CN111638330B - Biosensor for detecting salmonella typhimurium and application thereof - Google Patents

Biosensor for detecting salmonella typhimurium and application thereof Download PDF

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CN111638330B
CN111638330B CN202010522243.7A CN202010522243A CN111638330B CN 111638330 B CN111638330 B CN 111638330B CN 202010522243 A CN202010522243 A CN 202010522243A CN 111638330 B CN111638330 B CN 111638330B
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salmonella typhimurium
solution
biosensor
carbon quantum
aptamer
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CN111638330A (en
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吴薇
杨庆利
都晗
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Qingdao Agricultural University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • 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)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a biosensor for detecting salmonella typhimurium and application thereof, belonging to the technical field of harmful microorganism detection. The biosensor consists of a carbon quantum dot Td-CD modified by a Salmonella typhimurium aptamer and a magnetic bead Td-MB modified by the Salmonella typhimurium aptamer; the Salmonella typhimurium aptamer is assembled with other nucleic acids through sequence design to form a DNA regular tetrahedron structure, and then is used for modifying carbon quantum dots and magnetic beads respectively. The method for detecting the salmonella typhimurium by adopting the biosensor is simple and convenient to operate, short in time consumption, low in detection cost and free from carrying out complex pretreatment on a sample to be detected; the biosensor can effectively amplify signals of the salmonella typhimurium, can be used for detecting the salmonella typhimurium with low concentration in food, and realizes qualitative and quantitative analysis of the salmonella typhimurium.

Description

Biosensor for detecting salmonella typhimurium and application thereof
Technical Field
The invention belongs to the technical field of harmful microorganism detection, and particularly relates to a biosensor for detecting salmonella typhimurium and application thereof.
Background
Salmonella typhimurium (Salmonella typhimurium) is a common food-borne pathogenic bacterium, is a gram-negative anaerobic bacterium, is widely present in food, water sources and animals and plants, and contaminated food and water can cause gastroenteritis in mammals and humans. Common symptoms caused by infection with salmonella typhimurium include high fever, cough, diarrhea, abdominal cramps, and vomiting, and serious illness can lead to death, resulting in a great loss of food safety and human health. Therefore, accurate identification and detection of Salmonella typhimurium are particularly important.
At present, methods for detecting salmonella typhimurium include biological isolation culture methods, enzyme-linked immunoassay methods, PCR molecular detection methods and the like. The traditional biological separation culture method can obtain accurate detection results, but the detection process takes long time and needs four to seven days. The enzyme-linked immunoassay method has high detection specificity, is convenient and quick, but the detection process is very easily influenced by pH change, environmental temperature and the like, and the sensitivity is lower, so that the requirement on the detection sensitivity of the salmonella typhimurium is difficult to meet. The PCR molecular detection method is expensive, has high requirement on the operation specialty and is difficult to meet the actual detection requirement. Therefore, the method for detecting the salmonella typhimurium is sensitive, rapid and high in accuracy and has important significance
The food sample composition is complex, so the detection of the salmonella typhimurium in the food is easily influenced by the interference of a food matrix. In view of the above, the magnetic separation technology is a good method, and specific binding is generated between the coupling specific recognition molecules on the magnetic microspheres and pathogenic bacteria existing in food, and the formed compound is separated from other impurities under the action of magnetic force, so as to quickly enrich the pathogenic bacteria.
Aptamer (aptamer) is a DNA or RNA sequence selected, also called "chemical antibody", as a new generation of recognition molecule, with low molecular weight (15-50 bases), which has an affinity comparable to or even higher than that of protein antibodies, and compared with antibodies obtained by traditional biological methods, it has the advantages of easy synthesis and storage, and is widely used for recognition and detection due to its strong specificity, high affinity to biological molecules, good stability, and no immunogenicity and toxicity.
