CN104597027A - Raman multiple detection method based on silver nanoparticles tetrahedron - Google Patents
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Abstract
The invention discloses a raman multiple detection method based on a silver nanoparticles tetrahedron, belonging to the technical field of analytical chemistry. The silver nanoparticles tetrahedron is successfully assembled by a DNA-mediated nanoparticles self-assembly technology; three segments of NDAs containing to-be-detected articles SDM, AFMI and OTA nucleic acid adapter segments and three beacon molecules are introduced into the silver nanoparticles tetrahedron by utilizing the special steric configuration of tetrahedron; a raman multiple-detection system based on the silver nanoparticles tetrahedron is successfully built; when the to-be-detected articles exist, the steric configuration of the tetrahedron changes to cause change of raman signals so as to detect; the change of raman signals of the three beacon molecules corresponds to the concentration of three targets, so that simultaneous detection of the three targets SDM, AFM1 and OTA is achieved; the method has extremely high sensitivity and good specificity; the effects on the detection result caused by random aggregation of the nanoparticles are avoided; and application of a raman sensing technology in practice is facilitated.
Description
Technical field
The present invention relates to a kind of method based on Nano silver grain tetrahedron Raman Multiple detection, belong to technical field of analytical chemistry.
Background technology
1928, India scientist Raman Late Cambrian Raman scattering phenomenon, namely when photon and an interaction of molecules of a known energy or wavelength, frequency, caused the process of molecular vibration and energy loss.Mr. Raman has invented First Raman spectrometer subsequently, and therefore obtains Nobel Prize in physics.But because Raman signal is more weak, this technology is never widely used.Afterwards along with the invention of laser instrument, CCD and optical filter makes the performance of Raman spectrometer greatly improve, thus just there is Raman spectrometer universal and application on a large scale afterwards.Surface enhanced raman spectroscopy (SERS) is on the basis of original Raman scattering, utilizes the Electromagnetic enhancement effect on noble metal nanometer material (as gold, silver etc.) surface, makes the molecule being adsorbed on its surface produce the phenomenon of Raman enhancement effect.Generally, the Raman signal of Raman molecular can be strengthened 10 by SERS
6times, thus the individual molecule realizing Raman spectrum detects.In addition, due to SERS detect can well keep original state of sample, narrower by sample mechanism and impact privately, collection of illustrative plates peak width, there is unique molecular fingerprint collection of illustrative plates, in order to the advantage such as high temperature, hyperbaric environment, can be widely used in the fields such as pharmacy, drugs discriminating, biomedicine, the detection of food Hazard factor at present.
Sulfadimethoxine (SDM) is a class Prof. Du Yucang broad-spectrum antibacterial agent medicine, and being added in feed can getting fat domestic animal, prevention and therapy bacteriosis.This medicine easily remains in animal body and causes serious impact to health, as infringement cerebral nervous system, causes hemolytic anemia, allergic reaction, causes thyroid cancer etc.Given this, a lot of country makes regulation to the content of SDM in animal derived food, and the limitation of China is 100mg/mL.Ochratoxin A (OTA) is the secondary metabolite of some toxigenic bacterium strain of Eurotium and Penicillium, all more serious to the pollution of crops in the world, a kind of strong nephrotoxin and hepatotoxin, also there is inhibitive ability of immunity, direct harm humans is healthy, cause the damage of DNA, have teratogenesis, carcinogenic and mutagenic effect.Aflatoxins M1 (AFM1) belongs to the one in the similar compound of aflatoxin one class formation, occurs that the probability of aflatoxin is the highest in the food and feed of damp-heat area.Aflatoxins M1 harm is mainly manifested in carcinogenicity and mutagenicity, has destruction, liver cancer can be caused even dead to people and animal's liver tissue.
Summary of the invention
The object of the invention is to build a kind of Nano silver grain tetrahedron, and the multiple Raman spectrum being applied to sulfadimethoxine (SDM) and Aflatoxins M1 (AFM1), ochratoxin A (OTA) etc. detects.
