AU2021100518A4 - Fabrication of an aptamer biosensor based on quantum dots for detection and elimination of salmonella - Google Patents
Fabrication of an aptamer biosensor based on quantum dots for detection and elimination of salmonella Download PDFInfo
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- AU2021100518A4 AU2021100518A4 AU2021100518A AU2021100518A AU2021100518A4 AU 2021100518 A4 AU2021100518 A4 AU 2021100518A4 AU 2021100518 A AU2021100518 A AU 2021100518A AU 2021100518 A AU2021100518 A AU 2021100518A AU 2021100518 A4 AU2021100518 A4 AU 2021100518A4
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- aptamer
- quantum dots
- salmonella
- detection
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- 108091023037 Aptamer Proteins 0.000 title claims abstract description 68
- 241000607142 Salmonella Species 0.000 title claims abstract description 68
- 239000002096 quantum dot Substances 0.000 title claims abstract description 54
- 238000001514 detection method Methods 0.000 title claims abstract description 42
- 230000008030 elimination Effects 0.000 title claims abstract description 15
- 238000003379 elimination reaction Methods 0.000 title claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 title description 4
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical group O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims abstract description 27
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims abstract description 21
- 230000001954 sterilising effect Effects 0.000 claims abstract description 18
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 18
- 230000000295 complement effect Effects 0.000 claims abstract description 16
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 239000002953 phosphate buffered saline Substances 0.000 claims description 18
- 239000011248 coating agent Substances 0.000 claims description 17
- 238000000576 coating method Methods 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 10
- 235000013622 meat product Nutrition 0.000 claims description 10
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- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 3
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- 238000000151 deposition Methods 0.000 description 10
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 231100000570 acute poisoning Toxicity 0.000 description 5
- 230000000844 anti-bacterial effect Effects 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 231100000252 nontoxic Toxicity 0.000 description 5
- 230000003000 nontoxic effect Effects 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 208000004998 Abdominal Pain Diseases 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- CTENFNNZBMHDDG-UHFFFAOYSA-N Dopamine hydrochloride Chemical compound Cl.NCCC1=CC=C(O)C(O)=C1 CTENFNNZBMHDDG-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 208000005577 Gastroenteritis Diseases 0.000 description 2
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 description 2
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- NASVITFAUKYCPM-UHFFFAOYSA-N ethanol;tetraethyl silicate Chemical compound CCO.CCO[Si](OCC)(OCC)OCC NASVITFAUKYCPM-UHFFFAOYSA-N 0.000 description 2
- 229940044631 ferric chloride hexahydrate Drugs 0.000 description 2
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- -1 graphite carbon nitride quantum dots Chemical class 0.000 description 2
- 238000001027 hydrothermal synthesis Methods 0.000 description 2
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- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 2
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 2
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- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
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- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
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- 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"
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- A—HUMAN NECESSITIES
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- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/005—Preserving by heating
- A23B4/01—Preserving by heating by irradiation or electric treatment with or without shaping, e.g. in form of powder, granules or flakes
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- 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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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- 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"
- G01N2021/6432—Quenching
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- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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- G01N2333/255—Salmonella (G)
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Abstract
The invention discloses a preparation method and application of aptamer-quantum dots
biosensors for the detection and elimination of Salmonella, which belongs to the technical
field of detection and inactivation of food pathogenic bacteria. The biosensor of the
invention is to connect the aptamer C of Salmonella labeled by quantum dots on the surface
of Fe304 nanoparticles coated with poly dopamine (PDA), the complementary Strand A
modified with fluorescein was paired with the aforementioned aptamer C, and the Fe304
nanoparticles connected with aptamer C and strand A were used to detect Salmonella. The
detection sensitivity of the biosensor is 10 CFU/100 g, and the detection range is 10-104
CFU/100 g, which has good sensitivity and specificity; the sterilization degree is more than
95%, and the effect is remarkable.