The DNA regular tetrahedron structure is a DNA nano structure with excellent properties formed by assembling four different nucleic acids, and the nucleic acid aptamer is assembled with other 3 nucleic acids through sequence design to form the regular tetrahedron nucleic acid aptamer, so that the structure can overcome the defect that the nucleic acid aptamer is soft and not easy to fix. The quantum dot has small size, high fluorescence intensity, large Stokes shift, photobleaching resistance, long fluorescence life and good biocompatibility, is superior to other luminescent materials, and has attracted high attention since being used as a bioluminescent marker of living cells 20 years ago. The DNA regular tetrahedron structure is modified on the surface of the quantum dot, so that the accuracy and the sensitivity of detection can be improved.
Disclosure of Invention
Aiming at the problems in the prior art, the first purpose of the invention is to design a nano structure of a synthetic DNA regular tetrahedron aptamer for specific and efficient recognition of Salmonella typhimurium;
the second purpose of the invention is to synthesize non-toxic carbon nano-quantum dots, establish a signal amplification method based on the carbon quantum dot self-assembly microspheres, and improve the detection sensitivity of the salmonella typhimurium;
the third purpose of the invention is to prepare a magnetic separation quantum dot fluorescent sandwich sensor which takes a DNA regular tetrahedron aptamer nanostructure as a signal recognizer and a self-assembled carbon quantum dot microsphere as a signal amplifier, and a method for detecting salmonella typhimurium by using the sensor, so as to solve the problems of low sensitivity, complex detection method, high requirements on instruments and equipment and the like in the existing technology for detecting salmonella typhimurium.
In order to achieve the purpose, the invention adopts the following technical scheme:
a biosensor for detecting Salmonella typhimurium consists of a carbon quantum dot Td-CD modified by a Salmonella typhimurium aptamer and a magnetic bead Td-MB modified by a Salmonella typhimurium aptamer;
the DNA regular tetrahedron structure of the Salmonella typhimurium Aptamer for modifying the carbon quantum dots is Td-Aptamer1, and the DNA regular tetrahedron structure of the Salmonella typhimurium Aptamer for modifying the magnetic beads is Td-Aptamer 2.
Based on the above scheme, the nucleic acid sequence forming the Td-Aptamer1 is:
chain a 1: 5'-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ACT GAC AGA TTA AAA GTG GTT GGG CAA CTT CTG CTT GCG AA-3' (SEQ ID NO:1)
Chain B: 5'-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGATGC GAG GGT CCA ATA C-3' (SEQ ID NO:3), the 5 ' end of which is linked to a biotin molecule;
c chain: 5'-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3' (SEQ ID NO:4), with a biotin molecule attached to the 5 ' terminus;
chain D: 5'-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3' (SEQ ID NO:5), with a biotin molecule attached to the 5 ' end.
The nucleic acid sequence forming the Td-Aptamer2 is:
chain a 2: 5'-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ATA TGG CGG CGT CAC CCG ACG GGG ACT TGA CAT TAT GAC AG-3' (SEQ ID NO: 2);
chain B: 5'-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3' (SEQ ID NO:3), the 5 ' end of which is linked to a biotin molecule;
c chain: 5'-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3' (SEQ ID NO:4), with a biotin molecule attached to the 5 ' terminus;
chain D: 5'-TTC AGACTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCAT-3' (SEQ ID NO:5), with a biotin molecule attached to the 5 ' end.
On the basis of the scheme, the preparation method of the Td-Aptamer1 and the Td-Aptamer2 comprises the following steps: respectively mixing four nucleic acid sequences of the DNA regular tetrahedron structure in equimolar mode, dissolving the mixture in TE buffer solution, heating the solution after vortex mixing uniformly to 95 ℃ for 5min, and rapidly cooling the solution in ice water for 10min to 4 ℃ to obtain the DNA regular tetrahedron structure.
On the basis of the scheme, the preparation method of the carbon quantum dot Td-CD modified by the Salmonella typhimurium aptamer comprises the following steps:
adding the Td-aptamer1 and streptavidin-carbon quantum dots SA-CDs into a PBS buffer solution according to a molar ratio of 4:1, violently shaking for 1h at room temperature, rotating at 5000 rpm, centrifuging for 10min, discarding supernatant to obtain Td-CD microspheres, and dispersing the Td-CD microspheres in the PBS buffer solution to obtain a Td-CD solution.