Technical scheme of the present invention: a kind of method based on Nano silver grain tetrahedron Raman Multiple detection, utilize containing determinand SDM, AFM1, the DNA assembling of OTA aptamer fragment obtains Nano silver grain tetrahedron, when there is determinand, tetrahedral steric configuration changes and causes the change of Raman signal, and then detects; Processing step is:
(1) 10nm particle diameter Nano silver grain (AgNP) synthesis
Sodium borohydride reduction silver nitrate method synthesis particle diameter is adopted to be the Nano silver grain of 10nm.
(2) Nano silver grain modifying DNA
The Nano silver grain of above-mentioned synthesis and the DNA(DNA1 of sulfydryl modification, DNA2, DNA3, DNA4) carry out coupling formation AgNP-DNA1, AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complex.
(3) modification of Raman beacon molecule
By three kinds of Raman beacon molecules 4-aminothiophenol (4-ATP), 4-nitrobenzenethiol (NTP), 4-methoxybenzyl mercaptan (MATT) respectively corresponding modification obtain 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
By the AgNP-DNA1-ATP of above-mentioned preparation, AgNP-DNA2-NTP, AgNP-DNA3-MATT, AgNP-DNA4 mix, and utilize base pair complementarity to hybridize and obtain Nano silver grain tetrahedron.
Table 1 detects title and the sequence of object aptamer
Table 2 is for building the tetrahedral nucleotide sequence of Nano silver grain.
(5) based on the study on its developing of Nano silver grain tetrahedron Raman sensor
In the Nano silver grain tetrahedron system that step (4) is prepared, add SDM, AFM1, OTA standard solution of a series of variable concentrations, measure its Raman signal respectively, according to Raman signal intensity and the testing concentration Criterion curve of three kinds of different beacons.
Be specially:
(1) 10nm particle diameter Nano silver grain (AgNP) synthesis
The conical flask getting a cleaning is placed in ice bath, adds 20mL ultrapure water successively, and 5mL massfraction is the polyvinylpyrrolidone of 1% and the sodium borohydride aqueous solution of 0.6mL 0.01mol/L.Then by 5mL massfraction be 1% polyvinylpyrrolidone and 5mL massfraction be 1% silver nitrate aqueous solution simultaneously join in conical flask with the speed of 30mL/h, stir while adding, solution is by colourless yellowing.The Nano silver grain diameter of gained is 10nm.
(2) Nano silver grain modifying DNA
Get the Nano silver grain AgNP of the above-mentioned synthesis of 30 μ L 20nM in PCR pipe, after adding the DNA1 mixing of 1 μ L 10 μMs, 5 μ L 5 × tris-borate buffers and 1.25 μ L 2mol/L NaCl solution are added successively in system, rocked at room temperature reaction 12h, the centrifugal 10min of 13000r/min, remove supernatant, add ultrapure water to original volume, obtain AgNP-DNA1.Preparation method and the AgNP-DNA1 of AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complex are similar.
(3) modification of Raman beacon molecule
By three kinds of Raman beacon molecules 4-aminothiophenol (4-ATP), 4-nitrobenzenethiol (NTP), 4-methoxybenzyl mercaptan (MATT) respectively correspondence join in AgNP-DNA1, AgNP-DNA2, AgNP-DNA3 system, in solution, the final concentration of Raman beacon molecule is 3 μMs, beacon molecule joins reaction in system and spends the night, separately with centrifugal 10 min of 13000 r/min, remove supernatant, then in system, add 20 mM Tris-HCl damping fluids 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 gets 100 μ L and mixes in 1.5 mL centrifuge tubes, add 4 μ L 5M NaCl solution, concussion mixing, 90 DEG C of water-bath 5 min, in water vapor, slowly drop to room temperature again, namely prepare Nano silver grain tetrahedron.
