CN110632300B - Aptamer-based biosensor for detecting salmonella and preparation method and application thereof - Google Patents
Aptamer-based biosensor for detecting salmonella and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
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Abstract
The invention relates to the technical field of biosensors, in particular to a biosensor for detecting salmonella based on a nucleic acid aptamer, which is characterized in that based on the specific recognition of the nucleic acid aptamer and a target object, a hairpin probe HAP is opened, S1 is replaced from a composite probe S by utilizing a fulcrum-mediated strand displacement reaction, the replaced S can expose a sequence for forming a G-tetrad in a catalytic hairpin self-assembly (CHA) amplification mode, and the G-tetrad/heme DNase is formed in the presence of heme. The catalytic performance of G-tetrad/heme horseradish peroxidase is used for oxidizing cysteine into cystine, and charge transfer between cysteine and silver clusters through gold-sulfur bonds cannot be realized, so that fluorescence signal conduction is regulated and controlled, and an aptamer biosensor is constructed.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to a biosensor for detecting salmonella based on a nucleic acid aptamer, and also relates to a preparation method of the biosensor.
Background
Salmonella is a common food-borne pathogenic bacterium, and is a gram-negative intestinal bacterium parasitized in cells. The bacterium is widely existed in nature, not only can cause acute, chronic or recessive infection of livestock, poultry and other animals, but also can cause food poisoning of people by polluting food, thereby causing great threat to human beings. According to statistics, the food poisoning caused by salmonella is often listed as the top in various countries in the world of bacterial food poisoning. Salmonella causes 70 million deaths each year. If no action is taken to stop this global threat, it is expected that 1000 million people will die globally by the year 2050. Salmonella is the most common pathogenic bacterium in food poisoning in China, and accounts for the first place of food poisoning.
The currently reported methods for detecting salmonella include traditional culture methods, enzyme-linked immunosorbent assay, PCR techniques, etc. The traditional salmonella detection methods have long detection period, complicated procedures and expensive equipment, and can not meet the requirements far away. Therefore, a rapid, accurate, simple and trace analysis method is urgently needed to detect salmonella in food. In recent years, DNA biosensing detection technology has gained wide attention by virtue of its high sensitivity and specificity. The fluorescence technology has the remarkable advantages of high sensitivity, strong specificity, low price, no need of sample pretreatment and the like, and is more and more emphasized by people in the fields of biology, medicine and the like.
Disclosure of Invention
In order to solve the problems of low specificity and sensitivity, high cost and long detection period of the method for detecting the salmonella in the prior art, the invention provides the biosensor for detecting the salmonella, which has high specificity and sensitivity, low cost and high detection speed and is based on enzyme-free fluorescence signal conduction.
Another object of the present invention is to provide a method and use of the above biosensor for detecting Salmonella.
In order to achieve the purpose, the invention adopts the following technical scheme.
A biosensor for detecting salmonella comprises a hairpin probe HAP, a composite probe S, a nucleic acid chain S2, a nucleic acid chain C, a hairpin probe H1, a hairpin probe H2, a hairpin probe H3, cysteine, heme, salmonella and a buffer solution;
the HAP base series is shown as SEQ No. 1;
the S0 base series is shown as SEQ No. 2;
the S1 base series is shown as SEQ No. 3;
the S2 base series is shown as SEQ No. 4;
the H1 base series is shown as SEQ No. 5;
the H2 base series is shown as SEQ No. 6;
the H3 base series is shown as SEQ No. 7;
the C base series is shown as SEQ No. 8.
The detection of the salmonella is realized in a homogeneous solution, and the amplification of a signal is realized in an isothermal amplification mode without enzyme assistance, so that the high-sensitivity detection of the salmonella is realized, and a lower detection lower limit is obtained. In the homogeneous reaction, the reaction conditions were 37 ℃ and the reaction time was 90min.
The preparation method of the biosensor for detecting salmonella comprises the following steps:
(1) Constructing a composite probe S;
(2) Synthesis of DNA silver nanoclusters (AgNCs-DNA);
(3) Homogeneous reaction: adding salmonella and hairpin probe HAP into the homogeneous phase, simultaneously adding composite probe S, nucleic acid chain S2, H1, H2, H3, agNCs-DNA cysteine, heme and buffer solution, mixing uniformly and incubating;
(4) The fluorometer detects the intensity of the fluorescence.