1/3
DRAWINGS
Deposition of Modification of
olydop amn a chain Carbonquanlamdts
Complementary pairing of a
chain and C chain of aptamer V\A/________
I\J\A\IStrandA-FAM fluoreci
Near infrared radiation
\A StradAFAM florn
e-04 nanoparticles Fe0 nanopart cles \f\f\/\/ \Aptamer C- carbon quantum dots
W * Deposited PDA
\J\/\/\/\StrandA-FAM fluorescein * Carbon quantumdots Salmonella
Fig. 1
S80
70
S60
e50
40
30
20
10
0
Oh 1h 2h 3h
Deposition time
Fig. 2
Description
1/3
Deposition of Modification of olydop amn a chain Carbonquanlamdts
Complementary pairing of a chain and C chain of aptamer V\A/________ I\J\A\IStrandA-FAM fluoreci
Near infrared radiation
\A StradAFAM florn
e-04 nanoparticles W \J\/\/\/\StrandA-FAM * Fe0 nanopart cles \f\f\/\/ Deposited PDA fluorescein * Carbon quantumdots \Aptamer C- carbon quantum dots
Salmonella
Fig. 1
S80 70 S60 e50 40 30 20 10 0 Oh 1h 2h 3h Deposition time
Fig. 2
Technical field
[0001] The invention belongs to the technical field of detection and inactivation of
food pathogenic bacteria, specifically related to a fabrication of an aptamer biosensor
based on quantum dots for detection and elimination of Salmonella.
Technical background
[0002] Food pathogenic bacteria are pathogenic bacteria that can cause food
poisoning or spread by food. Pathogenic bacteria directly or indirectly contaminate
food and water. Human oral infection can lead to the prevalence of intestinal
infectious diseases, food poisoning and livestock and poultry infectious diseases.
Therefore, foodborne pathogens are an important source of food safety problems.
[0003] The impact of foodborne pathogens on human health: (1) acute poisoning: in
general, foodborne pathogens often cause acute poisoning, mild symptoms of acute
gastroenteritis, such as vomiting, nausea, abdominal pain, diarrhea, fever, etc., after
treatment can restore health; but severe symptoms of respiratory, circulatory, nervous
system, timely rescue can turn the crisis into safety, delay can be life-threatening.
Some acute poisoning, although by all means of treatment, but still left sequelae to the
poisoning. (2) Chronic poisoning or potential hazards: some deteriorated foods
contain less toxic substances, or due to their own toxic characteristics, they do not
cause acute poisoning, but long-term use can often cause chronic poisoning, and even
show carcinogenic, teratogenic and mutagenic effects. Besides acute poisoning, the use of rotten and moldy food has extremely serious potential hazards.
[0004] Salmonella is the most common pathogen causing food poisoning in
foodborne diseases in China, accounting for 70%-80% of the poisoning cases, and
more than 90% of the poisoning cases are caused by Salmonella contamination of
meat, eggs, milk and other livestock products. Salmonella infection is a zoonotic
infectious disease, which is mainly caused by eating contaminated food. After
ingesting toxic food, the symptoms generally appear within 12-14 hours, and some of
them have a long incubation period. The typical symptoms include fever, nausea,
vomiting, diarrhea and abdominal colic, as well as fatigue, muscle soreness, blurred
vision, restlessness and drowsiness, However, it usually gets better within 72 hours
after fever. Salmonella may also cause enteric typhoid, gastroenteritis and sepsis,
while infants, the elderly and patients with low immune function may have severe and
life-threatening bacteremia due to Salmonella entering the blood, and meningitis or
osteomyelitis can be found in a few patients.