(5) based on the study on its developing of Nano silver grain tetrahedron Raman sensor
Detect for while SDM, AFM1, OTA, three kinds of materials join in system one by one according to sequencing, often kind of material midfeather 30 min.The interpolation concentration of SDM is followed successively by 0,0.001,0.005,0.01,0.05,0.1,0.5 fM; The interpolation concentration of AFM1 is followed successively by 0,0.1,0.5,1,5,10,50 fM; The interpolation concentration of OTA is followed successively by 0,0.01,0.05,0.1,0.5,1,5 fM.Three kinds of materials all add and react the Raman spectrum terminating rear survey system, set up the concentration standard curve of SDM, AFM1, OTA respectively according to the intensity of the Raman signal of 4-ATP, NTP and MATT.The Raman spectrum test duration is 20 s, and excitation wavelength is 633 nm.
A universal method based on Nano silver grain tetrahedron Raman Multiple detection, by the change to the nucleic acid aptamer sequence used in Nano silver grain tetrahedron preparation process, can prepare the Raman Multiple detection sensor of corresponding determinand.
Beneficial effect of the present invention: first, the Raman enhancing effect of Nano silver grain is more obvious than other noble metals, is good Raman substrate material.Secondly, detection is based on the controllable adjustment to nano particle tetrahedron space structure, does not occur the random clustering phenomena of nano particle, decrease the interference of external environment in whole testing process; Again, object, by causing tetrahedral structure to change with aptamers identification, has good specificity; Finally, tetrahedron has six DNA limits, detects, both may be used for micromolecular detection, and also may be used for the macromolecular quantitative detections such as protein while can be used for multiple object.
Accompanying drawing explanation
Fig. 1 the present invention is based on the schematic diagram of Nano silver grain tetrahedron Raman detection.
Fig. 2 Nano silver grain is tetrahedral: (A) TEM schemes, (B) freezing electronics three-dimensional imaging figure, (C) steric configuration schematic diagram; Add the Nano silver grain after SDM 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 detect; (B) detect SDM, AFM1 and OTA based on the tetrahedral Multiple detection system of Nano silver grain, wherein the concentration of SDM is followed successively by 0 simultaneously, and 0.001,0.005,0.01,0.05,0.1,0.5 fM; The concentration of AFM1 is followed successively by 0, and 0.1,0.5,1,5,10,50 fM; The concentration of OTA is followed successively by 0, and 0.01,0.05,0.1,0.5,1,5 fM; (C) be from left to right followed successively by: the typical curve of SDM concentration and Raman signal intensity, the typical curve of AFM1 concentration and Raman signal intensity, the typical curve of OTA concentration and Raman signal intensity.
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 grain (AgNP) synthesis
The conical flask getting a cleaning is placed in ice bath, adds 20mL ultrapure water successively, and 5mL massfraction is the polyvinylpyrrolidone of 1% and the sodium borohydride aqueous solution of 0.6mL 0.01mol/L.Then by 5mL massfraction be 1% polyvinylpyrrolidone and 5mL massfraction be 1% silver nitrate aqueous solution simultaneously join in conical flask with the speed of 30mL/h, stir while adding, solution is by colourless yellowing.The Nano silver grain diameter of gained is 10nm.
(2) Nano silver grain modifying DNA
Get the Nano silver grain AgNP of the above-mentioned synthesis of 30 μ L 20nM in PCR pipe, after adding the DNA1 mixing of 1 μ L 10 μMs, 5 μ L 5 × tris-borate buffers and 1.25 μ L 2mol/L NaCl solution are added successively in system, rocked at room temperature reaction 12h, the centrifugal 10min of 13000r/min, remove supernatant, add ultrapure water to original volume, obtain AgNP-DNA1.Preparation method and the AgNP-DNA1 of AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complex are similar.
(3) modification of Raman beacon molecule
By three kinds of Raman beacon molecules 4-aminothiophenol (4-ATP), 4-nitrobenzenethiol (NTP) and 4-methoxybenzyl mercaptan (MATT) join AgNP-DNA1 respectively, AgNP-DNA2, in AgNP-DNA3 system, in solution, the final concentration of Raman beacon molecule is 3 uM, and beacon molecule joins reaction in system and spends the night, separately with centrifugal 10 min of 13000 r/min, remove supernatant, then in system, add 20 mM Tris-HCl damping fluids 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 gets 100 uL and mixes in 1.5 mL centrifuge tubes, add 4 μ L 5M NaCl solution, concussion mixing, 90 DEG C of water-bath 5 min, in water vapor, slowly drop to room temperature again, namely prepare Nano silver grain tetrahedron.
