Application of transition metal oxide

A technology of transition metals and oxides, which is applied in the detection and analysis of biochemical substances, can solve the problem of high detection cost and achieve the effects of high accuracy, good stability and low price

Inactive Publication Date: 2012-03-21
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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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.

Application Domain

Material analysis

Technology Topic

PopulationAntibody +14

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  • Application of transition metal oxide
  • Application of transition metal oxide
  • Application of transition metal oxide

Examples

  • Experimental program(10)

Example Embodiment

[0019] Example 1:
[0020] For the preparation of manganese dioxide nanosheets, see J. Am. Chem. Soc. 2008, 130, 15938-15943 related literature reports. Specifically: 20mL contains 0.6M TMA and 3% H 2 O 2 Add 10 ml of 0.3M manganese chloride solution to the solution. The resulting suspension was stirred at room temperature for 12 hours, then centrifuged, washed with Milli-Q water and absolute ethanol, and then freeze-dried. At the same time, other manganese dioxide nanoparticles, namely nanospheres.
[0021] For manganese dioxide nanoneedles and nanorods, refer to the hydrothermal method reported in J. Cry. Grow. 2008, 310, 716-722. Specifically: 2mM potassium permanganate manganese sulfate and 2mM manganese sulfate were dissolved in Milli-Q water (80 ml). These products are then transferred to the reactor, sealed, and heated at 160°C for a hydrothermal reaction time of 0.5-8 to 72 hours, then centrifuged, washed with Mili-Q water, and finally freeze-dried (see figure 2 ).
[0022] The manganese dioxide and the antibody are cross-linked by dextran (DT), chitosan (CS) or alginic acid (AA) to obtain an antibody-modified manganese dioxide complex.
[0023] Manganese dioxide is fixed by polymer modification and antibody:
[0024] Manganese dioxide nanowires (50 mg) and 25 mg of dextran (DT), chitosan (CS) or alginic acid (AA) were mixed in 50 mL Milli-Q water. The mixture was stirred at room temperature for 72 hours, until the suspension changed to yellow to brown, (indicating the formation of stable glucan, chitosan, alginic acid or manganese dioxide-plated nanoparticles), then centrifuged and washed away with Milli-Q water The excess polymer is finally freeze-dried.
[0025] The above-mentioned dextran, chitosan or alginic acid coated manganese dioxide nanowires are used as anti-sulfate reducing bacteria antibody immobilization carriers. Before coupling with antibody, 1mg mL -1 Dextran modified manganese oxide nanowires (10mL) and 2mg mL -1 Sodium periodate (1mL) mixed reaction. Activated manganese dioxide nanowire surface-modified dextran, then use 0.1mg mL -1 Incubate the anti-sulfate reducing bacteria antibody at 4℃ for 12 hours and fix the antibody, use 1mg mL -1 The reaction was terminated by sodium borohydride (2 mL). Antibody modified manganese dioxide nanowires are stored at 4℃
[0026] 1mg mL -1 Chitosan modified manganese oxide nanowires (10mL) and 2mg mL -1 Sodium periodate (1mL) mixed reaction. Activated manganese dioxide nanowire surface-modified chitosan, then use 0.1mg mL -1 Incubate the anti-sulfate reducing bacteria antibody at 4℃ for 12 hours and fix the antibody, use 1mg mL -1 The reaction was terminated by sodium borohydride (2 mL). Antibody-modified manganese dioxide nanowires are stored at 4℃
[0027] 1mg mL -1 Alginic acid modified manganese oxide nanowires (10mL) and 2mg mL -1 Sodium periodate (1mL) mixed reaction. Activated manganese dioxide nanowire surface-modified alginic acid, then use 0.1mg mL -1 Incubate the anti-sulfate reducing bacteria antibody at 4℃ for 12 hours and fix the antibody, use 1mg mL -1 The reaction was terminated by sodium borohydride (2 mL). Antibody modified manganese dioxide nanowires are stored at 4℃
[0028] One 100μL 0.1mg mL -1 Antibody modified manganese dioxide nanoparticles with 100μL 0.1mg mL -1 FITC-labeled protein A was used to determine the anti-SRB antibody, and the concentration of the antibody-modified nano-manganese dioxide solution was 0.2μL in culture. The resulting complex was washed with 0.1M PBS buffer containing 0.1% bovine serum albumin (pH 7.2) to remove excess fluorescein-labeled protein A, and fluorescence analysis and measurement suspension (see Image 6 ).

Example Embodiment

[0029] Example 2
[0030] Take 10 μg each of the manganese oxide nanosheets (a), manganese oxide nanospheres (b), manganese oxide nanowires (c), and manganese oxide nanocomposites (d) obtained in the above-mentioned embodiments, respectively, add 50 to 800 μMTMB, and react for 5 min. Then the absorbance of the reaction product is measured. Draw the curve of TMB concentration and absorbance, and then when passing the Michaelis equation, V=Vmax×[S]/(Km+[S]) to calculate the kinetic constant (see image 3 ).
[0031] Simultaneously detect the dependence of manganese dioxide nanowires and HRP on pH (1-12) and temperature (5-95℃) catalytic activity (see Figure 4 ), using hydrogen peroxide concentration (0-400mM) for the study. A series of studies on the pH and temperature stability of manganese dioxide and HRP of nanowires (see Figure 5 ).
[0032] The aforementioned kinetic experiment was carried out by changing the reaction conditions, and the reaction rate mechanism of TMB from 50 to 800 μM under the condition of no hydrogen peroxide concentration was studied. All measurements were observed at a wavelength of 652nm, using a Beckman DU650 spectrophotometer wavelength absorbance. The enzyme kinetics and kinetic parameters are estimated on the basis of the Michaelis equation, V=Vmax×[S]/(Km+[S]), where V is the current reaction rate, Vmax is the maximum reaction rate, [ s] is the substrate concentration and Km is the Michaelis constant. The Mie kinetics is a kind of enzyme kinetics, which introduces the irreversible enzyme reaction rate model for the reaction rate for the substrate concentration.

Example Embodiment

[0033] Example 3
[0034] Then the anti-sulfate reducing bacteria antibody (0.5mg mL -1 ) Add to the wells of the polystyrene multi-well plate and incubate again overnight at 4°C, then add 1% BSA to each well to block non-specific sites and wash three times with PBS (pH 7.4); finally add dropwise to each well SRB with different dilution concentrations was incubated in 96-well plates for two hours (from 1.8×10 1 cfu mL -1 To 1.8×10 8 cfu mL -1 ), then add the rabbit anti-mouse immunoglobulin labeled with HRP (Wuhan Boster Bioengineering Co., Ltd.) or the anti-sulfate reducing bacteria antibody labeled with manganese dioxide (0.1mg mL -1 ), at the end of the 1 hour incubation, the secondary antibody labeled with horseradish peroxidase or the antibody labeled with manganese dioxide binds to the peroxidase to measure the surface activity of the bacterial cell, and the substrate solution (50μM TMB, pH value 5.1). The manganese dioxide nanomaterial samples obtained in the above examples were added to the porous plate, 100μL per hole (10μg mL -1 ), and incubate at room temperature. After each incubation, the multiwell plate was washed four times with 0.05% Tween20 in PBS solution. After 10 minutes, the reaction stopped and the absorbance at 450 nm was measured. By measuring the SRB concentration from 1.8×10 1 cfu mL -1 To 1.8×10 8 cfu mL -1 , Then draw the curve of microbial concentration and absorbance value (such as Figure 7 Shown).

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