阅读说明：本技术 一种基于电致化学发光检测胶质芽孢杆菌的方法 (Method for detecting bacillus mucilaginosus based on electrochemiluminescence ) 是由 杜立志 邱彦国 田相利 张艾青 于 2021-08-20 设计创作，主要内容包括：本发明公开了一种基于电致化学发光检测胶质芽孢杆菌的方法,该方法依次利用GR-PTCA与胶质芽孢杆菌表面的氨基结合,引入胶质芽孢杆菌,然后利用胶质芽孢杆菌胞外的荚膜上的酸性多糖,引入NH-(2)-GO/Au纳米复合材料,最后将修饰的电极放入检测液中,进行扫描检测。本发明基于AuNCs-H-(2)O-(2)系统,提出了一种简单、高效、灵敏的ECL实验策略用于检测胶质芽孢杆菌,利用H-(2)O-(2)对AuNCs猝灭作用,及NH-(2)-GO引起ECL信号扩增,增加检测的灵敏度。本发明可以同时应用于其他细菌的检测,该方法为国内首创；本发明利用H-(2)O-(2)能够破坏AuNCs的内部结构,引起ECL信号猝灭,进而引起信号降低,用于检测胶质芽孢杆菌,该方法低毒性、环境友好性、可调控性较高。(The invention discloses a method for detecting bacillus mucilaginosus based on electrochemiluminescence, which comprises the steps of sequentially utilizing GR-PTCA to be combined with amino on the surface of the bacillus mucilaginosus, introducing the bacillus mucilaginosus, and then utilizing acidic polysaccharide on extracellular capsule of the bacillus mucilaginosus to introduce NH 2 And finally, putting the modified electrode into detection liquid for scanning detection. The invention is based on AuNCs-H 2 O 2 The system provides a simple, efficient and sensitive ECL experimental strategy for detecting the bacillus mucilaginosus, and utilizes H 2 O 2 Quenching of AuNCs, and NH 2 GO causes ECL signal amplification, increasing the sensitivity of detection. The invention can be simultaneously applied to the detection of other bacteria, and the method is initiated at home; the invention utilizes H 2 O 2 Can destroy the internal structure of AuNCs, cause ECL signal quenching and further cause signal reductionThe method is used for detecting the bacillus mucilaginosus, and has low toxicity, environmental friendliness and high controllability.)

1. A method for detecting Bacillus mucilaginosus based on electrochemiluminescence is characterized by comprising the following steps: the method comprises the following steps:

the method comprises the following steps: diluting the bacterial liquid to 10 degrees by using a bacillus mucilaginosus screening culture medium⁵-10 ⁶cfu ml ^-1Carrying out high-throughput screening, then carrying out re-screening to obtain a bacillus mucilaginosus strain, and subpackaging;

step two: preparing GR-PTCA (graphene-perylene-3, 4,9, 10-tetracarboxylic acid nano composite material), taking 10 mu L of 2 mg/mL^-1 The GR-PTCA dispersion is dripped on the surface of a GCE electrode, and N is added₂Naturally drying under the environment;

step three: dripping 10 mu L of NHS/EDC activated liquid on the surface of the electrode modified by GR-PTCA in the second step, culturing for 1.5h at normal temperature, washing off the NHS/EDC activated liquid which is not adsorbed on the surface of the electrode by using a washing liquid, dripping 10 mu L of the Bacillus mucilaginosus obtained by screening in the first step on the surface of the electrode activated by NHS/EDC, culturing for 2h at 37 ℃, and washing off the Bacillus mucilaginosus which is not adsorbed on the surface of the electrode by using a washing liquid;

step four: dripping 10 mu L of ethanolamine on the surface of the electrode, and culturing at normal temperature for 30 min to seal unreacted blank sites;

step five: take 10. mu.L of 5 mg/mL^-1NH2-GO/Au is dripped into the electrode table modified in the fourth stepCulturing for 1.5-2.5h at 37 ℃, and then washing off unadsorbed NH on the surface of the electrode by using a washing solution₂-GO/Au；

Step six: and (5) putting the electrode modified in the step five into detection liquid for scanning detection.

2. The method for detecting bacillus mucilaginosus based on electrochemiluminescence, which is characterized by comprising the following steps of: in the fourth step, the concentration of the ethanolamine is 8-10 mM.

3. The method for detecting bacillus mucilaginosus based on electrochemiluminescence, which is characterized by comprising the following steps of: in the sixth step, the scanning conditions are as follows: the voltage scanning range is-2-0V, and the scanning speed is as follows: 100 mV/s, photomultiplier: 600V.