[0005] Salmonella is the leading cause of death from food poisoning in the United
States. Americans are no stranger to this pathogen, and about 40000 cases of
Salmonella infection are reported in America every year. But the actual number of
infected people may be more than 20 times, because many light patients may not be
diagnosed. According to incomplete statistics, about 1000 people die of acute
Salmonella infection every year. The infection of pathogenic bacteria will not only
cause huge economic losses, but also seriously threaten people's health. Therefore, the
detection and sterilization technology of pathogenic bacteria in food is becoming
more and more important. The detection of Salmonella has always been based on
traditional detection methods. Although non selective and selective enrichment,
suspicious bacteria isolation and other conventional methods are classic and reliable,
the procedure is complex and cumbersome, which not only takes time and effort, but also has poor sensitivity and specificity, and high missed detection rate. However, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and other methods need to use the specified equipment, have the corresponding test conditions and skills, so it is difficult to promote in the grass-roots. Therefore, it is urgent to carry out in-depth research work in this field, establish a rapid and effective method to detect and kill Salmonella in food.
The invention content
[0006] In view of the problems existing in the prior art, the invention aims to provide a preparation method and application of aptamer-quantum dots biosensors for the detection and elimination of Salmonella; the aptamer quantum dots biosensors of the invention are aptamer C (with carbon quantum dots on the chain) connected with pathogenic bacteria on the surface of Fe304 nanoparticles deposited with PDA, and the complementary chain strand A modified with fluorescein was paired with the aptamer C, and pathogenic bacteria were detected by on-off fluorescence; NIR irradiation was carried out in the blue-green fluorescence region of quantum dots, and the enhanced photothermal effect of PDA coating on the surface of Fe304 nanoparticles was used to kill Salmonella. The biosensor and the sterilization method of the invention can solve the problems of low sensitivity, cumbersome detection method, high requirements for instruments and equipment, incomplete sterilization and the like in the existing detection and sterilization technology for Salmonella.
[0007] In order to achieve the above purpose, the invention adopts the following technical scheme: Aptamer-quantum dots biosensors for Salmonella detection and elimination is to connect aptamer C labeled with quantum dots on the surface of Fe304 nanoparticles coated with PDA, and pair strand A modified with fluorescein with aptamer C;
The nucleotide sequence of aptamer C of Salmonella is shown in SEQ ID No.1;
The nucleotide sequence of the complementary strand A is shown in SEQ ID
No.2.
[0008] The preparation method of aptamer quantum dots biosensors for Salmonella
detection and elimination is as follows:
(1) The aptamer C labeled by quantum dots was activated and connected with
Fe304 nanoparticles deposited with PDA coating. The 160 PL 2 PM quantum dots
labeled aptamer C was mixed with 30 pL 10 mg/L NHS and 30 pL 10 mg/L EDC for
activation. The activated aptamer C was mixed with Fe304 nanoparticles deposited
with PDA coating in the ratio of 3:1 by volume. After incubation at room temperature
for 30 min and gently shaking, the free aptamer C was removed by centrifugation at
,000 rpm for 25 min. The obtained conjugate was redispersed in 500 PL PBS buffer
after washing with 10 mM PBS at pH 7.0 for three times;
(2) The complementary Strand A modified with fluorescein was combined with
aptamer C of the conjugate obtained in step (1) by means of hydrogen bond through
base complementary pairing: 200 pL 2 pM Strand A was added into 500 pL PBS
buffer of the dispersed conjugate in step (1). After incubation at room temperature for
min and gently shaking, the free Strand A was removed by centrifugation at 10,000
rpm for 25 min. After washing with 10 mM PBS at pH 7.0 for three times, the
obtained conjugates were redispersed in 500 pL PBS buffer;
The fluorescein is any one of the fluorescein that can quench quantum dots such
as FAM or FITC;
On the basis of the above scheme, the quantum dots are any one of carbon
quantum dots, graphene quantum dots, non-toxic fluorescent quantum dots of
core-shell and composite materials, such as graphite carbon nitride quantum dots,
boron doped carbon dots, etc., preferably carbon quantum dots; using ascorbic acid as
carbon source, the carbon quantum dots with particle size of 2-3 nm and good
fluorescence performance were prepared by hydrothermal method in one step.
