Biosensor for detecting salmonella based on aptamer and preparation method thereof and application

Abstract

The invention relates to the technical field of biosensors, in particular to a biosensor for detecting salmonella based on an aptamer. Based on the specific recognition of the aptamer and a target, ahairpin probe HAP is opened, S1 is replaced from a composite probe S by using a fulcrum-mediated strand displacement reaction, a sequence for forming a G-quadruplex is exposed from the replaced S in acatalytic hairpin self-assembly (CHA) amplification manner, and the G-quadruplex / heme DNA enzyme is formed in the presence of heme. Cysteine is oxidized into cystine by using the catalytic performance of G-quadruplex / heme horseradish peroxidase, charge transfer between cysteine and silver clusters via a gold-sulfur bond cannot be realized, fluorescence signal conduction is regulated and controlled, the aptamer biosensor is constructed, and the biosensor only needs one step in reaction, so that the biosensor has the advantages of high detection speed, simplicity and convenience in operation, low price, low detection limit, high specificity and the like.

Description

technical field

[0001] The invention relates to the technical field of biosensors, in particular to a biosensor for detecting Salmonella based on nucleic acid aptamers, and also relates to a preparation method thereof. Background technique

[0002] Salmonella is a common foodborne pathogen, a Gram-negative, intracellular parasitic enteric bacterium. The bacteria widely exist in nature, not only can cause acute, chronic or latent infection of livestock, poultry and other animals, but also cause food poisoning by contaminating food, posing a great threat to human beings. According to statistics, among the types of bacterial food poisoning in various countries in the world, food poisoning caused by Salmonella often ranks first. Salmonella kills 700,000 people every year. Ten million people are expected to die globally by 2050 if no action is taken to stop this global threat. Salmonella is the most common pathogenic bacteria in food poisoning in my country, accounting for the...

Examples

Embodiment 1

[0062] The preparation method of described fluorescent biosensor comprises the following steps:

[0063] The synthetic operation steps of composite probe S are as follows:

[0064] Add 18 μL of sterilized water, 3 μL of 10×PB, 3 μL of 100 μM S0 probe and 6 μL of 100 μM S1 probe into the pre-prepared sterilized EP tube, shake for 30 seconds, and incubate at 95°C Incubate for 5 minutes, slowly cool down to room temperature and hybridize into probes, and store at -20°C for later use.

[0065] The steps for the synthesis of DNA silver nanoclusters (AgNCs-DNA) are as follows:

[0066] Add 15 μL of 100 μM Nucleic Acid Strand C and 73 μL of 20 mM PB (pH 7.0) buffer into the EP tube wrapped in foil paper, and then add 6 μL of 1.5 mM AgNO3 solution (to ensure the ratio of Ag+ to H3 6:1), shake for 1 min, and place at 4 °C for 30 min; after 30 min, continue to add 6 μL of 1.5 mM NaBH4 to the EP tube, shake for 1 min, and place at 4 °C in the dark for more than 6 h. ;

[0067] The ma...

Embodiment 2

[0074] The preparation method of described fluorescent biosensor comprises the following steps:

[0075] The synthetic operation steps of composite probe S are as follows:

[0076] Add 18 μL of sterilized water, 3 μL of 10×PB, 3 μL of 100 μM S0 probe and 6 μL of 100 μM S1 probe into the pre-prepared sterilized EP tube, shake for 30 seconds, and incubate at 95°C Incubate for 5 minutes, slowly cool down to room temperature and hybridize into probes, and store at -20°C for later use.

[0077] The steps for the synthesis of DNA silver nanoclusters (AgNCs-DNA) are as follows:

[0078] Add 15 μL of 100 μM Nucleic Acid Strand C and 73 μL of 20 mM PB (pH 7.0) buffer into the EP tube wrapped in foil paper, and then add 6 μL of 1.5 mM AgNO3 solution (to ensure the ratio of Ag+ to H3 6:1), shake for 1 min, and place at 4 °C for 30 min; after 30 min, continue to add 6 μL of 1.5 mM NaBH4 to the EP tube, shake for 1 min, and place at 4 °C in the dark for more than 6 h. ;

[0079] The ma...

Embodiment 3

[0086] The preparation method of described fluorescent biosensor comprises the following steps:

[0087] The synthetic operation steps of composite probe S are as follows:

[0088] Add 18 μL of sterilized water, 3 μL of 10×PB, 3 μL of 100 μM S0 probe and 6 μL of 100 μM S1 probe into the pre-prepared sterilized EP tube, shake for 30 seconds, and incubate at 95°C Incubate for 5 minutes, slowly cool down to room temperature and hybridize into probes, and store at -20°C for later use.