(5) based on the study on its developing of Nano silver grain tetrahedron Raman sensor
Detect for while SDM, AFM1, OTA, three kinds of materials join in system one by one according to sequencing, often kind of material midfeather 30 min.The interpolation concentration of SDM is followed successively by 0,0.001,0.005,0.01,0.05,0.1,0.5 fM; The interpolation concentration of AFM1 is followed successively by 0,0.1,0.5,1,5,10,50 fM; The interpolation concentration of OTA is followed successively by 0,0.01,0.05,0.1,0.5,1,5 fM.Three kinds of materials all add and react the Raman spectrum terminating rear survey system, set up the concentration standard curve of SDM, AFM1, OTA respectively according to the intensity of the Raman signal of 4-ATP, NTP and MATT.The Raman spectrum test duration is 20 s, and excitation wavelength is 633 nm.
Claims (6)
1. the method based on Nano silver grain tetrahedron Raman Multiple detection, it is characterized in that utilizing containing determinand SDM, AFM1, the DNA assembling of OTA aptamer fragment obtains Nano silver grain tetrahedron, when there is determinand, tetrahedral steric configuration changes and causes the change of Raman signal, and then detects; Processing step is:
(1) 10nm particle diameter Nano silver grain AgNP synthesizes:
Sodium borohydride reduction silver nitrate method synthesis particle diameter is adopted to be the AgNP of 10nm;
(2) Nano silver grain modifying DNA:
The Nano silver grain AgNP of above-mentioned synthesis respectively with the DNA1 of sulfydryl modification, DNA2, DNA3, DNA4 carry out coupling and form AgNP-DNA1, AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complex;
Build the tetrahedral nucleotide sequence of Nano silver grain:
DNA1：5’-SH-TTT ATT GAG GGC AAC GAG TGT TTA TAG ACT TTC CCT ATT AGA AGG TCT CAG GTG CGC GTT TCC AGC CAT ACC TTA GGT ACT TCT GCC-3’；
DNA2：5’ -SH -TTT CGC GCA CCT GAG ACC TTC TAA TAG GGT TTG CGA CAG TCG TTC AAC TAG AAT GCC CTT TGG GCT GTT CCG GGT GTG GCT CGT CGG-3’；
DNA3：5’ -SH -TTT ACT GCT AGA GAT TTT CCA CAT GGC TAT TT GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT ACA TTT CC GAC GAG CCA CAC CCG GAA CAG CCC-3’；
DNA4：5’ -SH -TTT GTC TAT AAA CAC TCG TTG CCC TCA ATT TT TGA CGA TCT CTA AAA GGT GTA CCG ATT TTG GGC ATT CTA G TTG AAC GAC TGT CGC-3’：
(3) modification of Raman beacon molecule:
By three kinds of Raman beacon molecule 4-aminothiophenol 4-ATP, 4-nitrobenzenethiol NTP, 4-methoxybenzyl mercaptan MATT correspondence is modified AgNP-DNA1, AgNP-DNA2, AgNP-DNA3 surface and is obtained AgNP-DNA1-ATP, AgNP-DNA2-NTP, AgNP-DNA3-MATT;
(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 mix, and utilize base pair complementarity to hybridize and obtain Nano silver grain tetrahedron;
(5) based on the study on its developing of Nano silver grain tetrahedron Raman sensor:
In the Nano silver grain tetrahedron system that step (4) is prepared, add SDM, AFM1, OTA standard solution of a series of variable concentrations, measure its Raman signal respectively, according to Raman signal intensity and the testing concentration Criterion curve of three kinds of different beacons.