The construction steps of the composite probe S in the step (1) are as follows:
adding sterilized water, 10 XPB, S0 probe and S1 probe into a prepared sterilized EP tube, shaking for 30S, incubating at 95 ℃ for 5min, slowly cooling to room temperature, hybridizing to obtain the probe, and storing at-20 ℃ for later use.
The step (2) of synthesizing the DNA silver nanoclusters (AgNCs-DNA) comprises the following operation steps:
mu.L of 100. Mu.M nucleic acid strand C and 73. Mu.L of 20 mM PB (pH 7.0) buffer were added to an EP tube wrapped in tinfoil, followed by 6. Mu.L of a 1.5 mM AgNO3 solution (ensuring Ag) + The ratio of H3 to H3 is 6);
30 After min, continuously adding 6 μ L of 1.5 mM NaBH4 into the EP tube, shaking for 1 min, and standing in the dark at 4 deg.C for more than 6h to obtain the final product.
The homogeneous reaction operation in the step (3) comprises the following steps:
3 μ L of Salmonella (5.0X 10) 5 cfu/mL), hairpin probe HAP (1.5. Mu.L, 500 nM), composite probe S (3. Mu.L, 1. Mu.M), nucleic acid strand S2 (3. Mu.L, 1. Mu.M), H1 (3. Mu.L, 1. Mu.M), H2 (3. Mu.L, 1. Mu.M), H3 (3. Mu.L, 1. Mu.M), agNCs-DNA (14. Mu.L, 4. Mu.M), cysteine 1. Mu.M, heme (3. Mu.L, 1. Mu.M), and buffer were added to the centrifuge tube, shaken for 30S, and then water-washed at 37 ℃ for 90min.
And (4) setting the excitation wavelength of the fluorometer to 570 nm.
The biosensor is used for detecting salmonella in food and water.
The sequences used in this application are:
HAP: 5’- AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGATTGGCATTACTAT
GGGTCTCACTATG-3’
S0: 5’-AAAAGAACCCATAGTGAGACCCATGAACCCATAGTGAGACCCATAGTAATG
CC-3’
S1: 5’- ATGGGTCTCACTATGGGTTCAACG-3’
S2: 5’- GTCTCACTATGGGTTCATGGGTCTCACTATGG-3’
H1: 5’-TGGGTAGGGCGGGTCGTTGAACCCATAGTGAGACCCATATGGGTCAAGA
CATGGGTCTCACTATGGGT-3’
H2: 5’-TGGGTAGGGCGGGTAGTGAGACCCATGTCTTGACCCATATGGGTTCAAC
GATGGGTCAAGACATGGGT-3’
H3: 5’-TGGGTAGGGCGGGTGTCTTGACCCATCGTTGAACCCATATGGGTCTCACT
ATGGGTTCAACGATGGGT-3’
C: 5’-CCCCCCCCCCCC-3’
the black italic part of hairpin probe HAP is the aptamer sequence of salmonella, and the black bold font of hairpin probe H1, H2, H3 is the split G tetrad sequence. Hybridizing the S0 probe and the S1 probe A1 and 2 to synthesize a composite probe S, wherein when a target exists, the target and an aptamer are specifically combined, so that a hairpin structure of HAP is opened, the opened HAP can bind an end foothold region of the composite probe S, the S1 is replaced from the composite probe S, a second foothold region is exposed, a nucleic acid strand S2 hybridizes with the nucleic acid strand S through the exposed foothold region, the other S1 and the opened HAP are further replaced, and the replaced opened HAP is subjected to next cycle to obtain a large amount of S1, the replaced S1 can open H1, the opened H1 can continue to open H2, the opened H2 can further open H3, and the opened H3 can replace S1, so that catalytic self-assembly amplification (CHA) is realized, a sequence of a G-tetrad is exposed, and a G-tetrad/heme DNA enzyme is formed in the presence of the hairpin. The catalytic performance of G-tetrad/heme horseradish peroxidase is used for oxidizing cysteine into cystine, and charge transfer between cysteine and silver clusters through gold-sulfur bonds cannot be realized, so that fluorescent signal conduction is regulated and controlled, and quantitative detection of salmonella is realized.