[0089] The steps for the synthesis of DNA silver nanoclusters (AgNCs-DNA) are as follows:

[0090] Add 15 μL of 100 μM Nucleic Acid Strand C and 73 μL of 20 mM PB (pH 7.0) buffer into the EP tube wrapped in foil paper, and then add 6 μL of 1.5 mM AgNO3 solution (to ensure the ratio of Ag+ to H3 6:1), shake for 1 min, and place at 4 °C for 30 min; after 30 min, continue to add 6 μL of 1.5 mM NaBH4 to the EP tube, shake for 1 min, and place at 4 °C in the dark for more than 6 h. ;

[0091] The ma...

technical field

[0001] The invention relates to the technical field of biosensors, in particular to a biosensor for detecting Salmonella based on nucleic acid aptamers, and also relates to a preparation method thereof. Background technique

[0002] Salmonella is a common foodborne pathogen, a Gram-negative, intracellular parasitic enteric bacterium. The bacteria widely exist in nature, not only can cause acute, chronic or latent infection of livestock, poultry and other animals, but also cause food poisoning by contaminating food, posing a great threat to human beings. According to statistics, among the types of bacterial food poisoning in various countries in the world, food poisoning caused by Salmonella often ranks first. Salmonella kills 700,000 people every year. Ten million people are expected to die globally by 2050 if no action is taken to stop this global threat. Salmonella is the most common pathogenic bacteria in food poisoning in my country, accounting for the...

Examples

Embodiment 1

[0062] The preparation method of described fluorescent biosensor comprises the following steps:

[0063] The synthetic operation steps of composite probe S are as follows:

[0064] Add 18 μL of sterilized water, 3 μL of 10×PB, 3 μL of 100 μM S0 probe and 6 μL of 100 μM S1 probe into the pre-prepared sterilized EP tube, shake for 30 seconds, and incubate at 95°C Incubate for 5 minutes, slowly cool down to room temperature and hybridize into probes, and store at -20°C for later use.

[0065] The steps for the synthesis of DNA silver nanoclusters (AgNCs-DNA) are as follows:

[0066] Add 15 μL of 100 μM Nucleic Acid Strand C and 73 μL of 20 mM PB (pH 7.0) buffer into the EP tube wrapped in foil paper, and then add 6 μL of 1.5 mM AgNO3 solution (to ensure the ratio of Ag+ to H3 6:1), shake for 1 min, and place at 4 °C for 30 min; after 30 min, continue to add 6 μL of 1.5 mM NaBH4 to the EP tube, shake for 1 min, and place at 4 °C in the dark for more than 6 h. ;

[0067] The ma...

Embodiment 2

[0074] The preparation method of described fluorescent biosensor comprises the following steps:

[0075] The synthetic operation steps of composite probe S are as follows:

[0076] Add 18 μL of sterilized water, 3 μL of 10×PB, 3 μL of 100 μM S0 probe and 6 μL of 100 μM S1 probe into the pre-prepared sterilized EP tube, shake for 30 seconds, and incubate at 95°C Incubate for 5 minutes, slowly cool down to room temperature and hybridize into probes, and store at -20°C for later use.

[0077] The steps for the synthesis of DNA silver nanoclusters (AgNCs-DNA) are as follows:

[0078] Add 15 μL of 100 μM Nucleic Acid Strand C and 73 μL of 20 mM PB (pH 7.0) buffer into the EP tube wrapped in foil paper, and then add 6 μL of 1.5 mM AgNO3 solution (to ensure the ratio of Ag+ to H3 6:1), shake for 1 min, and place at 4 °C for 30 min; after 30 min, continue to add 6 μL of 1.5 mM NaBH4 to the EP tube, shake for 1 min, and place at 4 °C in the dark for more than 6 h. ;

[0079] The ma...

Embodiment 3

[0086] The preparation method of described fluorescent biosensor comprises the following steps:

[0087] The synthetic operation steps of composite probe S are as follows:

[0088] Add 18 μL of sterilized water, 3 μL of 10×PB, 3 μL of 100 μM S0 probe and 6 μL of 100 μM S1 probe into the pre-prepared sterilized EP tube, shake for 30 seconds, and incubate at 95°C Incubate for 5 minutes, slowly cool down to room temperature and hybridize into probes, and store at -20°C for later use.

[0089] The steps for the synthesis of DNA silver nanoclusters (AgNCs-DNA) are as follows:

[0090] Add 15 μL of 100 μM Nucleic Acid Strand C and 73 μL of 20 mM PB (pH 7.0) buffer into the EP tube wrapped in foil paper, and then add 6 μL of 1.5 mM AgNO3 solution (to ensure the ratio of Ag+ to H3 6:1), shake for 1 min, and place at 4 °C for 30 min; after 30 min, continue to add 6 μL of 1.5 mM NaBH4 to the EP tube, shake for 1 min, and place at 4 °C in the dark for more than 6 h. ;

[0091] The ma...

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