CN104597027B - A kind of method based on Nano silver grain tetrahedron Raman Multiple detection - Google Patents
A kind of method based on Nano silver grain tetrahedron Raman Multiple detection Download PDFInfo
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Abstract
A kind of method based on Nano silver grain tetrahedron Raman Multiple detection, belongs to technical field of analytical chemistry.The present invention is successfully assembled using DNA mediations self-assembly technology and obtains Nano silver grain tetrahedron, utilize tetrahedral special space configuration, by introducing three sections of DNA and three kinds of beacon molecules containing determinand SDM, AFM1, OTA aptamer fragment in Nano silver grain tetrahedron, successfully construct based on the tetrahedral Raman Multiple detection system of Nano silver grain, when there is determinand, tetrahedral steric configuration, which changes, causes the change of Raman signal, and then is detected；The change of three kinds of beacon Molecular Raman signals corresponds to the concentration of three kinds of objects, it is achieved thereby that detecting while to three kinds of object SDM, AFM1 and OTA.This method has high sensitivity and good specificity, and avoids influence of the unordered aggregation of nano-particle to testing result, the advantageously application in Raman sensing detection technology in practice.
Description
Technical field
The present invention relates to a kind of method based on Nano silver grain tetrahedron Raman Multiple detection, belong to technique of analytical chemistry
Field.
Background technology
1928, India's scientist's Raman was found that Raman scattering phenomenon first, i.e., when a known energy or wavelength,
When the photon of frequency and an interaction of molecules, cause the process of molecular vibration and energy loss.Subsequent Mr. Raman invention
First Raman spectrometer, and therefore obtain Nobel Prize in physics.But it is due to that Raman signal is weaker, the technology is always
It is not widely used.Caused the performance of Raman spectrometer significantly with the invention of laser, CCD and optical filter later
Improve, thus just have later Raman spectrometer popularization and application on a large scale.SERS（SERS）It is in original
Have on the basis of Raman scattering, utilize noble metal nanometer material（Such as gold, silver）The Electromagnetic enhancement effect on surface so that inhale
The molecule for being attached to its surface produces the phenomenon of Raman enhancement effect.Generally, SERS can believe the Raman of Raman molecular
Number enhancing 106Times, so as to realize the individual molecule detection of Raman spectrum.Further, since SERS detections can be kept well
Original state of sample, not by sample mechanism and privately influenceed, collection of illustrative plates peak width is narrower, with unique molecular fingerprint collection of illustrative plates,
The advantages of can be used to high temperature, hyperbaric environment, is widely used in pharmacy, drugs discriminating, biomedical, food Hazard factor inspection at present
The fields such as survey.
Sulfadimethoxine（SDM）It is the artificial synthesized broad-spectrum antibacterial agent medicine of a class, addition can be with getting fat in feed
Domestic animal, prevents and treats bacteriosis.This medicine easily remains in animal body and serious shadow is caused to health
Ring, such as infringement cerebral nervous system, cause hemolytic anemia, allergic reaction triggers thyroid cancer etc..In consideration of it, many countries are right
SDM content makes regulation in animal derived food, and Chinese limitation is 100mg/mL.Ochratoxin A (OTA) is bent
Mould belongs to the secondary metabolite with some toxigenic bacteriums strain of Penicillium, and the pollution to crops in the world is all compared
Seriously, it is a kind of strong nephrotoxin and hepatotoxin, also with inhibitive ability of immunity, directly endangers human health, cause DNA's
Damage, has teratogenesis, a carcinogenic and mutagenic effect.Aflatoxins M1（AFM1）Belong to the class formation of aflatoxin one similar
, there is the probability highest of aflatoxin in damp-heat area food and feed in one kind in compound.Aflatoxins M1 is endangered
Evil is mainly manifested in carcinogenicity and mutagenicity, has destruction to people and animal's liver tissue, and liver cancer can be caused even dead.
The content of the invention
The purpose of the present invention is to build a kind of Nano silver grain tetrahedron, and applied to sulfadimethoxine（SDM）And
Aflatoxins M1（AFM1）, ochratoxin A（OTA）Deng multiple Raman spectrum detection.