[0009] Compared with the quantum dots synthesized by other materials, the quantum dots synthesized by carbon dots are safer and more nontoxic, and the carbon quantum dots have the advantages of good biocompatibility, excellent fluorescence (including light stability, light bleaching resistance and non scintillation), low cost-effectiveness, simple manufacturing method and high solubility in water. In addition, graphene quantum dots and non-toxic fluorescent quantum dots of core-shell and composite materials can also be used to detect Salmonella. The advantages of these quantum dots are non-toxic and safe.
[0010] On the basis of the above scheme, the method for labeling quantum dots on the aptamer C is as follows: 10 M carbon quantum dots and 1.6 M mercaptan modified aptamer C of Salmonella were mixed in 1 ml phosphate buffer solution (pH 7.4, 0.01 M); after stirring for 12 h, the prepared carbon quantum dots-aptamer conjugate was collected and centrifuged at 6000 rpm for 15 min and washed with 50% ethanol for 3 times to obtain aptamer C labeled by quantum dots. The aptamer C labeled with quantum dots was suspended in 0.01 M phosphate buffer solution (pH 7.4) for subsequent preparation of Salmonella detection reagent.
[0011] On the basis of the above scheme, the Fe304 nanoparticles are synthesized by the following method: 4.8 g ferric chloride hexahydrate and 2 g ferrous chloride tetrahydrate are dissolved in 60 ml H 2 0, then added to 90 mL sodium hydroxide solution, stirred for 30 min, and then magnetic separation and water washing are carried out to obtain black solid. Add 80 ml (1:1) ethanol water solution, 5 ml concentrated ammonia water and 1.5 g black solid into 14 ml tetraethyl orthosilicate ethanol solution slowly. Under the protection of nitrogen, stir the reaction at 50 °C for
4 h, and evaporate the ethanol. After the reaction continued for 4 h, the product was
washed to neutral, soaked in HCl for two days, dried to constant weight in a vacuum
drying oven at 50 °C to obtain Fe304 nanoparticles.
[0012] The reasons of selecting Fe304 to synthesize nanoparticles: Fe304
nanoparticles are the most common magnetic nanomaterials with low toxicity, good
chemical stability, high magnetic saturation strength, wide particle size distribution
and easy surface modification.
[0013] On the basis of the above scheme, the method of depositing PDA on Fe304
nanoparticles is as follows:
Fe304 nanoparticles (12 mg) and dopamine hydrochloride powder (7.5 mg) were
mixed in 30 ml Tris-HCl buffer (pH 8.5). The thickness of PDA coating after 1 h, 2 h
and 3 h deposition of Fe304 nanoparticles has little difference, so 1 h deposition is the
best time. The mixture was placed in an Eppendorf 5436 constant temperature
oscillator and shaken slowly but continuously at room temperature. The magnetic
beads deposited with PDA coating were separated by magnetic attraction and cleaned
with deionized water (three times) to obtain Fe304 nanoparticles deposited with PDA
coating.
[0014] The aptamer-quantum dots biosensors prepared by the above method can be
used in the detection and elimination of Salmonella in food.
[0015] On the basis of the above scheme, the food is solid food, preferably one of
meat products and fruit and vegetable products.
[0016] The method for detecting and eliminating Salmonella in food by using the aptamer-quantum dots biosensors is as follows: The aptamer quantum dots biosensors are dispersed in phosphate buffered saline to prepare a solution with a concentration of 0.32 pM, and the dispersion is added to the surface of food for detection of Salmonella. Salmonella specifically binds to aptamer C due to high affinity, and strand A is competed by bacteria, Quantum dots on the aptamer C will lose the quenching effect of fluorescein on the strand A and show fluorescence. The fluorescence of quantum dots can be used to determine the content of Salmonella.