The detection mode of the invention is fluorescence detection, and utilizes the chain displacement cyclic amplification and CHA amplification functions mediated by the fulcrum region to generate a large amount of G-quadruplet DNA enzyme, so as to realize the oxidation of the cysteine and ensure the obvious change of the fluorescence intensity. The detection of the target is performed by detecting the fluorescence intensity of the solution.
The invention utilizes the special identification of the aptamer to realize the high specificity detection of the target salmonella; using the chain displacement circulation amplification mediated by the fulcrum region to generate a large amount of secondary products S1; by utilizing the CHA amplification function, G-quadruplet DNA enzyme is continuously generated, the oxidation of cysteine is realized, the detection signal is amplified, the detection sensitivity is improved, and the ultra-sensitivity detection of the target salmonella is realized; the sensor has mild reaction conditions and high reaction speed; compared with other optical detection means, the detection method is simple and convenient to operate and short in detection period; the detection process is realized in a homogeneous phase, so that the complexity of operation is reduced; the process cost of the biosensor is effectively reduced without enzyme participation, and the method is suitable for the requirements of low cost in industrialization.
The invention has the beneficial effects that:
1. lower limit of detection
The specific recognition of the aptamer is utilized, and the combination of the aptamer and salmonella is utilized to realize the high-specificity detection of the target object; the chain displacement cyclic amplification mediated by the fulcrum region is utilized, the cyclic utilization of HAP is realized, a large amount of secondary products S1 are generated, the detection signal is amplified, and the detection sensitivity is improved; the CHA amplification function is utilized to continuously generate G-quadruplex DNA enzyme, thereby effectively improving the sensitivity of the sensor; the detection line can reach 0.45.
2. Simple method and stable performance
The construction of the sensor only needs one step, thereby effectively avoiding the pollution possibly caused by adding samples in multiple steps and having the advantages of simple and convenient operation, high reaction speed and the like; the main processes of the detection principle are realized in a homogeneous phase, so that the reaction speed is improved, the complexity of operation is reduced, and the rapid, simple and sensitive detection of the target object is realized.
3. Detection of salmonella in food and water
The process for manufacturing the biosensor has low cost and is suitable for the requirement of low price in industrialization. The method is suitable for food safety, detection of salmonella in water and practical application of biosensor industrialization.
Drawings
FIG. 1 is a schematic diagram of the experiment;
FIG. 2 is a graph showing the optimized detection results of the H1 concentration in example 1;
FIG. 3 is a graph showing the optimized detection results of hemoglobin concentration in example 2
FIG. 4 is a graph showing the results of the reaction time optimization assay in example 3.
FIG. 5 is a standard curve for the detection of Salmonella in example 4.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1
The preparation method of the fluorescence biosensor comprises the following steps:
the synthetic operation steps of the composite probe S are as follows:
mu.L of sterilized water, 3. Mu.L of 10 XPB, 3. Mu.L of 100. Mu.M S0 probe and 6. Mu.L of 100. Mu.M S1 probe were added to a previously prepared sterilized EP tube, shaken for 30S, incubated at 95 ℃ for 5min, slowly cooled to room temperature to hybridize as a probe, and stored at-20 ℃ until use.