On the basis of the scheme, the preparation method of the streptavidin-carbon quantum dots SA-CDs comprises the following steps:
weighing 5mg of amino-functionalized carbon quantum dots, dissolving the amino-functionalized carbon quantum dots in 1.5mL of absolute ethanol, uniformly dispersing by ultrasonic, dropwise adding a streptavidin solution into the solution, after performing ultrasonic treatment for 15min, violently shaking for 3h at room temperature, rotating at the speed of 12,000rpm, centrifuging for 20min, and discarding supernatant to obtain the streptavidin-modified carbon quantum dots.
On the basis of the scheme, the preparation method of the amino functionalized carbon quantum dot comprises the following steps:
weighing 0.4g of beta-cyclodextrin, dissolving in 20mL of ultrapure water, ultrasonically dispersing uniformly, transferring into a stainless steel high-pressure reaction kettle with a polytetrafluoroethylene lining, placing in a blast-type heating drying box, and carrying out hydrothermal reaction for 6h at the temperature of 200 ℃; naturally cooling to room temperature after the reaction is finished, and dialyzing the obtained yellow brown transparent liquid in ultrapure water for 24 hours by using a dialysis bag; and finally, performing rotary evaporation on a rotary evaporator at 45 ℃, and removing the solvent to obtain light yellow powder, namely the prepared amino functionalized carbon quantum dots.
On the basis of the scheme, the preparation method of the carbon quantum dot Td-MB modified by the Salmonella typhimurium aptamer comprises the following steps:
1mg of streptavidin functionalized magnetic beads and 1.6 mu M of Td-aptamer2 are mixed and dispersed in 500mL of Tris-HCl buffer solution, and vortex oscillation is carried out for 30 min; then, the magnetic beads were separated from the system with a magnet, the supernatant was discarded, and the magnetic beads were washed three times with 1mL of PBS buffer, and finally dispersed in the PBS buffer, i.e., Td-MB solution.
The biosensor for detecting the salmonella typhimurium is applied to detecting the salmonella typhimurium.
The salmonella typhimurium detection kit containing the biosensor.
The method for detecting the salmonella typhimurium by using the biosensor comprises the following steps:
(1) incubating the Td-MB solution, the Td-CD solution and the sample solution to be detected for 20 minutes at room temperature, and slowly shaking for full combination reaction; performing magnetic separation for the first time, and removing the supernatant to obtain an MB-Salmonella typhimurium-CD compound;
(2) resuspend MB-Salmonella typhimurium-CD complex with PBS, then add 4. mu.M Biotin molecular solution to mix and bind Td-MB and Td-CD for 30 minutes; then, carrying out secondary magnetic separation, discarding magnetic beads, and reserving supernatant solution;
(3) and (3) scanning the supernatant solution obtained in the step (2) by using a fluorescence spectrophotometer, measuring fluorescence intensity by using a fluorescence emission spectrum under the excitation wavelength of 470nm, introducing the measured fluorescence intensity into a standard curve, and calculating to obtain the concentration of the salmonella typhimurium in the sample to be measured.
The technical scheme of the invention has the advantages that:
the method for detecting the salmonella typhimurium by adopting the biosensor is simple and convenient to operate, does not need to carry out complex pretreatment on a sample to be detected, and has low detection cost;
the biosensor can effectively amplify signals of the salmonella typhimurium, can be used for detecting the salmonella typhimurium with low concentration, and has the sensitivity of 6 CFU.mL-1；
The method for detecting the salmonella typhimurium by adopting the biosensor has short time, the whole detection process does not exceed one hour, and the detection speed is high;
the biosensor can be used for detecting the salmonella typhimurium in food, and realizes qualitative and quantitative analysis of the salmonella typhimurium.
Drawings
Similar parts in the figures are denoted by the same reference numerals.
FIG. 1 is a schematic diagram of the detection principle of the method of the present invention;
FIG. 2 shows the results of the detection of various concentrations of Salmonella typhimurium;
FIG. 3 is a graph of the results of different food-borne pathogenic bacteria.
Detailed Description
In order to illustrate the invention more clearly, the invention is described in further detail below with reference to specific examples and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Unless otherwise defined, terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art.