4. The method for detecting bacillus mucilaginosus based on electrochemiluminescence, which is characterized by comprising the following steps of: in the sixth step, the detection solution is 0.1M K₂S₂O₈，0.1 M KCl，20 mM H₂O₂20 mM hydroquinone.

5. The method for detecting bacillus mucilaginosus based on electrochemiluminescence, which is characterized by comprising the following steps of: the NH₂The preparation method of-GO/Au comprises the following steps: under the condition of vigorous stirring, aminated graphene oxide and HAuCl₄Adding sodium borohydride to the well-mixed dispersion to make HAuCl₄Reduction to obtain NH₂-GO/Au composite.

Technical Field

The invention relates to the technical field of microbial detection, in particular to a method for detecting bacillus mucilaginosus based on Electrochemiluminescence (ECL).

Background

Bacillus mucilaginosus (Bacillus mucilaginosus) is a chemoheterotrophic aerobic bacterium capable of decomposing soil and silicate minerals, and is widely applied to many fields due to functional diversity. In agriculture, the strain can change potassium and phosphorus elements which are difficult to utilize by plants in soil into effective potassium and phosphorus for the plants to utilize, has certain nitrogen fixation capacity, can secrete substances such as gibberellin, growth hormone, enzymes and the like to promote the growth and development of the plants, and is a commonly used microbial fertilizer additive strain. In animal husbandry, protein, organic acid, etc. produced during the growth process of the strain can be used for producing feed supplement in feed industry.

Industrially, the hypertrophic capsule formed outside cells by bacillus mucilaginosus and produced small molecular organic acid can decompose silicate minerals to release impurities such as silicon, iron and the like, and is commonly used in the fields of bioleaching, metallurgy and the like in recent years. The main component of the capsule outside the mycosis is acidic polysaccharide, so that the bacillus has good flocculation activity and also shows good application in the aspect of sewage treatment.

The bacillus preparation accounts for a large proportion of the market share of the environment-friendly microbial inoculant, the environment-friendly microbial inoculant in China is large in import quantity at present, the accurate identification of the content of the microbial inoculant is a standard for measuring the quality safety of the microbial inoculant, and the traditional morphological observation, physiological and biochemical characteristic analysis and other technical accuracy are low, so that the detection requirements of a large amount of environment-friendly microbial inoculants entering the China cannot be met.

Disclosure of Invention

The invention provides a method for detecting bacillus mucilaginosus based on electrochemiluminescence, which aims to make up for the defects of the prior art.

The invention is realized by the following technical scheme: a method for detecting Bacillus mucilaginosus based on electrochemiluminescence comprises the following steps:

the method comprises the following steps: diluting the bacterial liquid to 10 degrees by using a bacillus mucilaginosus screening culture medium⁵-10⁶cfu ml-1, performing high-throughput screening, then re-screening to obtain a bacillus mucilaginosus strain, and subpackaging;

step two: preparation of GR-PTCA (graphene-perylene-3, 4,9, 10-tetracarboxylic acid nanocomposite)10 μ L of 2 mg.mL-1 GR-PTCA dispersion was dropped on the surface of the GCE electrode, and N was added₂Naturally drying under the environment;

step three: dripping 10 mu L of NHS/EDC activated liquid on the surface of the electrode modified by GR-PTCA in the second step, culturing for 1.5h at normal temperature, washing off the NHS/EDC activated liquid which is not adsorbed on the surface of the electrode by using a washing liquid, dripping 10 mu L of the Bacillus mucilaginosus obtained by screening in the first step on the surface of the electrode activated by NHS/EDC, culturing for 2h at 37 ℃, and washing off the Bacillus mucilaginosus which is not adsorbed on the surface of the electrode by using a washing liquid;

step four: dripping 10 mu L of ethanolamine on the surface of the electrode, and culturing at normal temperature for 30 min to seal unreacted blank sites;

step five: take 10. mu.L of 5 mg.mL-1 NH₂Dropwise adding GO/Au onto the electrode surface modified in the fourth step, culturing at 37 ℃ for 1.5-2.5h, and washing off unadsorbed NH on the electrode surface by using a washing solution₂-GO/Au；

Step six: and (5) putting the electrode modified in the step five into detection liquid for scanning detection.

Preferably, in the fourth step, the concentration of the ethanolamine is 8-10 mM.

Preferably, in the sixth step, the scanning conditions are as follows: the voltage scanning range is-2-0V, and the scanning speed is as follows: 100 mV/s, photomultiplier: 600V.

Preferably, in the sixth step, the detection solution is 0.1M K₂S₂O₈，0.1 M KCl，20 mM H₂O₂20 mM hydroquinone.