[0017] 1.5 W/cm2 808 nm NIR light was used for continuous irradiation in the area showing fluorescence; after sterilization, Fe304 nanoparticles were separated by putting food in magnetic field or under the action of magnet, and the strand A modified with fluorescein on the food surface was washed off with water.
[0018] The technical scheme of the invention has the following advantages:
[0019] In the invention, aptamer C of Salmonella modified with carbon quantum dots and complementary chain strand A modified with fluorescein are modified on Fe304 nanoparticles with PDA coating, Salmonella is specifically recognized and detected by switching fluorescence, and then Salmonella is killed by using the enhanced photothermal effect of PDA.
[0020] The method of the invention has the characteristics of specificity, high sensitivity and strong affinity by using the aptamer to recognize Salmonella, simple operation, no need for complex pretreatment of the sample to be tested, remarkable bactericidal effect.
[0021] In particular, when the invention is used for the detection of Salmonella in
food, the materials used are non-toxic and harmless, which can kill foodborne
pathogenic bacteria without causing adverse effects on food, and is conducive to
improving food quality.
[0022] The invention is applicable to the detection and elimination of Salmonella in
solid food, such as meat products, fruit and vegetable products, etc., and it has a wide
range of application and good practicability.
[0023] With the help of aptamer chain and quantum dots, the detection sensitivity of
the invention is 10 CFU/100 g, and the detection range is 10-104 CFU/100 g, which
has good sensitivity and specificity. The degree of sterilization is more than 95%, and
the effect is remarkable.
Illustration
[0024] Fig. 1 is a schematic diagram of the detection and sterilization principle of
the method of the invention;
[0025] Fig. 2 shows the effect of deposition time on the thickness of the PDA
coating deposited by Fe304 nanoparticles;
[0026] Fig. 3 shows the detection results of absorbance of different strains suspension;
[0027] Fig. 4 is the schematic diagram of Salmonella detection at different concentrations in meat products;
[0028] Fig. 5 is the standard curve of Salmonella detection at different concentrations;
[0029] Fig. 6 shows the comparison of fluorescence intensity for NIR sterilization of Fe304 nanoparticles before and after PDA deposition.
Specific implementation mode
[0030] Unless otherwise specified, the terms used in the present invention generally have the meanings generally understood by those skilled in the art.
[0031] The present invention is further described in detail with reference to specific embodiments and data. The following embodiments are only for illustrating the present invention, rather than limiting the scope of the present invention in any way.
[0032] The aptamer-quantum dots biosensors for detecting and killing Salmonella has the following working principle:
[0033] Referring to Fig. 1, Fe304 nanoparticles were synthesized firstly, and then
they were deposited by PDA. Through the deposited PDA coating, the photothermal
effect of nanoparticles could be enhanced, and the sterilization temperature could be
increased. The synthesized aptamer C was modified with carbon quantum dots
(showing blue-green fluorescence), which could be connected to Fe304 nanoparticles
by dehydration condensation of amino carboxyl group with PDA, and then the strand
A modified with fluorescein was complementary paired with the aptamer C. Due to
the fluorescence quenching effect of fluorescein, the carbon quantum dots on aptamer
C do not show blue-green fluorescence. The final modified Fe304 nanoparticles were
used for the detection of Salmonella in food. Because the high affinity aptamer C
would specifically bind to Salmonella, the strand A would be competed by bacteria,
and the carbon quantum dots on aptamer C would lose the fluorescence quenching
effect of fluorescein, showing blue-green fluorescence. The results show that NIR
irradiation can kill Salmonella by means of the photothermal effect enhanced by the
PDA coating on the surface of Fe304 nanoparticles.
