CN106370868A - SPR sensor for detecting microcystic toxin based on nucleic acid aptamer signal amplification strategy and preparation method and application thereof - Google Patents
SPR sensor for detecting microcystic toxin based on nucleic acid aptamer signal amplification strategy and preparation method and application thereof Download PDFInfo
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- CN106370868A CN106370868A CN201610846741.0A CN201610846741A CN106370868A CN 106370868 A CN106370868 A CN 106370868A CN 201610846741 A CN201610846741 A CN 201610846741A CN 106370868 A CN106370868 A CN 106370868A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000003053 toxin Substances 0.000 title claims abstract description 14
- 231100000765 toxin Toxicity 0.000 title claims abstract description 14
- 230000003321 amplification Effects 0.000 title abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 title abstract description 3
- 108091008104 nucleic acid aptamers Proteins 0.000 title abstract 2
- 238000001514 detection method Methods 0.000 claims abstract description 30
- 108091023037 Aptamer Proteins 0.000 claims abstract description 27
- 239000002086 nanomaterial Substances 0.000 claims abstract description 6
- 239000011521 glass Substances 0.000 claims abstract description 5
- 108020004414 DNA Proteins 0.000 claims description 95
- 239000000523 sample Substances 0.000 claims description 85
- 229910000510 noble metal Inorganic materials 0.000 claims description 47
- SRUWWOSWHXIIIA-UKPGNTDSSA-N Cyanoginosin Chemical compound N1C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](C)[C@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C(=C)N(C)C(=O)CC[C@H](C(O)=O)N(C)C(=O)[C@@H](C)[C@@H]1\C=C\C(\C)=C\[C@H](C)[C@@H](O)CC1=CC=CC=C1 SRUWWOSWHXIIIA-UKPGNTDSSA-N 0.000 claims description 45
- 108010067094 microcystin Proteins 0.000 claims description 45
- 239000000243 solution Substances 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 21
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 16
- 229910052737 gold Inorganic materials 0.000 claims description 16
- 239000010931 gold Substances 0.000 claims description 16
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- 239000002052 molecular layer Substances 0.000 claims description 13
- 230000000295 complement effect Effects 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 9
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- 241000195493 Cryptophyta Species 0.000 claims description 8
- 239000002082 metal nanoparticle Substances 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
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- 239000012498 ultrapure water Substances 0.000 claims description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 6
- 102000053602 DNA Human genes 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 6
- 239000002775 capsule Substances 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- ZYZCGGRZINLQBL-GWRQVWKTSA-N microcystin-LR Chemical compound C([C@H](OC)[C@@H](C)\C=C(/C)\C=C\[C@H]1[C@@H](C(=O)N[C@H](CCC(=O)N(C)C(=C)C(=O)N[C@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]([C@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1)C(O)=O)C(O)=O)C)C1=CC=CC=C1 ZYZCGGRZINLQBL-GWRQVWKTSA-N 0.000 claims description 4
- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical compound CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 claims description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 3
- 235000007164 Oryza sativa Nutrition 0.000 claims description 3
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 3
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 229910052802 copper Inorganic materials 0.000 claims description 3
- 239000010949 copper Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 235000009566 rice Nutrition 0.000 claims description 3
- 229910052709 silver Inorganic materials 0.000 claims description 3
- 239000004332 silver Substances 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
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- 238000012986 modification Methods 0.000 claims description 2
- 230000004048 modification Effects 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 5
- 238000012544 monitoring process Methods 0.000 abstract description 5
- 230000008859 change Effects 0.000 abstract description 2
- 108020003215 DNA Probes Proteins 0.000 abstract 2
- 239000003298 DNA probe Substances 0.000 abstract 2
- 239000010970 precious metal Substances 0.000 abstract 1
- 235000013339 cereals Nutrition 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 108010049746 Microcystins Proteins 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003094 microcapsule Substances 0.000 description 3
- 241000209094 Oryza Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
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- 108010073357 cyanoginosin LR Proteins 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
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- 235000020188 drinking water Nutrition 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- DIDLWIPCWUSYPF-UHFFFAOYSA-N microcystin-LR Natural products COC(Cc1ccccc1)C(C)C=C(/C)C=CC2NC(=O)C(NC(CCCNC(=N)N)C(=O)O)NC(=O)C(C)C(NC(=O)C(NC(CC(C)C)C(=O)O)NC(=O)C(C)NC(=O)C(=C)N(C)C(=O)CCC(NC(=O)C2C)C(=O)O)C(=O)O DIDLWIPCWUSYPF-UHFFFAOYSA-N 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000003911 water pollution Methods 0.000 description 2