Technical scheme：A kind of method based on Nano silver grain tetrahedron Raman Multiple detection, using containing
The DNA assemblings of determinand SDM, AFM1, OTA aptamer fragment obtain Nano silver grain tetrahedron, when there is determinand,
Tetrahedral steric configuration, which changes, causes the change of Raman signal, and then is detected；Processing step is：
（1）10nm particle diameter Nano silver grains（AgNP）Synthesis
Sodium borohydride reduction silver nitrate method is used to synthesize Nano silver grain of the particle diameter for 10nm.
（2）Nano silver grain modifying DNA
The Nano silver grain of above-mentioned synthesis and the DNA of sulfydryl modification（DNA1, DNA2, DNA3, DNA4）Carry out coupling shape
Into AgNP-DNA1, AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complexs.
（3）The modification of Raman beacon molecule
By three kinds of Raman beacon molecule 4- aminothiophenols（4-ATP）, 4- nitrobenzenethiols（NTP）, 4- methoxybenzyl sulphur
Alcohol（MATT）Modification is corresponded to respectively obtains AgNP-DNA1-ATP to AgNP-DNA1, AgNP-DNA2, AgNP-DNA3 surface,
AgNP-DNA2-NTP, AgNP-DNA3-MATT.
（4）The tetrahedral assembling of Nano silver grain
The AgNP-DNA1-ATP of above-mentioned preparation, AgNP-DNA2-NTP, AgNP-DNA3-MATT, AgNP-DNA4 are mixed,
Nano silver grain tetrahedron is obtained using base pair complementarity hybridization.
Table 1 detects the title and sequence of object aptamer
| Sequence (5’-3’) | |
| SDM | GAG GGC AAC GAG TGT TTA TAG A |
| AFM1 | ACT GCT AGA GAT TTT CCA CAT |
| OTA | GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA |
Table 2 is used to build the tetrahedral nucleotide sequence of Nano silver grain.
| Sequence (5’-SH- 3’) | |
| DNA1 | TTTATTGAGG GCAACGAGTG TTTATAGACT TTCCCTATTA GAAGGTCTCA GGTGCGCGTT TCCAGCCATA CCTTAGGTAC TTCTGCC |
| DNA2 | TTTCGCGCAC CTGAGACCTT CTAATAGGGT TTGCGACAGT CGTTCAACTA GAATGCCCTT TGGGCTGTTC CGGGTGTGGC TCGTCGG |
| DNA3 | TTTACTGCTA GAGATTTTCC ACATGGCTAT TTGATCGGGT GTGGGTGGCG TAAAGGGAGC ATACATTTCC GACGAGCCAC ACCCGGAACA GCCC |
| DNA4 | TTTGTCTATA AACACTCGTT GCCCTCAATT TTTGACGATC TCTAAAAGGT GTACCGATTT TGGGCATTCT AGTTGAACGA CTGTCGC |
（5）Structure and application based on Nano silver grain tetrahedron Raman sensor
To step（4）Added in the Nano silver grain tetrahedron system prepared the SDM of various concentrations a series of, AFM1,
OTA standard liquids, determine its Raman signal respectively, are set up according to the Raman signal intensity of three kinds of different beacons and testing concentration
Standard curve.
Specially：
（1）10nm particle diameter Nano silver grains（AgNP）Synthesis
Take a clean conical flask to be placed in ice bath, sequentially add 20mL ultra-pure waters, 5mL mass fractions are 1% polyethylene
The sodium borohydride aqueous solution of pyrrolidones and 0.6mL 0.01mol/L.Then by 5mL mass fractions be 1% polyvinylpyrrolidine
Ketone and 5mL mass fractions are added in conical flask with 30mL/h speed simultaneously for 1% silver nitrate aqueous solution, are stirred when adding
Mix, solution is by colourless yellowing.The a diameter of 10nm of Nano silver grain of gained.