The detection principle of the biosensor is shown in fig. 1, and specifically comprises the following steps:
(1) mixing and assembling four DNA strands A1 and B, C, D into a Td-Aptamer1, and then self-assembling the Td-Aptamer1 and SA-CD to form Td-CD microspheres;
(2) mixing and assembling four DNA chains A2 and B, C, D into a Td-Aptamer2, and then self-assembling the Td-Aptamer2 and streptavidin functionalized magnetic beads to form Td-MB;
(3) detection process
Adding Td-MB and Td-CD into a sample to be detected to ensure that the Td-MB and the Td-CD are combined with the salmonella typhimurium to react to form an MB-salmonella typhimurium-CD compound;
secondly, separating a magnetic frame, and removing supernatant to obtain an MB-Salmonella typhimurium-CD compound;
adding Biotin, wherein the free Biotin breaks Td-MB and Td-CD in the MB-Salmonella typhimurium-CD compound;
magnetic frame separation, discarding magnetic beads, and reserving supernatant solution (mixed solution of bacteria, quantum dots and DNA regular tetrahedron structures);
and fifthly, scanning the reacted solution by a fluorescence spectrophotometer, measuring fluorescence intensity by fluorescence emission spectrum under 470nm excitation wavelength, introducing the measured fluorescence intensity into a standard curve, and calculating to obtain the concentration of the salmonella typhimurium in the sample to be measured.
Example 1
The aptamer utilized by the invention specifically acts on the salmonella typhimurium.
The sequence of the Aptamer1 is as follows: 5'-CTG ACAGAT TAAAAG TGG TTG GGC AAC TTC TGC TTG CGAA-3'
The sequence of the Aptamer2 is as follows: 5'-TAT GGC GGC GTC ACC CGACGG GGA CTT GAC ATT ATG ACA G-3'
A biosensor for detecting Salmonella typhimurium consists of a carbon quantum dot Td-CD modified by a Salmonella typhimurium aptamer and a magnetic bead Td-MB modified by a Salmonella typhimurium aptamer;
the DNA regular tetrahedron structure of the Salmonella typhimurium Aptamer for modifying the carbon quantum dots is Td-Aptamer1, and the DNA regular tetrahedron structure of the Salmonella typhimurium Aptamer for modifying the magnetic beads is Td-Aptamer 2.
1.1 preparation of Td-Aptamer:
chain a 1: 5'-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ACT GAC AGA TTA AAA GTG GTT GGG CAA CTT CTG CTT GCG AA-3' (SEQ ID NO:1)
Chain a 2: 5'-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ATA TGG CGG CGT CAC CCG ACG GGG ACT TGA CAT TAT GAC AG-3' (SEQ ID NO:2)
Chain B: 5'-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3' (SEQ ID NO: 3); the 5' end is connected with biotin molecules.
C chain: 5'-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3' (SEQ ID NO: 4); the 5' end is connected with biotin molecules.
Chain D: 5'-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3' (SEQ ID NO: 5); the 5' end is connected with biotin molecules.
Taking 2 1.5mL EP tubes, mixing and dissolving four DNA chains A1 and A B, C, D and four DNA chains A2 and A B, C, D in 1mL TE (10mM Tris, 1mM EDTA, pH 8.0) buffer solution at the concentration of 2 mu M, and uniformly mixing by vortex;
② transferring the solution into a metal bath, and heating for 5min at 95 ℃;
③ rapidly cooling in ice water for 10min until the temperature of the solution is reduced to 4 ℃, and respectively storing the obtained solutions Td-Aptamer1 and Td-Aptamer2 at 4 ℃ for later use.
1.2 preparation of SA-CD:
preparing amino functionalized carbon quantum dots:
weighing 0.4g of beta-cyclodextrin, dissolving in 20mL of ultrapure water, performing ultrasonic dispersion uniformly, transferring the beta-cyclodextrin into a stainless steel high-pressure reaction kettle (50mL) with a polytetrafluoroethylene lining, placing the stainless steel high-pressure reaction kettle in a blast-type heating drying box, and performing hydrothermal reaction for 6 hours at the temperature of 200 ℃; naturally cooling to room temperature after the reaction is finished, and dialyzing the obtained yellow brown transparent liquid in ultrapure water for 24 hours by using a dialysis bag (MWCO 1000) to remove impurities such as excessive amino acid and the like; and finally, performing rotary evaporation on a rotary evaporator at 45 ℃, and removing the solvent to obtain light yellow powder, namely the prepared amino functionalized carbon quantum dots.