- 241000143060 Americamysis bahia Species 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
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- 102000004190 Enzymes Human genes 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- ZCHPKWUIAASXPV-UHFFFAOYSA-N acetic acid;methanol Chemical compound OC.CC(O)=O ZCHPKWUIAASXPV-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
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- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 231100000784 hepatotoxin Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000003987 high-resolution gas chromatography Methods 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
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- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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- 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
- G01N2333/405—Assays involving biological materials from specific organisms or of a specific nature from algae
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- General Health & Medical Sciences (AREA)
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Abstract
The invention belongs to the technical field of toxin detection and discloses an SPR sensor for detecting microcystic toxin based on a nucleic acid aptamer signal amplification strategy and a preparation method and application thereof. The SPR sensor comprises a glass chip, a microcystic toxin aptamer, a DNA probe 1, a DNA probe 2 and a precious metal nano material, wherein microcystic toxin detection can be performed by determining SPR signal change. The preparation of the SPR sensor is simple and facilitates establishment of a portable detecting instrument for field monitoring, and the cost is far lower than that of a large analysis and detection instrument.
Description
Technical field
The invention belongs to toxin detection technology field is and in particular to the detection based on aptamer signal amplification strategy is micro-
Surface plasma (spr) sensor of capsule Algae toxins and its preparation method and application.
Background technology
Microcystin (mcs) is a class ring-type heptapeptide class hepatotoxin of harmful blue-green alga bloom release, has strong cause
Cancer acts on, and is the important environmental factorss of the diseases such as induced hepatocellular carcinoma, gastroenteritis.In view of the toxicity of Microcystin and its harm, generation
Microcystin lr (mc-lr) has been classified as the noxious pollutant needing to control in drinking water by boundary's health organization (who).Blue-green alga bloom
The Microcystin secondary pollution causing and its harm to health attract attention, it has also become the whole world is common
The environmental science problem facing.
Analysis and detection technology currently for the Microcystin in water body mainly has Instrumental Analysis and sensor detection two
Big class, the former includes high resolution gas chromatography method (hpgc), high performance liquid chromatography (hplc) and liquid chromatograph mass spectrography (lc-
The method such as ms), has the advantages that detection sensitivity is high, selectivity is good, high specificity, but instrument detection is expensive, before sample
Complex disposal process, the degree of specialization to experiment condition and operating technology have high demands, detection time is longer, testing cost is high,
And equipment instrument is big, it is not easy to field monitoring and the monitoring realizing sudden pollution accident, there is certain application limitation.
Sensor detecting method includes the methods such as euzymelinked immunosorbent assay (ELISA), radioimmunoassays, boiled shrimps with shell in salt water method and bioprobe, and sensor detects
The pretreatment process of method relatively simplifies, and detection time is short, it is possible to achieve quick detection, and special to experiment condition and operation
Industry degree less demanding it is adaptable to the field quick detection of batch samples.But the enzyme being used is expensive, volatile
Live, the problems such as this kind of sensor detecting method generally existing sensitivity is low at present in addition, stability is poor, probe is expensive, because
And limit promoting the use of on a large scale of this technology.In recent years, molecular imprinting has specific recognition and knot to object
The ability closed, the molecular engram colorimetric sensor that it builds can realize quick detection Microcystin, but molecular engram film
Preparation process need to use the organic reagent having pollution to environment.Because the above analysis method is existed not with technology itself
Foot, seriously constrains and Microcystins in Water pollution is dynamically understood in time.Therefore, for China's water body low concentration, height
The pollution situation that toxicity, multiple organic and inorganic pollution coexist, develops quick, easy, highly sensitive, high specificity and scene makes
Detection Microcystin sensor, for the enforcement of monitoring Microcystins in Water pollution control measures, ensures drinking-water
Safety, alleviation water resource anxiety are significant.
