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Enzymatic Low Field NMR Immunosensor for Detection of Foodborne Pathogenic Bacteria

A low-field nuclear magnetic resonance, food-borne pathogenic bacteria technology, applied in the field of analytical chemistry and food safety, can solve the problems of incompetent sensitivity and accuracy, low sensitivity, complex food samples, etc., to achieve the limit of broadening the detection range, Simplify the magnetic signal analysis process, the effect of simple suspension stability

Active Publication Date: 2022-03-22
HUAZHONG AGRI UNIV
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  • Abstract
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Problems solved by technology

However, the traditional low-field nuclear magnetic resonance immunosensor also has the following problems: (1) the lack of effective signal amplification system, the sensitivity is low, especially the detection of trace food-borne pathogens, its sensitivity and accuracy are difficult to be competent; ( 2) The stability is relatively poor: some factors will lead to the aggregation of magnetic particles, resulting in false positive results, which in turn affects the reliability of the results; (3) The process of preparing super-parallel nano-sized magnetic particles with good stability is relatively complicated and costly. It is also relatively high, and the repeatability and stability need to be further improved. Therefore, it is particularly important to develop a magnetic signal probe with better stability, lower cost, and better operability.
Fe(III) / Fe(II), Cu(II) / Cu(I) are common paramagnetic ions, and the transformation of their different valence states can lead to the transverse relaxation time (T 2 ) or longitudinal relaxation time (T 1 ), but the T 1 or T 2 The signal change is limited, because the two groups of paramagnetic ions with different valence states have magnetic signals themselves, but the magnetic signal changes from weak to strong, so the change of the magnetic signal is not significant, and it is difficult to use trace food sources in complex food samples Analysis of Sexual Pathogens

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  • Enzymatic Low Field NMR Immunosensor for Detection of Foodborne Pathogenic Bacteria
  • Enzymatic Low Field NMR Immunosensor for Detection of Foodborne Pathogenic Bacteria
  • Enzymatic Low Field NMR Immunosensor for Detection of Foodborne Pathogenic Bacteria

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Embodiment 1

[0051] Alkaline phosphatase (ALP) is a labeling enzyme widely used in immunoassays. At the same time, ALP can catalyze its substrate (ascorbyl phosphate) to produce reducing ascorbic acid (AA). The redox reaction mediated by AA Can achieve MnO 4 - to Mn 2+ transformation, resulting in T 2 signal change. Given MnO 4 - The strong oxidation of AA and the strong reduction of AA, the redox reaction catalyzed by ALP can realize the ultrasensitive response to AA. Based on this principle, a magnetic immunosensor can be constructed to detect different targets.

[0052] 1)T 2 Magnetic sensor response to ascorbic acid (AA)

[0053] Add 100 μL of ascorbic acid solutions with different concentrations (1, 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 μM) to 100 μL of 0.5 mM KMnO 4 solution, reacted for 5 minutes, and measured the T 2 signal, the result of which is figure 2 Shown, T 2 The change in value becomes larger with the increase of ascorbic acid ...

Embodiment 2

[0064] The detection of embodiment 2 Salmonella

[0065] (1) Preparation of MNPs-capture antibody conjugates: Take 500 μL 1000nm COOH-MNPs, wash twice with pure water, and resuspend with 2 mL pure water; add 100 μL EDC (10 mg / mL) and 50 μL NHS (10 mg / mL ) activation for 30 minutes; resuspend with 2mL PBS (pH=7.4) after magnetic separation; add 0.2mg Salmonella capture antibody (Ab 1 ), reacted with gentle shaking for 3 hours, added 500 μL of 3% BSA for 30 minutes to block unbound sites; washed 4 to 5 times with PBST, and finally resuspended with 2 mL of PBS and stored at 4°C for later use.

[0066] (2) Biotinylation of Salmonella detection antibody: first, EZ-Link TM Sulfo-NHS-LC-LC-Biotin was diluted to 1mg / mL with dimethylformamide, and the Salmonella detection antibody (Ab 2 ) was diluted to 1 mg / mL with PBS; then the two were mixed at a molar ratio of 30:1 (biotin / detection antibody), and the reaction was shaken at room temperature for 4 hours to realize the biotinylatio...

Embodiment 3

[0074] Embodiment 3 detects the specificity investigation of Salmonella

[0075] (1) Dilute the cultured Salmonella, Escherichia coli, Staphylococcus aureus, Listeria and Vibrio parahaemolyticus to 10% respectively with normal saline 5 CFU / mL, take 400 μL each to a 1.5 mL centrifuge tube, then add 100 μL MNPs-Salmonella capture antibody solution, mix well and react gently at 37°C for 30 minutes.

[0076] (2) Magnetic separation, washing with PBST for 3 times, then adding biotinylated Salmonella detection antibody to each centrifuge tube, and reacting gently at 37°C for 30 minutes.

[0077] (3) The excess biotinylated antibody was removed by magnetic separation, and the resulting complex was washed 3 times with PBST.

[0078] (4) Add a certain amount of streptavidin-labeled alkaline phosphatase to the complex obtained in (3), react gently at 37°C for 30 minutes, magnetically separate, wash the complex 4 times with PBST, and finally use Resuspend the complex in 100 μL pure wat...

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Abstract

The invention discloses a method for detecting food-borne pathogenic bacteria by an enzymatic low-field nuclear magnetic resonance immunosensor, the main content of which is to use Mn(VII) / Mn(II) as a signal readout system, combined with immune analysis technology, to construct A magnetic immunosensor based on antibody-antigen recognition. In this method, alkaline phosphatase (ALP) is applied to the sensor as an immunolabeling enzyme, and ascorbyl phosphate is used as the substrate of ALP. ALP can remove ascorbyl phosphate Phosphorylation, and then converted to reducing ascorbic acid, and ascorbic acid can convert Mn(VII) to Mn(II), and then cause a magnetic signal (transverse relaxation time, T 2 ) change from scratch, T 2 The amount of change was positively correlated with the food-borne pathogenic bacteria in the tested samples. The invention broadens the limit of the detection range of the sensor based on the superparamagnetic nano-magnetic particle magnetic relaxation time, greatly improves the analysis performance, and further provides a powerful tool for the early prevention and control of food safety.

Description

technical field [0001] The invention belongs to the fields of analytical chemistry and food safety, in particular, the invention relates to a method for detecting pathogenic bacteria by an alkaline phosphatase-catalyzed low-field nuclear magnetic resonance immunosensor. Background technique [0002] Foodborne pathogens are an important factor leading to food safety problems. Rapid and highly sensitive detection of foodborne pathogens is of great significance to ensure food safety. At present, the main detection methods are microbial culture method, immunological detection method and PCR molecular diagnosis method. The microbial culture method requires a long time for cultivation, and the operation steps are cumbersome, so it is not suitable for on-site and rapid detection. Immunological detection methods mainly include enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatographic test strip method. The enzyme-linked immunosorbent assay requires multi-s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N24/08G01N33/569G01N33/543
CPCG01N24/087G01N33/56911G01N33/54326G01N33/54346
Inventor 陈翊平王知龙
Owner HUAZHONG AGRI UNIV
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