Example 1
[0034] The aptamer-quantum dots biosensors for Salmonella detection and
elimination is to connect aptamer C (SEQ ID NO.1) labeled with carbon quantum dots
on the surface of Fe304 nanoparticles coated with PDA, and to couple strand A (SEQ
ID NO.2) modified with fluorescein with aptamer C;
The 5'of the Salmonella aptamer Aptamer C is modified with a carboxyl group,
and the 3'is modified with -SH, as follows:
5'-COOH-AGCAGCACAG-GCGATCCAAGCTTCTTCA-SH-3' The complementary chain Strand A modified with fluorescein FAM is as
follows:
3'-CGCTAGGTTCGAAGAAGT-FAM-5'
Using ascorbic acid as carbon source, the carbon quantum dots with particle size of 2-3 nm and good fluorescence performance were prepared by hydrothermal method in one step.
[0035] The preparation method of aptamer-quantum dots biosensors for Salmonella detection and elimination is as follows: (1) Labeling quantum dots on aptamer C: 10 M carbon quantum dots were mixed with 16 M mercaptan modified aptamer C of Salmonella (purchased from Bioengineering Co., Ltd.) in 1 ml phosphate buffer solution (pH 7.4, 0.01 M); after stirring for 12 h, the prepared carbon quantum dots-aptamer conjugate was collected and centrifuged at 6000 rpm for 15 min. The aptamer C labeled with quantum dots was obtained by washing with 50% ethanol for 3 times, the aptamer C labeled with quantum dots was suspended in 0.01 M phosphate buffer solution (pH 7.4) for subsequent preparation of Salmonella detection and elimination reagent. (2) Strand A, which modified fluorescein FAM, purchased from bioengineering (Shanghai) Co., Ltd. (3) Synthesis of Fe304 nanoparticles: 4.8 g ferric chloride hexahydrate and 2 g ferrous chloride tetrahydrate are dissolved in 60 ml H 2 0, then added to 90 mL sodium hydroxide solution, stirred for 30 min, and then magnetic separation and water washing are carried out to obtain black solid. Add 80 ml (1:1) ethanol water solution, ml concentrated ammonia water and 1.5 g black solid into 14 ml tetraethyl orthosilicate ethanol solution slowly. Under the protection of nitrogen, stir the reaction at 50 °C for 4 h, and evaporate the ethanol. After the reaction continued for 4 h, the product was washed to neutral, soaked in HCl for two days, dried to constant weight in a vacuum drying oven at 50 °C to obtain Fe304 nanoparticles. (4) Deposite PDA on Fe304 nanoparticles: Fe304 nanoparticles (12 mg) and dopamine hydrochloride powder (7.5 mg) were mixed in 30 ml Tris-HCl buffer (pH 8.5). As shown in Fig. 2, the thickness of PDA coating after 1h, 2 h and 3 h deposition of Fe304 nanoparticles has little difference, all of them are about 70 nm, so
1 h deposition is the best time. The mixture was placed in an Eppendorf 5436 constant
temperature oscillator and shaken slowly but continuously at room temperature. The magnetic beads deposited with PDA coating were separated by magnetic attraction and cleaned with deionized water (three times) to obtain Fe304 nanoparticles deposited with PDA coating. (5) The aptamer C labeled by quantum dots was activated and connected with Fe304 nanoparticles deposited with PDA coating. The 160 PL 2 PM quantum dots labeled aptamer C was mixed with 30 pL 10 mg/L NHS and 30 pL 10 mg/L EDC for activation. The activated aptamer C was mixed with Fe304 nanoparticles deposited with PDA coating in the ratio of 3:1 by volume. After incubation at room temperature for 30 min and gently shaking, the free aptamer C was removed by centrifugation at ,000 rpm for 25 min. The obtained conjugate was redispersed in 500 PL PBS buffer after washing with 10 mM PBS at pH 7.0 for three times; the temperature is 4 °C. The number of aptamers modified on each Fe304 nanoparticle was estimated by the concentration of Fe304 nanoparticles and aptamers. The concentration of aptamer modified on Fe304 nanoparticles was determined according to the decrease of absorbance at 260 nm. (6) The complementary strand A modified with fluorescein FAM is combined with the aptamer C by base complementary pairing and hydrogen bonding. The strand A at
a concentration of 2 pM and a dosage of 200 pL is mixed with 500 PL PBS buffer solution. After incubation at room temperature for 30 min and gently shaking, the free Strand A was removed by centrifugation at 10,000 rpm for 25 min. After washing with mM PBS at pH 7.0 for three times, the obtained conjugates were redispersed in
500 pL PBS buffer. Due to the fluorescence quenching effect of fluorescein FAM, the carbon quantum dots on aptamer C no longer show blue-green fluorescence.