Content of the invention
In view of this, an object of the present invention is to provide the detection microcapsule amplifying strategy based on aptamer signal
The spr sensor of Algae toxins；The second object of the present invention is to provide the method preparing described spr sensor；The mesh of the present invention
Three be provide spr sensor detection Microcystin in application；The fourth object of the present invention is to provide utilization
The method that described spr sensor detects capsule Algae toxins.
For achieving the above object, the following technical scheme of present invention offer:
1st, amplify the spr sensor of the detection Microcystin of strategy, described sensor bag based on aptamer signal
Include glass-chip, Microcystin aptamers, dna probe 1, dna probe 2 and noble metal nanometer material；Described glass-chip
One layer of noble metal nano layer of area load, described dna probe 1 passes through 5 ' terminal modified sulfydryls and described glass-chip area load
Noble metal nano layer pass through Covalent bonding together；Described dna probe 2 is passed through 3 ' terminal modified sulfydryls and is tied with noble metal nanometer material
Close；5 ' ends of described dna probe 1 are complementary with 3 ' ends of described dna probe 2 to be combined；Described Microcystin aptamers one end with
3 ' ends of described dna probe 1 are complementary to be combined, and the other end is complementary with 5 ' ends of described dna probe 2 to be combined.
Further, described dna probe 1 is single-stranded dna as shown in seq id no.1 for the base sequence；Described dna probe 2
For single-stranded dna as shown in seq id no.2 for the base sequence；Described Microcystin aptamers are base sequence such as seq id
Single-stranded dna shown in no.3.
Further, the thickness of described noble metal nano layer is 10-20nm.
Further, the particle diameter of described noble metal nanometer material is 30-50nm.
Further, described noble metal includes gold, silver or copper.
2nd, the preparation method of spr sensor, comprises the steps:
(1) prepare glass-chip: it is positively charged amino that glass slide cleaning is made surface, be then immersed in citric acid stablize expensive
Make noble metal nano in metal nano solution on the slide after electrostatic force is deposited on process, obtain glass-chip；
(2) glass-chip modified by dna probe 1: step (1) gained glass-chip is immersed in probe containing dna 1 solution, makes
Described dna probe 1 is combined with the noble metal nano layer of glass-chip area load by covalent bond；
(3) noble metal nano particles modified by dna probe 2: dna probe 2 is added in noble metal nano solution, makes described
Dna probe 2 is combined with noble metal nanometer material surface by covalent bond；
(4) spr sensor preparation: the glass-chip immersion dna probe 2 that dna probe 1 in step (2) is modified is modified expensive
In metal nano solution, it is subsequently adding Microcystin aptamers, obtain spr sensor.
Further, comprise the steps:
(1) prepare glass-chip: slide first passes through ammonia: hydrogen peroxide: ultra-pure water volume ratio is that the mixing of 1:1:1 is molten
Liquid cleans 1h, is then placed in 10min in the ethanol solution of 10% aminopropyl trimethoxysilane in anhydrous conditions, then with ultrapure
Water cleans, and makes surface be positively charged amino, and being finally immersed in citric acid and stablizing 2h in noble metal nano solution makes noble metal receive
Rice grain pattern is crossed on the slide after electrostatic force is deposited on process, obtains glass-chip；
(2) glass-chip modified by dna probe 1: step (1) gained glass-chip is immersed 1h in probe containing dna 1 solution,
Described dna probe 1 is made to be combined with the noble metal nano layer of glass-chip area load by covalent bond；
(3) noble metal nano particles modified by dna probe 2: dna probe 2 is added 1h in noble metal nano solution, makes described
Dna probe 2 is combined with noble metal nanometer material surface by covalent bond, then carries out ultrafiltration, removes unconjugated dna probe 2；
(4) spr sensor preparation: dna probe 1 in step (2) is modified your gold glass-chip immersion dna probe 2 modifies
Belong in nanometer solution, be subsequently adding Microcystin aptamers, obtain spr sensor.
