbance numerical value, then draw the curve of microorganism concn and absorbance.
Then by sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation again that add above-mentioned polystyrene porous plate, then in every hole, add 1%BSA to seal non-specific site and wash (pH value 7.4) three times with PBS; The last SRB that drips again different dilute concentrations in every hole cultivates two hours (from 1.8 * 10 in 96 orifice plates
1cfu mL
-1to 1.8 * 10
8cfu mL
-1), then add the anti-rat immune globulin of rabbit (Wuhan Boster Biological Technology Co., Ltd.) with HRP mark of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of vanadium oxide mark
-1), within 1 hour, hatch finally, the antibody of the second antibody of horseradish peroxidase-labeled or vanadium oxide mark is combined in to the bacterial cell determination of surface activity of peroxidase, add substrate solution (TMB of 50 μ M, pH value 5.1).Above-described embodiment gained vanadium oxide nano material sample is added respectively on porous plate to every hole 100 μ L (10 μ g mL
-1), and at room temperature hatch.After each hatching, porous plate carries out four times at the PBS solution with containing 0.05%Tween20 and cleans.After 10 minutes, reaction stops, and measures the absorbance at 450nm place.By measuring SRB concentration from 1.8 * 10
1cfu mL
-1to 1.8 * 10
8cfumL
-1absorbance numerical value, then draw the curve of microorganism concn and absorbance.
Then by sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation again that add above-mentioned polystyrene porous plate, then in every hole, add 1%BSA to seal non-specific site and wash (pH value 7.4) three times with PBS; The last SRB that drips again different dilute concentrations in every hole cultivates two hours (from 1.8 * 10 in 96 orifice plates
1cfu mL
-1to 1.8 * 10
8cfu mL
-1), then add the anti-rat immune globulin of rabbit (Wuhan Boster Biological Technology Co., Ltd.) with HRP mark of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of cobalt oxide mark
-1), within 1 hour, hatch finally, the antibody of the second antibody of horseradish peroxidase-labeled or cobalt oxide mark is combined in to the bacterial cell determination of surface activity of peroxidase, add substrate solution (2 of 50 μ M, 2 '-azine-(3-acetyl phenyl thiazole sulfonic acid-6), pH value 5.1).Above-described embodiment gained cobalt oxide nano material sample is added respectively on porous plate to every hole 100 μ L (10 μ g mL
-1), and at room temperature hatch.After each hatching, porous plate carries out four times at the PBS solution with containing 0.05%Tween20 and cleans.After 10 minutes, reaction stops, and measures the absorbance at 425nm place.By measuring SRB concentration from 1.8 * 10
1cfu mL
-1to 1.8 * 10
8cfu mL
-1absorbance numerical value, then draw the curve of microorganism concn and absorbance.
Then by sulfate reducing bacteria resisting antibody (0.5mg mL
-1) plate hole 4 ℃ of the night incubation again that add above-mentioned polystyrene porous plate, then in every hole, add 1%BSA to seal non-specific site and wash (pH value 7.4) three times with PBS; The last SRB that drips again different dilute concentrations in every hole cultivates two hours (from 1.8 * 10 in 96 orifice plates
1cfu mL
-1to 1.8 * 10
8cfu mL
-1), then add the anti-rat immune globulin of rabbit (Wuhan Boster Biological Technology Co., Ltd.) with HRP mark of dilution 1/2000 or sulfate reducing bacteria resisting antibody (the 0.1mg mL of cobalt oxide mark
-1), within 1 hour, hatch finally, the antibody of the second antibody of horseradish peroxidase-labeled or cobalt oxide mark is combined in to the bacterial cell determination of surface activity of peroxidase, add substrate solution (dopamine of 50 μ M, pH value 5.1).Above-described embodiment gained cobalt oxide nano material sample is added respectively on porous plate to every hole 100 μ L (10 μ g mL
-1), and at room temperature hatch.After each hatching, porous plate carries out four times at the PBS solution with containing 0.05%Tween20 and cleans.After 10 minutes, reaction stops, and measures the absorbance at 300nm place.By measuring SRB concentration from 1.8 * 10
1cfu mL
-1to 1.8 * 10
8cfu mL
-1absorbance numerical value, then draw the curve of microorganism concn and absorbance.
Claims (3)
1. an application for transition metal oxide, is characterized in that: transition metal oxide is the content size for detection of specific recognition biomolecule as signal mark;
Described transition metal oxide is manganese oxide, cobalt oxide, nickel oxide or vanadium oxide.
2. by the application of transition metal oxide claimed in claim 1, it is characterized in that: described transition metal oxide is nanoscale, comprises nano wire, nanosphere, porous nanometer material, size is 1-1000nm.
3. by the application of transition metal oxide claimed in claim 1, it is characterized in that: the substrate of described transition metal oxide catalysis is dopamine, o-phenylenediamine, 2,2 '-azine-(3-acetyl phenyl thiazole sulfonic acid-6), tetramethyl benzidine, 4-AA or phenol are coupled substrate pair.
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| CN106248930B (en) * | 2016-07-22 | 2017-12-08 | 厦门大学 | One kind is without enzymophathy substance colorimetric analysis detection sensor |
| CN106940314B (en) * | 2017-03-09 | 2019-06-21 | 首都师范大学 | A kind of color developing agent of ascorbic acid detection and its application |
| CN108226473B (en) * | 2018-04-27 | 2020-09-11 | 中国科学院青岛生物能源与过程研究所 | Application of transition metal catalytic oxidation reduction fluorescence regulation and control system in detection of biological protein marker |
| CN110987833A (en) * | 2019-11-04 | 2020-04-10 | 河北农业大学 | Glucose biosensor material, preparation method and application |
| CN111077124B (en) * | 2019-12-30 | 2022-08-02 | 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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