Example 2
[0036] The method for detecting and killing Salmonella comprises the following steps:
The aptamer-quantum dots biosensors are dispersed in phosphate buffered saline
to prepare a solution with a concentration of 0.32 pM, and the dispersion is added to
the surface of food for detection of Salmonella. Salmonella specifically binds to
aptamer C due to high affinity, and strand A is competed by bacteria, Quantum dots on
the aptamer C will lose the quenching effect of fluorescein FAM on the strand A and
show blue-green fluorescence. The fluorescence of quantum dots can be used to
determine the content of Salmonella.
[0037] In the blue-green fluorescent region of carbon quantum dots, the NIR light of
1.5 w/cm2 808 nm was used for continuous irradiation; after sterilization, Fe304
nanoparticles were separated by putting food in magnetic field or under the action of
magnet, and the strand A modified with fluorescein FAM on the food surface was
washed with water.
Sensitivity and specificity
[0038] Specificity
[0039] Salmonella, Escherichia coli, Vibrio parahaemolyticus and blank control
group are respectively used to test the detection specificity of the biosensor of the
invention: 104 CFU/100 g Salmonella, Escherichia coli, Vibrio parahaemolyticus and
sterile suspension are detected by the method of example 2, and the obtained
experimental results are shown in Fig. 3.
[0040] It can be seen from Fig. 3 that the results of detecting three kinds of bacteria
by using the biosensor of the invention show that the Salmonella suspension has a strong absorbance signal compared with the blank control group, while the absorbance of Escherichia coli and Vibrio parahaemolyticus is equivalent to that of the blank control group. It can be seen that the biosensor has good specificity.
[0041] Sensitivity
[0042] The meat product in thin section state is sterilized by ultraviolet firstly, and then the mixed Salmonella suspension is inoculated on the surface of the meat product in sterile state, so that the surface of the meat product has different Salmonella concentrations, such as 10,102,103,104 CFU/100 g. the detection method of example 2
is used to evaluate the sensitivity of the biosensor in example 1 of the present invention. It can be seen from Fig. 4 and Fig. 5 that with the increase of Salmonella concentration, the blue fluorescence intensity of quantum dots detected by fluorescence detector gradually increases. When the bacterial concentration range is to 104 CFU/100 g, the detection sensitivity of the biosensor is 10 CFU/100 g, the standard curve is y=0.182x+0.08, and the correlation coefficient is 0.9874. It can be seen that the biosensor has good linear relationship and high sensitivity.
Germicidal efficacy
[0043] The detection method of example 2 was used to detect Salmonella in meat products, as shown in Fig. 6. The first column diagram before NIR sterilization showed strong fluorescence.
[0044] If the Fe304 nanoparticles without PDA deposition were used to detect Salmonella, and the NIR sterilization was carried out in the fluorescent region. After sterilization, Fe304 nanoparticles connected with aptamer C (modified with quantum dots) were separated by putting meat products in magnetic field or under the action of magnet, and strand A with fluorescein FAM on the food surface was washed off with water, and then detected the bactericidal effect, as shown in the second column diagram of Fig. 6, it can be found that the fluorescence intensity becomes weak to a certain extent.