3rd, application in detection Microcystin for the described spr sensor.
4th, utilize described spr sensor detect capsule Algae toxins method it is characterised in that: described spr sensor is added
Enter in the solution containing Microcystin, measure spr signal intensity.
Spr sensor is utilized to detect that the principle of capsule Algae toxins is as follows in the present invention: dna probe 1 and dna probe 2 are with microcapsule
Algae toxins aptamers form y type dna diploid as bridge by base pair complementarity, thus forming one on glass-chip surface
The new noble metal nano layer of layer, obtains higher and more rich spr signal, and object Microcystin is adapted to Microcystin
Body specifically binds, and changes its structure, after being allowed to lose bridge beam action cannot glass-chip surface formed one layer new
Noble metal nano layer, and then this spr sensor measures the detection that spr signal intensity carries out Microcystin.
The beneficial effects of the present invention is: spr sensor of the present invention amplifies strategy based on aptamer signal
Realize the detection of Microcystin, using the exclusive office of the surface plasma body resonant vibration of noble metal film and noble metal nano structure
Field surface plasmon resonance principles, by measure spr signal intensity reach quickly, the purpose of Sensitive Detection Microcystin.
Spr transducer production method of the present invention is simple, detection range width, selectivity height, good stability, simple to operate, the green nothing of reagent
Pollution, disclosure satisfy that the real-time analysis to optical signal it is easy to build field monitoring Portable detection instrument, and the glass of sensor
Chip can be reused, and cost is far below large-scale analysis and detecting instrument.
Brief description
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, present invention offer drawings described below:
Fig. 1 is the sensor mechanism schematic diagram of Microcystin sensor；
Fig. 2 is the schematic diagram of oligonucleotide used in Microcystin lr mensure；
Fig. 3 is the test result figure to Microcystin for the Microcystin sensor.
Specific embodiment
The preferred embodiments of the present invention will be described in detail below.The experiment of unreceipted actual conditions in embodiment
Method, generally according to normal condition or according to the condition proposed by manufacturer.
Embodiment 1
Amplify the preparation method of the spr sensor of the detection Microcystin of strategy based on aptamer signal, specifically
The step of preparation method is as follows:
(1) prepare glass-chip: slide is first used ammonia: hydrogen peroxide: ultrapure water volume ratio is the mixed solution of 1:1:1
Cleaning 1h, is then placed in the ethanol solution of the aminopropyl trimethoxysilane that volume fraction is 10% in anhydrous conditions
10min, then cleaned with ultra-pure water, make surface be positively charged amino, be finally immersed in citric acid and stablize 2h in gold nano solution
Make gold nano grain on the slide after electrostatic force is deposited on process, obtain glass-chip；
(2) glass-chip modified by dna probe 1: by the immersion of step (1) gained glass-chip containing 0.03 μm of dna probe 1
1h in solution, is made dna probe 1 be combined with the gold nano grain on glass-chip surface by au-s key, is cleaned with ultra-pure water, natural
Dry stand-by；Wherein dna probe 1 is that 5 ' terminal modified sulfydryl dna are single-stranded, and base sequence is 5 '-sh-ctgtgacggtaatt-3 '
(seq id no.1)；
(3) gold nano grain modified by dna probe 2: it is 1h in 30-50nm gold nano solution that dna probe 2 is added particle diameter,
Make dna probe 2 be covalently bonded in gold nano-material surface by au-s key, then carry out ultrafiltration, remove unconjugated dna probe
2；Wherein dna probe 2 is single-stranded for the dna of 3 ' terminal modified sulfydryls, and base sequence is 3 '-sh-gacactggtatggt-5 ' (seq
id no.2)；
(4) spr sensor preparation: dna probe 1 in step (2) is modified glass-chip immersion dna probe 2 and modifies Jenner
In rice solution, it is subsequently adding Microcystin aptamers, obtains spr sensor；Wherein Microcystin aptamers are that dna is single-stranded,
Base sequence is 5 '-ggcgccaaacaggaccatgacaattacccataccacctcattatgccccatctccg c-3'(seq
id no.3).