Preparation of SA-CD:
taking a 5mL centrifuge tube, weighing 5mg of the prepared amino functionalized carbon quantum dots, dissolving in 1.5mL absolute ethyl alcohol, and performing ultrasonic dispersion uniformly; dropwise adding 1mL of streptavidin solution into the solution, and carrying out ultrasonic treatment for 15 min; violently shaking the mixture on a vortex mixer for 3h at room temperature, centrifuging the mixture in a high-speed centrifuge for 20min at the rotating speed of 12000 rpm, and removing supernatant to obtain streptavidin-modified carbon quantum dots; another 5mL centrifuge tube was prepared, and 1.0mg of the above carbon quantum dots were dispersed in 1mL HEPES (10mM, pH7.0) buffer solution to give a concentration of 1 mg. mL-1The SA-CD solution is evenly subjected to ultrasonic treatment for standby.
1.3Td-Aptamer1 self-assembles with SA-CD to form Td-CD microspheres:
taking the molar ratio of Td-aptamer 1200 mu L to SA-CD 100 mu L to 4:1 adding into 1ml PBS buffer solution;
placing the mixed solution on a vortex oscillation instrument at room temperature and oscillating for 1 hour;
and thirdly, centrifuging the mixture for 10min by a high-speed centrifuge at the rotating speed of 5000 rpm, discarding the supernatant to obtain Td-CD microspheres, and dispersing the Td-CD microspheres in a PBS buffer solution to obtain a Td-CD solution for later use.
1.4Td-Aptamer2 self-assembled with streptavidin functionalized magnetic beads to form Td-MB:
firstly, 1mg of streptavidin functionalized magnetic beads and 2200 microliter of 1.6 MuM Td-aptamer are mixed and dispersed in 500mL of Tris-HCl buffer solution (10mM Tris-HCl, 1mM EDTA,1M Na Cl, 0.01-0.1% Tween-20), and vortex and shake for 30 min.
Secondly, separating the magnetic beads from the system by using a magnet, and discarding the supernatant to obtain Td-MB magnetic beads;
③ washing the obtained Td-MB three times with 1mL of PBS buffer, and finally dispersing the Td-MB in the PBS buffer, namely, Td-MB solution.
Example 2
A method for detecting Salmonella typhimurium comprises the following steps:
2.1 standard curve plotting:
adding 1mL of drinking water containing no Salmonella typhimurium into 6 EP tubes of 1.5mL, respectively, adding Salmonella typhimurium bacterial liquid to make the concentration of the Salmonella typhimurium bacterial liquid 10CFU/mL and 102CFU/mL，103CFU/mL，104CFU/mL，105CFU/mL，106CFU/mL. Taking another 6 EP tubes with the volume of 1.5mL, respectively adding 100 mu L of bacterial liquid, 1mL of Td-MB solution and 1mL of Td-CD solution, incubating for 20 minutes at room temperature, and slowly shaking to fully combine the reaction; separating a magnetic frame, and removing supernatant to obtain an MB-Salmonella typhimurium-CD compound; resuspending the complex with 1.5mL of PBS, adding 2mL of Biotin molecular solution with a certain concentration, mixing and combining for 30 minutes; then, carrying out secondary magnetic bead separation, discarding magnetic beads, and keeping a supernatant solution; and finally, scanning the reacted solution by a fluorescence spectrophotometer, measuring fluorescence intensity by a fluorescence emission spectrum under the excitation wavelength of 470nm, and obtaining a standard curve chart 2. The lower detection limit of the biosensor reaches 6 CFU/mL.