（2）Nano silver grain modifying DNA
The Nano silver grain AgNP of the 30 above-mentioned synthesis of μ L 20nM is taken in PCR pipes, adds 10 μM of 1 μ L's
After DNA1 is mixed, 5 μ 5 × tris- of L borate buffers are added into system successively and 1.25 μ L 2mol/L NaCl are molten
Liquid, rocked at room temperature reaction 12h, 13000r/min centrifugation 10min, removes supernatant, plus ultra-pure water is to original volume, obtains AgNP-
DNA1.The preparation method of AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complex is similar with AgNP-DNA1.
（3）The modification of Raman beacon molecule
By three kinds of Raman beacon molecule 4- aminothiophenols（4-ATP）, 4- nitrobenzenethiols（NTP）, 4- methoxybenzyl sulphur
Alcohol（MATT）Correspond to and be added in AgNP-DNA1, AgNP-DNA2, AgNP-DNA3 system respectively, Raman beacon molecule in solution
Final concentration be 3 μM, beacon molecule is added in system reaction and stayed overnight, and each centrifuges 10 min with 13000 r/min,
Supernatant is removed, then 20 mM Tris-HCl buffer solutions are added into system and returns to original volume.Prepare AgNP-DNA1-
ATP, AgNP-DNA2-NTP, AgNP-DNA3-MATT complex.
（4）The tetrahedral assembling of Nano silver grain
By the AgNP-DNA1-ATP of above-mentioned preparation, AgNP-DNA2-NTP, AgNP-DNA3-MATT, AgNP-DNA4 respectively takes
100 μ L are well mixed in 1.5 mL centrifuge tubes, add 4 μ L 5M NaCl solution, and concussion is mixed, 90 DEG C of water-baths 5
Min, then be slowly decreased to room temperature in vapor, that is, prepare Nano silver grain tetrahedron.
（5）Structure and application based on Nano silver grain tetrahedron Raman sensor
Detected while for SDM, AFM1, OTA, three kinds of materials are added in system one by one according to sequencing, every kind of
The min of material midfeather 30.SDM addition concentration is followed successively by 0,0.001,0.005,0.01,0.05,0.1,0.5 fM；
AFM1 addition concentration is followed successively by 0,0.1,0.5,1,5,10,50 fM；OTA addition concentration is followed successively by 0,0.01,0.05,
0.1,0.5,1,5 fM.Three kinds of materials all add and react terminate after survey system Raman spectrum, respectively according to 4-ATP,
The intensity of NTP and MATT Raman signal sets up SDM, AFM1, OTA concentration standard curve.The Raman spectrum testing time
For 20 s, excitation wavelength is 633 nm.
A kind of universal method based on Nano silver grain tetrahedron Raman Multiple detection, by Nano silver grain four sides
The change of the nucleic acid aptamer sequence used in production procedure, you can the Raman Multiple detection for preparing correspondence determinand is passed
Sensor.
Beneficial effects of the present invention：First, the Raman enhancing effect of Nano silver grain is fine than other noble metals substantially
Raman substrate material.Secondly, detection is, based on the controllable adjustment to nano-particle tetrahedron space structure, to be detected entirely
Do not occur the random clustering phenomena of nano-particle in journey, reduce the interference of external environment；Again, object passes through with fitting
Part identification causes tetrahedral structure to change, with good specificity；Finally, tetrahedron has six DNA sides, can
Detected while for multiple objects, both can be used for the detection of small molecule, can be used for determining for the macromoleculars such as protein
Amount detection.
Brief description of the drawings
Schematic diagram of Fig. 1 present invention based on Nano silver grain tetrahedron Raman detection.
Fig. 2 Nano silver grains are tetrahedral：(A) TEM schemes, (B) freezing electronics three-dimensional imaging figure, (C) steric configuration
Schematic diagram；The Nano silver grain added after SDM is tetrahedral：(D) TEM schemes, (E) freezing electronics three-dimensional imaging figure, (F)
Steric configuration schematic diagram.