2. the method based on Nano silver grain tetrahedron Raman Multiple detection according to claim 1, it is characterized in that 10nm particle diameter Nano silver grain AgNP synthesizes: the conical flask getting a cleaning is placed in ice bath, add 20mL ultrapure water successively, 5mL massfraction is the polyvinylpyrrolidone of 1% and the sodium borohydride aqueous solution of 0.6mL 0.01mol/L, then by 5mL massfraction be 1% polyvinylpyrrolidone and 5mL massfraction be 1% silver nitrate aqueous solution simultaneously join in conical flask with the speed of 30mL/h, stir while adding, solution is by colourless yellowing, obtain 10nm particle diameter Nano silver grain AgNP.
3. the method based on Nano silver grain tetrahedron Raman Multiple detection according to claim 1, it is characterized in that Nano silver grain modifying DNA: get the Nano silver grain AgNP that synthesizes described in 30 μ L 20nM in PCR pipe, after adding the DNA1 mixing of 1 μ L 10 μMs, 5 μ L 5 × tris-borate buffers and 1.25 μ L 2mol/L NaCl solution are added successively in system, rocked at room temperature reaction 12h, the centrifugal 10min of 13000r/min, remove supernatant, add ultrapure water to original volume, obtain AgNP-DNA1; Preparation method and the AgNP-DNA1 of AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complex are similar.
4. the method based on Nano silver grain tetrahedron Raman Multiple detection according to claim 1, it is characterized in that the modification of Raman beacon molecule: by three kinds of Raman beacon molecule 4-aminothiophenol 4-ATP, 4-nitrobenzenethiol NTP, 4-methoxybenzyl mercaptan MATT respectively correspondence joins AgNP-DNA1, AgNP-DNA2, in AgNP-DNA3 system, in solution, the final concentration of Raman beacon molecule is 3 μMs, beacon molecule joins reaction in system and spends the night, separately with centrifugal 10 min of 13000 r/min, remove supernatant, in system, add 20 mM Tris-HCl damping fluids again return to original volume, prepare AgNP-DNA1-ATP, AgNP-DNA2-NTP, AgNP-DNA3-MATT complex.
5. the method based on Nano silver grain tetrahedron Raman Multiple detection according to claim 1, it is characterized in that 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 get 100 μ L and mix in 1.5 mL centrifuge tubes, add 4 μ L 5M NaCl solution, concussion mixing, 90 DEG C of water-bath 5 min, more slowly room temperature is dropped in water vapor, namely prepare Nano silver grain tetrahedron.
6. the method based on Nano silver grain tetrahedron Raman Multiple detection according to claim 1, it is characterized in that the study on its developing based on Nano silver grain tetrahedron Raman sensor: detect for while SDM, AFM1, OTA, three kinds of materials join in system one by one according to sequencing, often kind of material midfeather 30 min; The interpolation concentration of SDM is followed successively by 0,0.001,0.005,0.01,0.05,0.1,0.5 fM; The interpolation concentration of AFM1 is followed successively by 0,0.1,0.5,1,5,10,50 fM; The interpolation concentration of OTA is followed successively by 0,0.01,0.05,0.1,0.5,1,5 fM; Three kinds of materials all add and react the Raman spectrum terminating rear survey system, set up the concentration standard curve of SDM, AFM1, OTA respectively according to the intensity of the Raman signal of 4-ATP, NTP and MATT; The Raman spectrum test duration is 20 s, and excitation wavelength is 633 nm.
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| CN105738342A (en) * | 2016-02-26 | 2016-07-06 | 中国人民解放军军事医学科学院军事兽医研究所 | SERS method for in-situ-synthesizing nano-silver by serving aptamer as support |
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| CN111198177A (en) * | 2019-12-18 | 2020-05-26 | 江南大学 | Preparation method and application of gold space tetrahedron structure of ratio-type surface-enhanced Raman spectrum |
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| CN115728284A (en) * | 2022-10-28 | 2023-03-03 | 南京师范大学 | Method for determining interaction between aptamer and small molecule target based on surface enhanced Raman spectroscopy |
| 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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| WO2016109911A1 (en) | 2016-07-14 |
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