Preferably, the NH is₂The preparation method of-GO/Au comprises the following steps: under the condition of vigorous stirring, aminated graphene oxide and HAuCl₄Adding sodium borohydride to the well-mixed dispersion to make HAuCl₄Reduction to obtain NH₂-GO/Au composite.

Compared with the prior art, the invention has the advantages that:

1. the invention is based on AuNCs-H₂O₂The system provides a simple, efficient and sensitive ECL experimental strategy for detecting the bacillus mucilaginosus, and utilizes H₂O₂Quenching of AuNCs, and the acidic polysaccharide, NH, component of the extracellular capsule of Bacillus mucilaginosus₂-GO/Au nanocomposites are introduced into the system, using NH₂GO causes ECL signal amplification. The invention can be simultaneously applied to the detection of other bacteria, and the method is initiated at home;

2. the invention utilizes H₂O₂The method can destroy the internal structure of AuNCs, causes ECL signal quenching and further causes signal reduction, is used for detecting the bacillus mucilaginosus, and has low toxicity, environmental friendliness and high controllability.

Drawings

The invention will be further described with reference to the accompanying drawings.

FIG. 1 is an ECL profile in example 2 of the present invention;

FIG. 2 is an ECL profile in example 3 of the present invention;

FIG. 3 is an ECL profile in example 4 of the present invention;

FIG. 4 is an ECL profile in example 5 of the present invention.

Detailed Description

The detailed description is only a part of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Example 1

Preparation of graphene-perylene-3, 4,9, 10-tetracarboxylic acid (GR-PTCA) nanocomposite

Firstly, 0.005g of aminated graphene oxide, which is obtained by purchasing the aminated graphene oxide, is weighed and dispersed in 5 mL of secondary distilled water, and then 1mg/mL of uniform solution is obtained after ultrasonic treatment. Then 1 g of perylene-3, 4,9, 10-tetracarboxylic dianhydride is weighed and dissolved in 10 mL of 2% KOH solution, and the reaction is carried out in a water bath at 80 ℃ for 1 h. After the reaction was completed, the pH was adjusted with hydrochloric acid to maintain it at a weakly acidic pH. The red precipitate was then collected by centrifugation and dried in vacuo to give PTCA. And mixing the 0.1% chitosan solution and 1mg/mL graphene in equal volume to obtain a chitosan-graphene solution. And adding 4975 mu L of secondary water into 10 mL of chitosan-graphene solution, performing ultrasonic dispersion, adding 25 mu L of PTCA solution with the concentration of 20 mg/mL, mixing, performing ultrasonic treatment, and centrifuging at 12000 r for 10 min to obtain the GR-PTCA nanocomposite.

Preparation of NH2-GO/Au nano composite material

Weighing 2mg of aminated graphene oxide (NH)₂-GO) dissolved in 2mL of redistilled water to make up a 1mg/mL GO solution, then 2g of HAuCl4 are weighed and dissolved in a 200mL volumetric flask to make up 1% HAuCl₄Solution 0.2mL of 1mg/mL NH was added to a round bottom flask containing 20mL of distilled water₂GO solution, after stirring for 10 minutes, 0.1mL of 1% HAuCl was added rapidly₄The solution was stirred vigorously for 10 minutes and 0.035mg NaBH was added₄. And finally stirring for 30 minutes to obtain the composite material of the GO modified nano-gold.

Bacillus mucilaginosus screening

Suspending four different bacterial liquids in a screening medium, and respectively adjusting the bacterial concentration to 10⁵-10⁶cfu ml^-1After fully shaking up, subpackaging in 96-hole culture plates, 200 mul per hole; culturing at 32 deg.C for 7 days, and rescreening; and (4) obtaining bacillus mucilaginosus after re-screening, wherein the bacillus mucilaginosus with salt tolerance is adopted in all the implementation cases.