Fig. 3（A）Nano silver grain tetrahedron is used for the Raman finger-print that SDM, AFM1 and OTA are detected；（B）It is based on
The tetrahedral Multiple detection system of Nano silver grain detects SDM, AFM1 and OTA simultaneously, and wherein SDM concentration is followed successively by 0,
0.001,0.005,0.01,0.05,0.1,0.5 fM；AFM1 concentration is followed successively by 0,0.1,0.5,1,5,10,50 fM；OTA
Concentration be followed successively by 0,0.01,0.05,0.1,0.5,1,5 fM；（C）From left to right it is followed successively by：SDM concentration and Raman signal
The standard of the standard curve of the standard curve of intensity, AFM1 concentration and Raman signal intensity, OTA concentration and Raman signal intensity is bent
Line.
Fig. 4 detects the Raman spectrum of SDM in milk based on Nano silver grain tetrahedron.
Embodiment
Embodiment 1
（1）10nm particle diameter Nano silver grains（AgNP）Synthesis
Take a clean conical flask to be placed in ice bath, sequentially add 20mL ultra-pure waters, 5mL mass fractions are 1% polyethylene
The sodium borohydride aqueous solution of pyrrolidones and 0.6mL 0.01mol/L.Then by 5mL mass fractions be 1% polyvinylpyrrolidine
Ketone and 5mL mass fractions are added in conical flask with 30mL/h speed simultaneously for 1% silver nitrate aqueous solution, are stirred when adding
Mix, solution is by colourless yellowing.The a diameter of 10nm of Nano silver grain of gained.
（2）Nano silver grain modifying DNA
The Nano silver grain AgNP of the 30 above-mentioned synthesis of μ L 20nM is taken in PCR pipes, adds 10 μM of 1 μ L's
After DNA1 is mixed, 5 μ 5 × tris- of L borate buffers are added into system successively and 1.25 μ L 2mol/L NaCl are molten
Liquid, rocked at room temperature reaction 12h, 13000r/min centrifugation 10min, removes supernatant, plus ultra-pure water is to original volume, obtains AgNP-
DNA1.The preparation method of AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complex is similar with AgNP-DNA1.
（3）The modification of Raman beacon molecule
By three kinds of Raman beacon molecule 4- aminothiophenols（4-ATP）, 4- nitrobenzenethiols（NTP）With 4- methoxybenzyls
Mercaptan（MATT）It is added separately in AgNP-DNA1, AgNP-DNA2, AgNP-DNA3 systems, Raman beacon molecule in solution
Final concentration is 3 uM, and beacon molecule is added to reaction in system and stayed overnight, each centrifuges 10 min with 13000 r/min, go
Except supernatant, then add 20 mM Tris-HCl buffer solutions into system and return to original volume.Prepare AgNP-DNA1-
ATP, AgNP-DNA2-NTP, AgNP-DNA3-MATT complex.
（4）The tetrahedral assembling of Nano silver grain
By the AgNP-DNA1-ATP of above-mentioned preparation, AgNP-DNA2-NTP, AgNP-DNA3-MATT, AgNP-DNA4 respectively takes
100 uL are well mixed in 1.5 mL centrifuge tubes, add 4 μ L 5M NaCl solution, and concussion is mixed, 90 DEG C of water-baths 5
Min, then be slowly decreased to room temperature in vapor, that is, prepare Nano silver grain tetrahedron.
（5）Structure and application based on Nano silver grain tetrahedron Raman sensor
Detected while for SDM, AFM1, OTA, three kinds of materials are added in system one by one according to sequencing, every kind of
The min of material midfeather 30.SDM addition concentration is followed successively by 0,0.001,0.005,0.01,0.05,0.1,0.5 fM;
AFM1 addition concentration is followed successively by 0,0.1,0.5,1,5,10,50 fM;OTA addition concentration is followed successively by 0,0.01,0.05,
0.1,0.5,1,5 fM.Three kinds of materials all add and react terminate after survey system Raman spectrum, respectively according to 4-ATP,
The intensity of NTP and MATT Raman signal sets up SDM, AFM1, OTA concentration standard curve.The Raman spectrum testing time
For 20 s, excitation wavelength is 633 nm.