5 ' ends of dna probe 1 are complementary with 3 ' ends of described dna probe 2, and Microcystin aptamers serve as a connection, its
One end is complementary with 3 ' ends of dna probe 1, and the other end is complementary with 5 ' ends of dna probe 2, forms y type by base pair complementarity
Dna diploid, its structure as shown in figure 1, thus the gold nano grain modifying dna probe 2 introduces glass-chip top layer, due to
The gold nano grain on glass-chip top layer increases, and the spr signal that it produces will be higher more rich.When object Microcystin with
After the specific binding of Microcystin aptamers, the structure of Microcystin aptamers changes therewith, leads to its function served as bridge to lose
Lose, thus the gold nano grain modified by dna probe 2 can not be introduced on glass-chip surface, and then change spr signal, pass through
This principle carries out the detection of Microcystin.
Prepared spr sensor forms structure as shown in Figure 2, including glass-chip, Microcystin aptamers, dna
Probe 1, dna probe 2 and gold nano-material；One layer of gold nano layer of area load of wherein glass-chip, described dna probe 1 leads to
Cross 5 ' terminal modified sulfydryls and pass through au-s key covalent bond with glass-chip surface；Described dna probe 2 passes through 3 ' terminal modified mercaptos
Base is combined with gold nano-material and is combined with glass-chip surface；5 ' ends of described dna probe 1 and 3 ' ends of described dna probe 2
Complementary combination；Complementary combination, the other end and described dna are held with the 3 ' of described dna probe 1 in described Microcystin aptamers one end
5 ' ends of probe 2 are complementary to be combined.
In above-described embodiment, gold nano can be silver nanoparticle or copper nanometer, and the noble metal nano on glass-chip surface
Granule can from dna probe 2 modify noble metal nano particles different it is also possible to identical.
Embodiment 2
Microcystin in the spr sensor detection water of the detection Microcystin amplifying strategy based on aptamer signal
The method of cellulose content, comprises the steps:
(1) preparation of line sensor is entered according to the method and step preparing spr sensor in embodiment 1；
(2) sensor of step (1) is immersed respectively the pbs buffer solution containing 0 μm, 10 μm, 100 μm Microcystin
In, then measure spr signal intensity, as shown in figure 3, as seen from the figure, sensor is not before being washed with water, though containing difference for result
The Microcystin of concentration, but because the noble metal nano particles that the dna probe 2 of injection is modified rest on its surface, so three
Group response value increase simultaneously, but water washed after, with Microcystins increase glass-chip surface combine dna probe
The noble metal nano particles of 2 modifications are fewer, and corresponding spr response value is also less.The microcapsule of variable concentrations can be measured based on this
Algae toxins.Meanwhile, sensor is repeatable after the methanol-acetic acid mixed liquor for 9:1 cleans through volume ratio utilizes.
Finally illustrate, preferred embodiment above only in order to technical scheme to be described and unrestricted, although logical
Cross above preferred embodiment the present invention to be described in detail, it is to be understood by those skilled in the art that can be
In form and various changes are made to it, without departing from claims of the present invention limited range in details.
Claims (9)
1. the spr sensor of detection Microcystin amplifying strategy based on aptamer signal is it is characterised in that described biography
Sensor includes glass-chip, Microcystin aptamers, dna probe 1, dna probe 2 and noble metal nanometer material；Described glass
One layer of noble metal nano layer of the area load of chip, described dna probe 1 passes through 5 ' terminal modified sulfydryls and described glass-chip table
The noble metal nano layer of face load passes through Covalent bonding together；Described dna probe 2 passes through 3 ' terminal modified sulfydryls and noble metal nano
Material combines；5 ' ends of described dna probe 1 are complementary with 3 ' ends of described dna probe 2 to be combined；Described Microcystin aptamers
One end is complementary with 3 ' ends of described dna probe 1 to be combined, and the other end is complementary with 5 ' ends of described dna probe 2 to be combined.