The operation steps of the synthesis of DNA silver nanoclusters (AgNCs-DNA) are as follows:
mu.L of 100. Mu.M nucleic acid strand C and 73. Mu.L of 20 mM PB (pH 7.0) buffer were added to an EP tube wrapped in tinfoil, followed by 6. Mu.L of 1.5 mM AgNO3 solution (ensuring a ratio of Ag + to H3 of 6; 30 After min, continuously adding 6 mu L of 1.5 mM NaBH4 into the EP tube, shaking for 1 min, and placing in the dark at 4 ℃ for more than 6h to obtain the product;
the main steps of the reaction process in the homogeneous solution are as follows:
3 μ L of Salmonella bacteria (A), (B), (C)5.0×10 5 cfu/mL), hairpin probe HAP (1.5. Mu.L, 500 nM), composite probe S (3. Mu.L, 1. Mu.M), nucleic acid strand S2 (3. Mu.L, 1. Mu.M), H1 (final concentrations 0.4. Mu.M, 0.6. Mu.M, 0.8. Mu.M, 1.0. Mu.M, 1.2. Mu.M, 1.4. Mu.M), H2 (3. Mu.L, 1. Mu.M), H3 (3. Mu.L, 1. Mu.M), agNCs-DNA (14. Mu.L, 4. Mu.M), cysteine 1. Mu.M, hemoglobin (3. Mu.L, 1. Mu.M), and buffer were added to a centrifuge tube, shaken for 30S, and then incubated at 37 ℃ for 90min.
The fluorescence intensity detection by the fluorescence instrument mainly comprises the following steps:
the solution after the homogeneous reaction (30. Mu.L) was diluted to 100. Mu.L, and the fluorescence peak intensity was detected at 635nm using a fluorometer.
The excitation wavelength of the fluorescence instrument is set to be 570nm, the emission wavelength is 635nm, the detection range is 570nm-800nm, the change of a fluorescence signal is read, and a target object is detected.
The results are shown in FIG. 2, from which it can be seen that the peak of the detected fluorescence intensity decreases as the concentration of H1 increases, and that the fluorescence intensity tends to stabilize when the concentration exceeds 1.0. Mu.M. Therefore, the optimal final concentration of H1 is 1.0. Mu.M.
Example 2
The preparation method of the fluorescence biosensor comprises the following steps:
the synthetic operation steps of the composite probe S are as follows:
mu.L of sterilized water, 3. Mu.L of 10 XPB, 3. Mu.L of 100. Mu.M S0 probe and 6. Mu.L of 100. Mu.M S1 probe were added to a previously prepared sterilized EP tube, shaken for 30S, incubated at 95 ℃ for 5min, slowly cooled to room temperature to hybridize as a probe, and stored at-20 ℃ until use.
The operation steps of the synthesis of DNA silver nanoclusters (AgNCs-DNA) are as follows:
mu.L of 100. Mu.M nucleic acid strand C and 73. Mu.L of 20 mM PB (pH 7.0) buffer were added to an EP tube wrapped in tinfoil, followed by 6. Mu.L of 1.5 mM AgNO3 solution (ensuring a ratio of Ag + to H3 of 6; 30 After min, continuously adding 6 mu L of 1.5 mM NaBH4 into the EP tube, shaking for 1 min, and placing in the dark at 4 ℃ for more than 6h to obtain the product;
the main steps of the reaction process in the homogeneous solution are as follows:
3 μ L of Salmonella (5.0X 10) 5 cfu/mL), hairpin probe HAP (1.5. Mu.L, 500 nM), composite probe S (3. Mu.L, 1. Mu.M), nucleic acid strand S2 (3. Mu.L, 1. Mu.M), H1 (3. Mu.L, 1. Mu.M), H2 (3. Mu.L, 1. Mu.M), agNCs-DNA (14. Mu.L, 4. Mu.M), cysteine 1. Mu.M, 3. Mu.L of different concentrations of heme (final concentrations of 0.01. Mu.M, 0.05. Mu.M, 0.1. Mu.M, 0.5. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M, respectively), buffer were added to the centrifuge tubes, shaken for 30S, and then bathed for 90min at 37 ℃.
The fluorescence intensity detection by the fluorescence instrument mainly comprises the following steps:
the solution after the homogeneous reaction (30. Mu.L) was diluted to 100. Mu.L, and the fluorescence peak intensity was detected at 635nm using a fluorometer.
The excitation wavelength of the fluorometer is set to 570nm, the emission wavelength is 635nm, the detection range is 570nm-800nm, the change of a fluorescence signal is read, and the target object is detected.