DNA1：5’-SH-TTTATTGAGG GCAACGAGTG TTTATAGACT TTCCCTATTA GAAGGTCTCA
GGTGCGCGTT TCCAGCCATA CCTTAGGTAC TTCTGCC-3’；
DNA2：5’ -SH -TTTCGCGCAC CTGAGACCTT CTAATAGGGT TTGCGACAGT CGTTCAACTA
GAATGCCCTT TGGGCTGTTC CGGGTGTGGC TCGTCGG-3’；
DNA3：5’ -SH -TTTACTGCTA GAGATTTTCC ACATGGCTAT TTGATCGGGT GTGGGTGGCG
TAAAGGGAGC ATACATTTCC GACGAGCCAC ACCCGGAACA GCCC-3’；
DNA4：5’ -SH - TTTGTCTATA AACACTCGTT GCCCTCAATT TTTGACGATC TCTAAAAGGT
GTACCGATTT TGGGCATTCT AGTTGAACGA CTGTCGC -3’。
Claims (5)
1. the method that one kind is based on Nano silver grain tetrahedron Raman Multiple detection SDM, AFM1, OTA, synthesizes 10nm particle diameters first
Nano silver grain AgNP；It is characterized in that being assembled using the DNA containing determinand SDM, AFM1, OTA aptamer fragment
To Nano silver grain tetrahedron, when there is determinand, tetrahedral steric configuration, which changes, causes the change of Raman signal,
And then detected；Processing step is：
（1）Nano silver grain modifying DNA：
The Nano silver grain AgNP of above-mentioned synthesis DNA1 respectively with sulfydryl modification, DNA2, DNA3, DNA4 carry out coupling formation
AgNP-DNA1, AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complex；
Build the tetrahedral nucleotide sequence of Nano silver grain：
DNA1：5’-SH-TTTATTGAGG GCAACGAGTG TTTATAGACT TTCCCTATTA GAAGGTCTCA
GGTGCGCGTT TCCAGCCATA CCTTAGGTAC TTCTGCC-3’；
DNA2：5’ -SH -TTTCGCGCAC CTGAGACCTT CTAATAGGGT TTGCGACAGT CGTTCAACTA
GAATGCCCTT TGGGCTGTTC CGGGTGTGGC TCGTCGG-3’；
DNA3：5’ -SH -TTTACTGCTA GAGATTTTCC ACATGGCTAT TTGATCGGGT GTGGGTGGCG
TAAAGGGAGC ATACATTTCC GACGAGCCAC ACCCGGAACA GCCC-3’；
DNA4：5’ -SH - TTTGTCTATA AACACTCGTT GCCCTCAATT TTTGACGATC TCTAAAAGGT
GTACCGATTT TGGGCATTCT AGTTGAACGA CTGTCGC -3’：
（2）The modification of Raman beacon molecule：
By three kinds of MATT pairs of Raman beacon molecule 4- aminothiophenols 4-ATP, 4- nitrobenzenethiol NTP, 4- methoxybenzyl mercaptan
AgNP-DNA1 should be modified, AgNP-DNA2, AgNP-DNA3 surfaces obtain AgNP-DNA1-ATP, AgNP-DNA2-NTP,
AgNP-DNA3-MATT；
（3）The tetrahedral assembling of Nano silver grain：
The AgNP-DNA1-ATP of above-mentioned preparation, AgNP-DNA2-NTP, AgNP-DNA3-MATT, AgNP-DNA4 are mixed, utilized
Base pair complementarity hybridization obtains Nano silver grain tetrahedron；
（4）Structure and application based on Nano silver grain tetrahedron Raman sensor：
To step（3）SDM, AFM1, OTA mark of a series of various concentrations are added in the Nano silver grain tetrahedron system prepared
Quasi- solution, determines its Raman signal respectively, and standard is set up with testing concentration according to the Raman signal intensity of three kinds of different beacons
Curve.