Example 2

Take 10. mu.L of 2 mg/mL^-1 The GR-PTCA dispersion is dripped on the surface of a GCE electrode, and N is added₂Naturally drying under the environment; then, 10 mu L of NHS/EDC activated liquid is dripped on the surface of the electrode modified by GR-PTCA in the step two, the electrode is cultured for 1.5h at normal temperature, carboxyl is activated, then the NHS/EDC activated liquid which is not adsorbed on the surface of the electrode is washed away by using a washing liquid, then 10 mu L of bacillus mucilaginosus obtained by screening a first group of bacterium liquid is dripped on the surface of the electrode activated by NHS/EDC, the electrode is cultured for 2h at 37 ℃, and then the bacillus mucilaginosus which is not adsorbed on the surface of the electrode is washed away by using the washing liquid; then, 10 mu L of 8-10 mM ethanolamine is dropwise added on the surface of the electrode, and the mixture is cultured for 30 min at normal temperature to seal the unreacted blank sites; take 10. mu.L of 5 mg/mL^-1 NH₂Dripping GO/Au onto the modified electrode surface, culturing at 37 deg.C for 1.5-2.5h due to extracellular capsule of Bacillus mucilaginosusThe main component is acidic polysaccharide, which can be combined with amino on the surface of NH2-GO/Au nano composite material to be able to combine NH₂Loading GO/Au nano composite material on the surface of the electrode, and then washing away unadsorbed NH on the surface of the electrode by using a washing solution₂-GO/Au; finally, the modified electrode is put into detection liquid for scanning detection, and the detection liquid is 0.1M K₂S₂O₈，0.1 M KCl，20 mM H₂O₂20 mM hydroquinone, scanning conditions: the voltage scanning range is-2-0V, and the scanning speed is as follows: 100 mV/s, photomultiplier: 600V.

ECL spectra of the first bacterial suspension test are shown in FIG. 1

Embodiment 3

Take 10. mu.L of 2 mg/mL^-1 Dripping GR-PTCA dispersion liquid on the surface of a GCE electrode, and naturally drying in an N2 environment; then, 10 mu L of NHS/EDC activated liquid is dripped on the surface of the electrode modified by GR-PTCA in the step II, the electrode is cultured for 1.5h at normal temperature, carboxyl is activated, then the NHS/EDC activated liquid which is not adsorbed on the surface of the electrode is washed away by using a washing solution, then 10 mu L of bacillus mucilaginosus obtained by screening a second group of bacterium liquid is dripped on the surface of the electrode activated by NHS/EDC, the electrode is cultured for 2h at 37 ℃, and then the bacillus mucilaginosus which is not adsorbed on the surface of the electrode is washed away by using the washing solution; then dripping 10 mu L of 8mM ethanolamine on the surface of the electrode, and culturing for 30 min at normal temperature to seal unreacted blank sites; take 10. mu.L of 5 mg/mL^-1 NH₂Dropwise adding GO/Au onto the modified electrode surface, culturing at 37 ℃ for 1.5-2.5h, wherein the extracellular capsule of Bacillus mucilaginosus contains acidic polysaccharide capable of reacting with NH₂The amino groups on the surface of the GO/Au nanocomposite material are combined to be capable of bonding NH₂Loading GO/Au nano composite material on the surface of the electrode, and then washing away unadsorbed NH on the surface of the electrode by using a washing solution₂-GO/Au; finally, the modified electrode is put into detection liquid for scanning detection, and the detection liquid is 0.1M K₂S₂O₈，0.1 M KCl，20 mM H₂O₂20 mM hydroquinone, scanning conditions: the voltage scanning range is-2-0V, and the scanning speed is as follows: 100 mV/s, photomultiplier: 600V.

ECL spectrum of the second group of bacteria liquid detection is shown in figure 2

Example 4

Take 10. mu.L of 2 mg/mL^-1 The GR-PTCA dispersion is dripped on the surface of a GCE electrode, and N is added₂Naturally drying under the environment; then, 10 mu L of NHS/EDC activated liquid is dripped on the surface of the electrode modified by GR-PTCA in the step two, the electrode is cultured for 1.5h at normal temperature, carboxyl is activated, then the NHS/EDC activated liquid which is not adsorbed on the surface of the electrode is washed away by using a washing solution, then 10 mu L of bacillus mucilaginosus obtained by screening a third group of bacterium liquid is dripped on the surface of the electrode activated by NHS/EDC, the electrode is cultured for 2h at 37 ℃, and then the bacillus mucilaginosus which is not adsorbed on the surface of the electrode is washed away by using the washing solution; then 10 mu L10 mM ethanolamine is dripped on the surface of the electrode, and the mixture is cultured for 30 min at normal temperature to seal the unreacted blank sites; take 10. mu.L of 5 mg/mL^-1 NH₂Dropwise adding GO/Au onto the modified electrode surface, culturing at 37 ℃ for 1.5-2.5h, wherein the extracellular capsule of Bacillus mucilaginosus contains acidic polysaccharide capable of reacting with NH₂The amino groups on the surface of the GO/Au nanocomposite material are combined to be capable of bonding NH₂Loading GO/Au nano composite material on the surface of the electrode, and then washing away unadsorbed NH on the surface of the electrode by using a washing solution₂-GO/Au; finally, the modified electrode is put into detection liquid for scanning detection, and the detection liquid is 0.1M K₂S₂O₈，0.1 M KCl，20 mM H₂O₂20 mM hydroquinone, scanning conditions: the voltage scanning range is-2-0V, and the scanning speed is as follows: 100 mV/s, photomultiplier: 600V.