2.2 sample detection
Taking a 1.5mL EP tube, adding 100 μ L of a sample to be detected, taking 1mL of the Td-MB solution prepared in the example 1 and 1mL of the Td-CD solution prepared in the example 1, incubating for 20 minutes at room temperature, and slowly shaking to fully combine the reaction; magnetic frame separation, carrying out first separation, and removing supernatant to obtain MB-Salmonella typhimurium-CD compound; resuspend the complex with PBS, then add 4 μ M concentration of Biotin molecular solution to mix and bind for 30 minutes; then, carrying out secondary magnetic bead separation, discarding magnetic beads, and keeping a supernatant solution; and finally, scanning the reacted solution by a fluorescence spectrophotometer, measuring the fluorescence intensity by fluorescence emission spectrum under the excitation wavelength of 470nm, introducing the measured fluorescence intensity into a standard curve, and calculating to obtain the concentration of the salmonella typhimurium in the sample to be measured.
Specific detection of the biosensor described in example 1
Three kinds of interference food-borne pathogenic bacteria including staphylococcus aureus, listeria monocytogenes and escherichia coli are selected to be tested together with salmonella typhimurium, so as to verify the specificity of the aptamer sensor, and the concentration of each bacterium is 102CFU/mL. The specificity results are shown in figure 3: as can be seen from the fluorescence value, the value of the salmonella typhimurium is obviously higher than that of other bacteria, so that the sensor has high specificity to the salmonella typhimurium.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Sequence listing
<110> Qingdao agricultural university
<120> biosensor for detecting salmonella typhimurium and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 95
<212> DNA
<213> Artificial sequence (Salmonella typhimurium)
<400> 1
acattcctaa gtctgaaaca ttacagcttg ctacacgaga agagccgcca tagtactgac 60
agattaaaag tggttgggca acttctgctt gcgaa 95
<210> 2
<211> 95
<212> DNA
<213> Artificial sequence (Salmonella typhimurium)
<400> 2
acattcctaa gtctgaaaca ttacagcttg ctacacgaga agagccgcca tagtatatgg 60
cggcgtcacc cgacggggac ttgacattat gacag 95
<210> 3
<211> 55
<212> DNA
<213> Artificial sequence (Salmonella typhimurium)
<400> 3
tatcaccagg cagttgacag tgtagcaagc tgtaatagat gcgagggtcc aatac 55
<210> 4
<211> 55
<212> DNA
<213> Artificial sequence (Salmonella typhimurium)
<400> 4
tcaactgcct ggtgataaaa cgacactacg tgggaatcta ctatggcggc tcttc 55
<210> 5
<211> 55
<212> DNA
<213> Artificial sequence (Salmonella typhimurium)
<400> 5
ttcagactta ggaatgtgct tcccacgtag tgtcgtttgt attggaccct cgcat 55

Claims (9)

1. A biosensor for detecting Salmonella typhimurium is characterized by consisting of a carbon quantum dot Td-CD modified by a Salmonella typhimurium aptamer and a magnetic bead Td-MB modified by a Salmonella typhimurium aptamer;
the DNA regular tetrahedron structure of the Salmonella typhimurium Aptamer for modifying the carbon quantum dots is Td-Aptamer1, and the DNA regular tetrahedron structure of the Salmonella typhimurium Aptamer for modifying the magnetic beads is Td-Aptamer 2;
the nucleic acid sequence forming the Td-Aptamer1 is:
chain a 1: 5'-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ACT GAC AGA TTA AAA GTG GTT GGG CAA CTT CTG CTT GCG AA-3'
Chain B: 5'-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3', connecting biotin molecules at the 5 ' end;
c chain: 5'-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3', connecting biotin molecules at the 5 ' end;
chain D: 5'-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3', connecting biotin molecules at the 5 ' end;
the nucleic acid sequence forming the Td-Aptamer2 is:
chain a 2: 5'-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ATA TGG CGG CGT CAC CCG ACG GGG ACT TGA CAT TAT GAC AG-3', respectively;
chain B: 5'-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3', connecting biotin molecules at the 5 ' end;
c chain: 5'-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3', connecting biotin molecules at the 5 ' end;
chain D: 5'-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3', the 5 ' end is connected with biotin molecule.