The results are shown in FIG. 3, from which it can be seen that the peak of the detected fluorescence intensity decreases as the concentration of hemoglobin increases, and that the fluorescence intensity tends to stabilize when the concentration exceeds 1.0. Mu.M. Therefore, the optimal final concentration of hemoglobin is 1.0. Mu.M.
Example 3
The preparation method of the fluorescence biosensor comprises the following steps:
the synthetic operation steps of the composite probe S are as follows:
mu.L of sterilized water, 3. Mu.L of 10 XPB, 3. Mu.L of 100. Mu.M S0 probe and 6. Mu.L of 100. Mu.M S1 probe were added to a previously prepared sterilized EP tube, shaken for 30S, incubated at 95 ℃ for 5min, slowly cooled to room temperature to hybridize as a probe, and stored at-20 ℃ until use.
The operation steps of the synthesis of DNA silver nanoclusters (AgNCs-DNA) are as follows:
mu.L of 100. Mu.M nucleic acid strand C and 73. Mu.L of 20 mM PB (pH 7.0) buffer were added to an EP tube wrapped in tinfoil, followed by 6. Mu.L of 1.5 mM AgNO3 solution (ensuring a ratio of Ag + to H3 of 6; 30 After min, continuously adding 6 mu L of 1.5 mM NaBH4 into the EP tube, shaking for 1 min, and placing in the dark at 4 ℃ for more than 6h to obtain the NaBH-NaBH suspension;
the main steps of the reaction process in the homogeneous solution are as follows:
3 μ L of Salmonella (5.0X 10) 5 cfu/mL), hairpin probe HAP (1.5. Mu.L, 500 nM), composite probe S (3. Mu.L, 1. Mu.M), nucleic acid strand S2 (3. Mu.L, 1. Mu.M), H1 (3. Mu.L, 1. Mu.M), H2 (3. Mu.L, 1. Mu.M), agNCs-DNA (14. Mu.L, 4. Mu.M), cysteine 1. Mu.M, heme (3. Mu.L, 1. Mu.M), buffer were added to the centrifuge tube, shaken for 30S, washed with water at 37 ℃ for 30 min,45 min,60min,75 min,90 min,105 min,120min.
The fluorescence intensity detection by the fluorescence instrument mainly comprises the following steps:
the solution after the homogeneous reaction (30. Mu.L) was diluted to 100. Mu.L, and the fluorescence peak intensity was detected at 635nm using a fluorometer.
The excitation wavelength of the fluorescence instrument is set to be 570nm, the emission wavelength is 635nm, the detection range is 570nm-800nm, the change of a fluorescence signal is read, and a target object is detected.
The results are shown in FIG. 4, from which it can be seen that the peak value of the detected fluorescence intensity decreases with the lapse of the reaction time, and the fluorescence intensity tends to stabilize after the reaction time exceeds 90min. The optimum homogeneous reaction time is 90min.
Example 4
The preparation method of the fluorescence biosensor comprises the following steps:
the synthetic operation steps of the composite probe S are as follows:
mu.L of sterilized water, 3. Mu.L of 10 XPB, 3. Mu.L of 100. Mu.M S0 probe and 6. Mu.L of 100. Mu.M S1 probe were added to a previously prepared sterilized EP tube, shaken for 30S, incubated at 95 ℃ for 5min, slowly cooled to room temperature to hybridize as a probe, and stored at-20 ℃ until use.
The operation steps of the synthesis (AgNCs-DNA) of the DNA silver nanocluster are as follows:
mu.L of 100. Mu.M nucleic acid strand C and 73. Mu.L of 20 mM PB (pH 7.0) buffer were added to an EP tube wrapped in tinfoil, followed by 6. Mu.L of 1.5 mM AgNO3 solution (ensuring a ratio of Ag + to H3 of 6; 30 After min, continuously adding 6 mu L of 1.5 mM NaBH4 into the EP tube, shaking for 1 min, and placing in the dark at 4 ℃ for more than 6h to obtain the NaBH-NaBH suspension;
the main steps of the reaction process in the homogeneous solution are as follows:
3 μ L of Salmonella (5.0X 10) 5 ，1.0×10 5 ，5.0×10 4 ，1.0×10 4 ，5.0×10 3 ，1.0×10 3 ，5.0×10 2 ，1.0×10 2 50, 10 cfu/mL), hairpin probe HAP (1.5. Mu.L, 500 nM), composite probe S (3. Mu.L, 1. Mu.M), nucleic acid strand S2 (3. Mu.L, 1. Mu.M), H1 (3. Mu.L, 1. Mu.M), H2 (3. Mu.L, 1. Mu.M), H3 (3. Mu.L, 1. Mu.M), agNCs-DNA (14. Mu.L, 4. Mu.M), cysteine 1. Mu.M, heme (3. Mu.L, 1. Mu.M), and buffer were added to the centrifuge tube, shaken for 30S, and then incubated at 37 ℃ for 90min.