2. the method according to claim 1 based on Nano silver grain tetrahedron Raman Multiple detection SDM, AFM1, OTA,
It is characterized in that Nano silver grain modifying DNA：The Nano silver grain AgNP synthesized described in 30 μ L 20nM is taken in PCR pipes,
After the DNA1 mixings for adding 10 μM of 1 μ L, 5 μ 5 × tris- of L borate buffers and 1.25 μ are added into system successively
L 2mol/L NaCl solution, rocked at room temperature reaction 12h, 13000r/min centrifugation 10min, removes supernatant, plus ultra-pure water
To original volume, AgNP-DNA1 is obtained；The preparation method and AgNP-DNA1 of AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complex
It is similar.
3. the method according to claim 1 based on Nano silver grain tetrahedron Raman Multiple detection SDM, AFM1, OTA,
It is characterized in that the modification of Raman beacon molecule：By three kinds of Raman beacon molecule 4- aminothiophenol 4-ATP, 4- nitrobenzenethiols
NTP, 4- methoxybenzyl mercaptan MATT are corresponded to be added in AgNP-DNA1, AgNP-DNA2, AgNP-DNA3 system respectively, solution
The final concentration of middle Raman beacon molecule is 3 μM, and beacon molecule is added to reaction in system and stayed overnight, each with 13000 r/
Min centrifuges 10 min, removes supernatant, then adds 20 mM Tris-HCl buffer solutions into system and return to original volume, makes
It is standby to obtain AgNP-DNA1-ATP, AgNP-DNA2-NTP, AgNP-DNA3-MATT complexs.
4. the method according to claim 1 based on Nano silver grain tetrahedron Raman Multiple detection SDM, AFM1, OTA,
It is characterized in that the tetrahedral assembling of Nano silver grain：By AgNP-DNA1-ATP, the AgNP-DNA2-NTP of above-mentioned preparation,
AgNP-DNA3-MATT, AgNP-DNA4 respectively take 100 μ L to be well mixed in 1.5 mL centrifuge tubes, add 4 μ L 5M
NaCl solution, concussion is mixed, 90 DEG C of min of water-bath 5, then is slowly decreased to room temperature in vapor, that is, prepares nano grain of silver
Sub- tetrahedron.
5. the method according to claim 1 based on Nano silver grain tetrahedron Raman Multiple detection SDM, AFM1, OTA,
It is characterized in that structure and application based on Nano silver grain tetrahedron Raman sensor：While for SDM, AFM1, OTA
Detection, three kinds of materials are added in system one by one according to sequencing, every kind of min of material midfeather 30；SDM addition is dense
Degree is followed successively by 0,0.001,0.005,0.01,0.05,0.1,0.5 fM；AFM1 addition concentration is followed successively by 0,0.1,0.5,1,
5,10,50 fM；OTA addition concentration is followed successively by 0,0.01,0.05,0.1,0.5,1,5 fM；Three kinds of materials are all added simultaneously
The Raman spectrum of system is surveyed in reaction after terminating, respectively according to 4-ATP, and the intensity of NTP and MATT Raman signal is set up
SDM, AFM1, OTA concentration standard curve；The Raman spectrum testing time is 20 s, and excitation wavelength is 633 nm.
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| PCT/CN2015/000721 WO2016109911A1 (en) | 2015-01-09 | 2015-10-28 | Multiple raman detection method based on silver nanoparticle tetrahedron |
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| CN104597027B (en) * | 2015-01-09 | 2017-07-25 | 江南大学 | A kind of method based on Nano silver grain tetrahedron Raman Multiple detection |
| CN105738342B (en) * | 2016-02-26 | 2018-09-25 | 中国人民解放军军事医学科学院军事兽医研究所 | It is a kind of using aptamers as the SERS methods of holder fabricated in situ nano silver |
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| CN115728284B (en) * | 2022-10-28 | 2024-04-26 | 南京师范大学 | Method for determining interaction between aptamer and small molecular target based on surface-enhanced Raman spectroscopy |
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