2. spr sensor as claimed in claim 1 is it is characterised in that described dna probe 1 is base sequence such as seq id
Single-stranded dna shown in no.1；Described dna probe 2 is single-stranded dna as shown in seq id no.2 for the base sequence；Described Microcystis aeruginosa
Toxin aptamers are single-stranded dna as shown in seq id no.3 for the base sequence.
3. spr sensor as claimed in claim 1 is it is characterised in that the thickness of described noble metal nano layer is 10-20nm.
4. spr sensor as claimed in claim 1 is it is characterised in that the particle diameter of described noble metal nanometer material is 30-
50nm.
5. spr sensor as claimed in claim 1 is it is characterised in that described noble metal includes gold, silver or copper.
6. the preparation method of the spr sensor described in any one of Claims 1 to 5 is it is characterised in that comprise the steps:
(1) prepare glass-chip: it is positively charged amino that glass slide cleaning is made surface, is then immersed in citric acid and stablizes noble metal
Make noble metal nano in nanometer solution on the slide after electrostatic force is deposited on process, obtain glass-chip；
(2) glass-chip modified by dna probe 1: step (1) gained glass-chip is immersed in probe containing dna 1 solution, makes described
Dna probe 1 is combined with the noble metal nano layer of glass-chip area load by covalent bond；
(3) noble metal nano particles modified by dna probe 2: dna probe 2 is added in noble metal nano solution, so that described dna is visited
Pin 2 is combined with noble metal nanometer material surface by covalent bond；
(4) spr sensor preparation: noble metal modified by the glass-chip immersion dna probe 2 that dna probe 1 in step (2) is modified
In nanometer solution, it is subsequently adding Microcystin aptamers, obtain spr sensor.
7. preparation method as claimed in claim 6 is it is characterised in that comprise the steps:
(1) prepare glass-chip: slide first passes through ammonia: hydrogen peroxide: ultra-pure water volume ratio is that the mixed solution of 1:1:1 is clear
Wash 1h, be then placed in 10min in the ethanol solution of 10% aminopropyl trimethoxysilane in anhydrous conditions, more clear with ultra-pure water
Wash, make surface be positively charged amino, be finally immersed in citric acid stablize 2h in noble metal nano solution make noble metal nano lead to
Cross on the slide after electrostatic force is deposited on process, obtain glass-chip；
(2) glass-chip modified by dna probe 1: step (1) gained glass-chip is immersed 1h in probe containing dna 1 solution, makes institute
State dna probe 1 to be combined with the noble metal nano layer of glass-chip area load by covalent bond；
(3) noble metal nano particles modified by dna probe 2: dna probe 2 is added 1h in noble metal nano solution, makes described dna
Probe 2 is combined with noble metal nanometer material surface by covalent bond, then carries out ultrafiltration, removes unconjugated dna probe 2；
(4) spr sensor preparation: dna probe 1 in step (2) is modified glass-chip immersion dna probe 2 modification noble metal and receives
In rice solution, it is subsequently adding Microcystin aptamers, obtains spr sensor.
8. application in detection Microcystin for the spr sensor described in any one of Claims 1 to 5.