The main steps of detecting fluorescence intensity by a fluorescence instrument are as follows:
the solution after the homogeneous reaction (30. Mu.L) was diluted to 100. Mu.L, and the fluorescence peak intensity was detected at 635nm using a fluorometer.
The excitation wavelength of the fluorescence instrument is set to be 570nm, the emission wavelength is 635nm, the detection range is 570nm-800nm, the change of a fluorescence signal is read, and a target object is detected.
The results are shown in FIG. 5, from which it can be seen that the peak of the detected fluorescence intensity decreases with increasing Salmonella concentration, when the concentration exceeds 5.0X 10 5 After cfu/mL, the fluorescence intensity tends to be stable. Therefore, the optimal final concentration of Salmonella is 5.0X 10 5 cfu/mL。
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Sequence listing
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Claims (6)
1. A biosensor for detecting salmonella is characterized by comprising a hairpin probe HAP, a composite probe S, a nucleic acid chain S2, a nucleic acid chain C, a hairpin probe H1, a hairpin probe H2, a hairpin probe H3, cysteine, heme, salmonella and buffer solution;
the composite probe S is composed of an S0 probe, an S1 probe 1:2 into a double strand;
the HAP base series is shown as SEQ No. 1;
the S0 base series is shown as SEQ No. 2;
the S1 base series is shown as SEQ No. 3;
the S2 base series is shown as SEQ No. 4;
the H1 base series is shown as SEQ No. 5;
the H2 base series is shown as SEQ No. 6;
the H3 base series is shown as SEQ No. 7;
the C base series is shown as SEQ No. 8;
the preparation method of the biosensor comprises the following steps:
(1) Constructing a composite probe S;
(2) Synthesizing DNA silver nanocluster AgNCs-DNA;
(3) Homogeneous reaction: uniformly mixing salmonella, a hairpin probe HAP, a composite probe S, a nucleic acid chain S2, H1, H2, H3, agNCs-DNA, cysteine, heme and a buffer solution, and then incubating;
(4) The fluorometer detects the intensity of the fluorescence.
2. The biosensor for detecting Salmonella according to claim 1, wherein the composite probe S of step (1) is constructed by the steps of:
and (3) shaking sterilized water, 10 XPB, an S0 probe and an S1 probe for 30S, incubating for 5min at 95 ℃, slowly cooling to room temperature, hybridizing to obtain a probe, and storing at-20 ℃ for later use.
3. The biosensor for detecting Salmonella according to claim 1, wherein the step (2) of synthesizing DNA silver nanocluster AgNCs-DNA comprises the following steps:
mixing the nucleic acid chain C and PB buffer solution, and then adding AgNO 3 Shaking the solution for 1 min, and standing at 4 deg.C for 30 min;
adding NaBH 4 Shaking for 1 min, and standing at 4 deg.C in dark for more than 6 hr.
4. The salmonella detection biosensor of claim 1, wherein the step (3) of homogeneous reaction comprises the following steps:
mixing salmonella, hairpin probe HAP, composite probe S, nucleic acid chain S2, H1, H2, H3, agNCs-DNA, cysteine, heme and buffer solution, shaking for 30S, and bathing for 90min at 37 ℃.
5. The salmonella detection biosensor of claim 1, wherein the step (4) fluorometer sets the excitation wavelength at 570 nm.
6. Use of the biosensor of claim 1 for detecting salmonella in food and water.
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