2. The biosensor of claim 1, wherein the Td-Aptamer1 and Td-Aptamer2 are prepared by: respectively mixing four nucleic acid sequences of the DNA regular tetrahedron structure in equimolar mode, dissolving the mixture in TE buffer solution, heating the solution after vortex mixing uniformly to 95 ℃ for 5min, and rapidly cooling the solution in ice water for 10min to 4 ℃ to obtain the DNA regular tetrahedron structure.
3. The biosensor for detecting Salmonella typhimurium according to claim 1, wherein the Salmonella typhimurium aptamer-modified carbon quantum dot Td-CD is prepared by:
adding the Td-aptamer1 and streptavidin-carbon quantum dots SA-CDs into a PBS buffer solution according to a molar ratio of 4:1, violently shaking for 1h at room temperature, rotating at 5000 rpm, centrifuging for 10min, discarding supernatant to obtain Td-CD microspheres, and dispersing the Td-CD microspheres in the PBS buffer solution to obtain a Td-CD solution.
4. The biosensor for detecting Salmonella typhimurium according to claim 3, wherein the streptavidin-carbon quantum dots SA-CDs are prepared by a method comprising:
weighing 5mg of amino-functionalized carbon quantum dots, dissolving the amino-functionalized carbon quantum dots in 1.5mL of absolute ethanol, uniformly dispersing by ultrasonic, dropwise adding a streptavidin solution into the solution, after performing ultrasonic treatment for 15min, violently shaking for 3h at room temperature, rotating at the speed of 12,000rpm, centrifuging for 20min, and discarding supernatant to obtain the streptavidin-modified carbon quantum dots.
5. The biosensor for detecting Salmonella typhimurium according to claim 4, wherein the amino functionalized carbon quantum dots are prepared by a method comprising the following steps:
weighing 0.4g of beta-cyclodextrin, dissolving in 20mL of ultrapure water, ultrasonically dispersing uniformly, transferring into a stainless steel high-pressure reaction kettle with a polytetrafluoroethylene lining, placing in a blast-type heating drying box, and carrying out hydrothermal reaction for 6h at the temperature of 200 ℃; naturally cooling to room temperature after the reaction is finished, and dialyzing the obtained yellow brown transparent liquid in ultrapure water for 24 hours by using a dialysis bag; and finally, performing rotary evaporation on a rotary evaporator at 45 ℃, and removing the solvent to obtain light yellow powder, namely the prepared amino functionalized carbon quantum dots.
6. The biosensor for detecting Salmonella typhimurium according to claim 1, wherein the Salmonella typhimurium aptamer-modified carbon quantum dot Td-MB is prepared by:
1mg of streptavidin functionalized magnetic beads and 1.6 mu M of Td-aptamer2 are mixed and dispersed in 500mL of Tris-HCl buffer solution, and vortex oscillation is carried out for 30 min; then, the magnetic beads were separated from the system with a magnet, the supernatant was discarded, and the magnetic beads were washed three times with 1mL of PBS buffer, and finally dispersed in the PBS buffer, i.e., Td-MB solution.
7. Use of the biosensor for detecting Salmonella typhimurium according to any one of claims 1 to 6 for detecting Salmonella typhimurium in food.
8. A Salmonella typhimurium detection kit comprising the biosensor according to any one of claims 1 to 6.
9. A method for detecting Salmonella typhimurium in food using the biosensor according to any one of claims 1 to 6, characterized by the steps of:
(1) incubating the Td-MB solution, the Td-CD solution and the sample solution to be detected for 20 minutes at room temperature, and slowly shaking for full combination reaction; performing magnetic separation for the first time, and removing the supernatant to obtain an MB-Salmonella typhimurium-CD compound;
(2) resuspending MB-Salmonella typhimurium-CD complex with PBS, adding 4 μm Biotin molecular solution, mixing and combining for 30 min; then, carrying out secondary magnetic separation, discarding magnetic beads, and reserving supernatant solution;
(3) and (3) scanning the supernatant solution obtained in the step (2) by using a fluorescence spectrophotometer, measuring fluorescence intensity by using a fluorescence emission spectrum under the excitation wavelength of 470nm, introducing the measured fluorescence intensity into a standard curve, and calculating to obtain the concentration of the salmonella typhimurium in the sample to be measured.
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