9. utilize any one of claim 1～5 described in spr sensor detect capsule Algae toxins method it is characterised in that: by institute
State spr sensor to add in the solution containing Microcystin, measure spr signal intensity.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108344863A (en) * | 2018-02-08 | 2018-07-31 | 中国科学院重庆绿色智能技术研究院 | A kind of new method of the nano-pore detection Microcystin based on aptamers-nanogold sensing |
| CN109696430A (en) * | 2019-03-01 | 2019-04-30 | 长江师范学院 | A method of measurement Microcystins Concentration |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009107803A1 (en) * | 2008-02-27 | 2009-09-03 | ソニー株式会社 | Method for quantification of target substance |
| CN102628802A (en) * | 2012-04-17 | 2012-08-08 | 王利兵 | Method for detecting biotoxins in foods based on surface plasma resonance technology |
| CN103940815A (en) * | 2014-05-05 | 2014-07-23 | 福州大学 | Nucleic acid colorimetric sensor with Y-shaped structure and application of sensor |
| CN104914151A (en) * | 2015-05-08 | 2015-09-16 | 首都师范大学 | Forming method of ampicillin and sulfadimethoxine electrochemical sensor self-assembled passivation layer, and electrochemical sensor thereof |
| CN105779597A (en) * | 2016-03-28 | 2016-07-20 | 大连理工大学 | Unimolecular recognition method based on split aptamer bi-metal nanoprobe |
-
2016
- 2016-09-23 CN CN201610846741.0A patent/CN106370868B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009107803A1 (en) * | 2008-02-27 | 2009-09-03 | ソニー株式会社 | Method for quantification of target substance |
| CN102628802A (en) * | 2012-04-17 | 2012-08-08 | 王利兵 | Method for detecting biotoxins in foods based on surface plasma resonance technology |
| CN103940815A (en) * | 2014-05-05 | 2014-07-23 | 福州大学 | Nucleic acid colorimetric sensor with Y-shaped structure and application of sensor |
| CN104914151A (en) * | 2015-05-08 | 2015-09-16 | 首都师范大学 | Forming method of ampicillin and sulfadimethoxine electrochemical sensor self-assembled passivation layer, and electrochemical sensor thereof |
| CN105779597A (en) * | 2016-03-28 | 2016-07-20 | 大连理工大学 | Unimolecular recognition method based on split aptamer bi-metal nanoprobe |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108344863A (en) * | 2018-02-08 | 2018-07-31 | 中国科学院重庆绿色智能技术研究院 | A kind of new method of the nano-pore detection Microcystin based on aptamers-nanogold sensing |
| CN109696430A (en) * | 2019-03-01 | 2019-04-30 | 长江师范学院 | A method of measurement Microcystins Concentration |
| CN109696430B (en) * | 2019-03-01 | 2021-07-27 | 长江师范学院 | Method for measuring concentration of microcystin |
| CN110734962A (en) * | 2019-11-06 | 2020-01-31 | 江苏开放大学（江苏城市职业学院） | method for detecting food toxin based on aptamer |
| CN110734962B (en) * | 2019-11-06 | 2021-10-19 | 江苏开放大学（江苏城市职业学院） | Method for detecting food toxin based on aptamer |
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| CN111298836B (en) * | 2020-03-06 | 2023-03-31 | 军事科学院军事医学研究院环境医学与作业医学研究所 | DNA hydrogel based on biological mimic enzyme signal amplification and application thereof |
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| CN112014453B (en) * | 2020-07-24 | 2023-01-31 | 南京师范大学 | Method for detecting microcystin based on annular allosteric DNA electrochemical reaction |
| CN112014453A (en) * | 2020-07-24 | 2020-12-01 | 南京师范大学 | Method for detecting microcystin based on annular allosteric DNA electrochemical reaction |
| CN112834757A (en) * | 2020-12-31 | 2021-05-25 | 中国科学院重庆绿色智能技术研究院 | C-reactive protein detection method based on composite solid-state nanopore monomolecular technology |
| CN112834757B (en) * | 2020-12-31 | 2023-02-28 | 中国科学院重庆绿色智能技术研究院 | C-reactive protein detection method based on composite solid-state nanopore monomolecular technology |
| CN114395558A (en) * | 2022-01-17 | 2022-04-26 | 南通大学 | Magnetic bead-DNA probe, MC-LR detection biosensor, preparation method and application |
| CN114561451A (en) * | 2022-02-23 | 2022-05-31 | 中国地质大学（武汉） | Precisely modified nano-pore membrane and preparation method and application thereof |
| CN114561451B (en) * | 2022-02-23 | 2023-12-05 | 中国地质大学（武汉） | Precise modified nano pore canal membrane and preparation method and application thereof |
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