[0045] If the Fe304 nanoparticles deposited with PDA were used to detect Salmonella, and the NIR sterilization was carried out in the fluorescent region. After sterilization, Fe304 nanoparticles connected with aptamer C (modified with quantum dots) were separated by putting meat products in magnetic field or under the action of magnet, and strand A with fluorescein FAM on the food surface was washed off with water, and then detected the bactericidal effect, as shown in the third column figure in Fig. 6, the column figure after NIR sterilization shows weak fluorescence.
[0046] It shows that the invention has good bactericidal effect, and the photothermal effect caused by PDA can enhance the bactericidal effect.
[0047] The above description is only a better embodiment of the invention, and is not to limit the invention in other forms. Any person familiar with the art may use the above disclosed technical content to change or modify it into an equivalent embodiment of the same change. Those skilled in the art can apply it to the detection and killing of other pathogenic bacteria and toxins. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the invention without departing from the content of the technical scheme of the invention still belong to the protection scope of the technical scheme of the invention.
Claims (5)
1. The aptamer-quantum dots biosensors for Salmonella detection and elimination are characterized in that Salmonella aptamer C labeled with quantum dots is connected on the surface of Fe304 nanoparticles coated with PDA, and the complementary strand A modified with fluorescein is paired with aptamer C; the nucleotide sequence of aptamer C of Salmonella is shown in SEQ ID No.1; the nucleotide sequence of the complementary strand A is shown in SEQ ID No.2.
2. The preparation method of aptamer-quantum dots biosensors for Salmonella detection and elimination according to claim 1, which is characterized by the following steps: Step1: the aptamer C labeled by quantum dots was activated and connected with Fe304 nanoparticles deposited with PDA coating. The 160 pL 2 pM quantum dots labeled aptamer C was mixed with 30 pL 10 mg/L NHS and 30 pL 10 mg/L EDC for activation, the activated aptamer C was mixed with Fe304nanoparticles deposited with PDA coating in the ratio of 3:1 by volume. After incubation at room temperature for 30 min and gently shaking, the free aptamer C was removed by centrifugation at 10,000 rpm for 25 min, the obtained conjugate was redispersed in 500 pL PBS buffer after washing with 10 mM PBS at pH 7.0 for three times; Step2: the complementary Strand A modified with fluorescein was combined with aptamer C of the conjugate obtained in step 1 by means of hydrogen bond through base complementary pairing: 200 pL 2 pM Strand A was added into 500 pL PBS buffer of the dispersed conjugate in step 1; after incubation at room temperature for 30 min and gently shaking, the free strand A was removed by centrifugation at 10,000 rpm for 25 min, the obtained conjugate was redispersed in 500 pL PBS buffer after washing with 10 mM PBS at pH 7.0 for three times.
3. The application of aptamer-quantum dots biosensors prepared by the method of claim 2 in the detection and elimination of Salmonella in food.
4. The application according to claim 3, which is characterized in that the food is solid food, preferably one of meat products and fruit and vegetable products.
5. The method for detecting and eliminating Salmonella in food using the aptamer quantum dots biosensors of claim 1, which is characterized by the following steps: the aptamer-quantum dots biosensors described in claim 1 are dispersed in phosphate
buffered saline to prepare a solution with a concentration of 0.32 PM, and the dispersion is added to the surface of food for detection of Salmonella. Salmonella specifically binds to aptamer C due to high affinity, and strand A is competed by bacteria, Quantum dots on the aptamer C will lose the quenching effect of fluorescein on the strand A and show fluorescence, the fluorescence of quantum dots can be used to determine the content of Salmonella; in the fluorescent region of carbon quantum dots, the near-infrared (NIR) light of 1.5 w/cm2 808 nm was used for continuous irradiation; after sterilization, Fe304 nanoparticles were separated by putting food in magnetic field or under the action of magnet, and the strand A modified with fluorescein on the food surface was washed with water.
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