The ECL spectrum of the third group of bacteria liquid detection is shown in figure 3

Example 5

Take 10. mu.L of 2 mg/mL^-1 The GR-PTCA dispersion is dripped on the surface of a GCE electrode, and N is added₂Naturally drying under the environment; then 10 mu L of NHS/EDC activated liquid is dripped on the surface of the electrode modified by GR-PTCA in the step two, the electrode is cultured for 1.5h at normal temperature, carboxyl is activated, then the NHS/EDC activated liquid which is not adsorbed on the surface of the electrode is washed away by using a washing solution, then 10 mu L of Bacillus mucilaginosus obtained by screening a fourth group of bacteria liquid is dripped on the surface of the electrode activated by NHS/EDC, the electrode is cultured for 2h at 37 ℃, and then the electrode is cultured for 2h by using the washing solutionWashing away non-adsorbed bacillus mucilaginosus on the surface of the electrode; then dripping 10 mu L of 8mM ethanolamine on the surface of the electrode, and culturing for 30 min at normal temperature to seal unreacted blank sites; take 10. mu.L of 5 mg/mL^-1 NH₂Adding GO/Au dropwise onto the modified electrode surface, culturing at 37 deg.C for 1.5-2.5h, since the extracellular capsule of Bacillus mucilaginosus contains acidic polysaccharide capable of reacting with NH₂The amino groups on the surface of the GO/Au nanocomposite material are combined to be capable of bonding NH₂Loading GO/Au nano composite material on the surface of the electrode, and then washing away unadsorbed NH on the surface of the electrode by using a washing solution₂-GO/Au; finally, the modified electrode is put into detection liquid for scanning detection, and the detection liquid is 0.1M K₂S₂O₈，0.1 M KCl，20 mM H₂O₂20 mM hydroquinone, scanning conditions: the voltage scanning range is-2-0V, and the scanning speed is as follows: 100 mV/s, photomultiplier: 600V.

ECL chromatogram of fourth bacterial liquid detection is shown in FIG. 4

Example 6

Firstly, preparing standard solutions of salt-tolerant bacillus mucilaginosus bacterial solutions with different concentration gradients.

Take 10. mu.L of 2 mg/mL^-1 Dripping GR-PTCA dispersion liquid on the surface of a GCE electrode, and naturally drying in an N2 environment; then, 10 mu L of NHS/EDC activated liquid is dropwise added to the surface of the electrode modified by GR-PTCA in the second step, the electrode is cultured for 1.5h at normal temperature, carboxyl is activated, then the NHS/EDC activated liquid which is not adsorbed on the surface of the electrode is washed away by using a washing liquid, then 10 mu L of salt-resistant bacillus mucilaginosus liquid standard liquid with different concentration gradients is respectively dropwise added to the surface of the electrode activated by NHS/EDC, the electrode is cultured for 2h at 37 ℃, and then the non-adsorbed bacillus mucilaginosus on the surface of the electrode is washed away by using the washing liquid; then dripping 10 mu L of 8mM ethanolamine on the surface of the electrode, and culturing for 30 min at normal temperature to seal unreacted blank sites; take 10. mu.L of 5 mg/mL^-1 NH₂Dropwise adding GO/Au onto the modified electrode surface, culturing at 37 ℃ for 1.5-2.5h, wherein the extracellular capsule of Bacillus mucilaginosus contains acidic polysaccharide capable of reacting with NH₂The amino groups on the surface of the GO/Au nanocomposite material are combined to be capable of bonding NH₂Loading GO/Au nano composite material on the surface of the electrode, and then washing away unadsorbed NH on the surface of the electrode by using a washing solution₂-GO/Au; finally, the modified electrode is put into detection liquid for scanning detection, and the detection liquid is 0.1M K₂S₂O₈，0.1 M KCl，20 mM H₂O₂20 mM hydroquinone, scanning conditions: the voltage scanning range is-2-0V, and the scanning speed is as follows: 100 mV/s, photomultiplier: 600V.

And (3) constructing a standard curve according to the measured ECL light intensity values of the salt-tolerant bacillus mucilaginosus bacterial liquid with different concentration gradients, and then substituting the ECL light intensity values measured in the examples 2-5 into the standard curve to calculate the concentration of the salt-tolerant bacillus mucilaginosus bacterial liquid.