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Method for identifying and detecting bacteria based on platform of functionalized magnetic nano material and gold nanocluster broad-spectrum probe

A gold nanocluster and magnetic nanotechnology, which can be used in the analysis of materials, material excitation analysis, material analysis by optical means, etc., and can solve problems such as limiting applications.

Inactive Publication Date: 2021-12-31
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these probes suffer from photobleaching and complicated preparation processes, which limit their wide application

Method used

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  • Method for identifying and detecting bacteria based on platform of functionalized magnetic nano material and gold nanocluster broad-spectrum probe
  • Method for identifying and detecting bacteria based on platform of functionalized magnetic nano material and gold nanocluster broad-spectrum probe
  • Method for identifying and detecting bacteria based on platform of functionalized magnetic nano material and gold nanocluster broad-spectrum probe

Examples

Experimental program
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preparation example Construction

[0035] Preparation of FcMBL@AuNC;

[0036] Biotinylation of FcMBL was as follows. First, FcMBL protein was incubated with PLP solution at pH 6.4 for 12 hours at room temperature. The PLP-treated protein was dialyzed 4 times against 50 mM sodium phosphate at 4°C. The resulting protein was incubated with aminobiotin for 12 hours. Following the previously reported procedure, AuNCs were synthesized with streptavidin with a small modification of glutathione (31 mM, 8 mL) and HAuCl 4The solution (25mM, 1mL) was mixed, and the pH was adjusted to 8.0±0.2 with NaOH solution (1M), and sodium borohydride (1mg / mL, 2mL) was added, and reacted at room temperature for 24h. The carboxyl groups on glutathione-modified AuNCs were covalently bound to the primary amines of streptavidin using EDC / NHS. Briefly, 20 mg AuNCs were washed three times with 10 mm PBS. EDC (10mg / mL, 100μL) and NHS (10mg / mL, 100μL) were used to activate glutathione-modified AuNCs carboxy-terminus for 30min, and strept...

Embodiment 1

[0038] Example 1 A method for identifying and detecting methicillin-resistant Staphylococcus aureus based on a platform of functionalized magnetic nanomaterials and gold nanoclusters with broad-spectrum probes , the description is to explain rather than limit the present invention. The method for identifying and detecting methicillin-resistant Staphylococcus aureus based on a platform of functionalized magnetic nanomaterials and gold nanocluster broad-spectrum probes of the present invention comprises the following steps:

[0039] 1. Different concentrations of methicillin-resistant Staphylococcus aureus (10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 and 10 9 CFU / mL, 100μL) in a centrifuge tube;

[0040] 2. Mix different concentrations of methicillin-resistant Staphylococcus aureus with APT-MB (1.25 μM, 50 μL), and incubate at 37°C for 10 minutes to complete the specific recognition of bacteria;

[0041] 3. The enriched bacteria were washed three times in PBS solution with...

Embodiment 2

[0052] Example 2 A method for identifying and detecting carbapenem-resistant Klebsiella pneumoniae based on a platform of functionalized magnetic nanomaterials and gold nanoclusters with broad-spectrum probes

[0053] The method for identifying and detecting carbapenem-resistant Klebsiella pneumoniae based on a platform of functionalized magnetic nanomaterials and gold nanocluster broad-spectrum probes of the present invention comprises the following steps: 1. Carbapenem-resistant Klebsiella pneumoniae (10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 and 10 9 CFU / mL, 100μL) in a centrifuge tube;

[0054] 2. Different concentrations of carbapenem-resistant Klebsiella pneumoniae solutions were mixed with APT-MB (1.25 μM, 50 μL), and incubated at 37°C for 10 minutes to complete the specific recognition of bacteria;

[0055] 3. The enriched bacteria were washed three times in PBS solution with pH 7 to remove non-target molecules in the system;

[0056] 4. Add FcMBL@AuNC (0.25mg / ...

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Abstract

The invention provides a method for identifying and detecting bacteria based on a platform of a functionalized magnetic nano material and a gold nanocluster broad-spectrum probe. The method comprises the following steps: (1) carrying out mixed incubation on bacteria, aptamer functionalized magnetic beads and a gold nanocluster broad-spectrum probe; (2) carrying out magnetic separation to remove supernate mainly comprising the free gold nanocluster broad-spectrum probe; (3) separating the magnetic beads and the bacteria from the obtained precipitate under the action of sodium hydroxide; (4) obtaining the bacteria with the gold nanocluster broad-spectrum probe; and (5) detecting the bacteria by a fluorescence method. According to the invention, rapid detection of bacteria in a complex sample can be realized, and rapid identification and quantitative determination can be carried out on the sample with the bacteria content of about 20CFU / mL or more.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a method for identifying and detecting bacteria based on a platform of functionalized magnetic nanomaterials and gold nanocluster broad-spectrum probes [0002] technical background [0003] The emergence of multidrug-resistant bacteria with the widespread use of antibiotics is one of the most important threats to public health and food safety. For example, Staphylococcus aureus, a common Gram-positive bacterium clinically, is a common and susceptible bacterium, including mild skin, tissue and respiratory tract infections. Methicillin is one of the broad-spectrum antibiotics commonly used to treat bacterial infections. Clinically, in order to quickly cure patients with bacterial infections, a large number of antibiotics are used or even misuse of antibiotics occurs, which has caused a substantial increase in methicillin-resistant bacteria. Among them, methicillin...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428
Inventor 余绍宁孙姣姣施海梅
Owner NINGBO UNIV