CN107290284B - The method that the gold nanorod chiral dimer of thorn-like platinum package is used for DNA damage detection - Google Patents
The method that the gold nanorod chiral dimer of thorn-like platinum package is used for DNA damage detection Download PDFInfo
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Abstract
A method of the gold nanorod chiral dimer preparing thorn-like platinum package is detected for DNA damage, belongs to technical field of analytical chemistry and technical field of food safety.Step of the present invention are as follows: (1) synthesis of gold nanorods；(2) synthesis of thorn-like type platinum cladding gold nanorods；(3) thorn-like type platinum coats gold nanorods surface orientation nucleic acid function；(4) the gold nanorod chiral dimer of building thorn-like platinum package；(5) advance damage of the biotoxin to DNA；(6) detection of the gold nanorod chiral dimer of thorn-like platinum package to DNA damage.Plasma chirality Spectroscopy signal can be successfully applied to bio-sensing detection field.It carries out assembling the gold nanorods dimer generation visible light/near infrared region circular dichroism signal for forming thorn-like platinum package using nucleic acid and then is used for the detection of biotoxin and its metabolin to DNA damage.
Description
Technical field
A kind of gold nanorod chiral dimer preparing thorn-like platinum package is used for the detection method of DNA damage, belongs to analysis
Technical field of chemistry and technical field of food safety.
Background technique
DNA is the important inhereditary material of life entity, but DNA damage is recurrent thing in vivo.External source
The chemistry or biological substance and its metabolin of property, such as biotoxin and its metabolin, often make the DNA in cell
At oxidation (base oxidation, base fall off or the fracture of single, double chain) and/or base modification (base adduct, base methylation or shape
At pyrimidine dimer) etc. including structural damage.The DNA of such damage easily causes gene mutation even to cause body
Canceration.Therefore, carry out DNA damage detection to all close phase such as environmental monitoring and food safety (especially cross-border food safety)
It closes, is badly in need of development at present and is directed to the high specific of DNA damage product, highly sensitive novel Fast Detection Technique, realize exogenous
Chemistry and biological substance accurate and quantitative analysis is carried out to the degree of DNA damage.
The generation of circular dichroism is since absorption of the chiral material in system to left and right circularly polarized light is different.Currently,
Circular dichroism is mainly the Spectroscopy signal for passing through measurement large biological molecule, to solve to its second level or tertiary structure
Analysis.In recent years, chiral nanometer assembling superstructure becomes research hotspot problem concerned by people, especially since we were in 2009
Since successfully constructing the plasma nanometer chirality tetrahedral structure with similar organic chiral carbon, plasma chirality optical activity
The concern of a large amount of scientific circles and industry is successfully attracted.
The circular dichroism signal of biomolecule is mainly situated in ultraviolet region, the circular dichroism light of natural biomolecule
Spectrum signal is located at visible light region.And plasma chirality circular dichroism signal is different from the circular dichroism of biomolecule
Signal, the ultraviolet region being not only often in traditional biomolecule have a stronger Spectroscopy signal, also can
Light-exposed/near infrared region produces extremely strong circular dichroism signal, and more importantly, plasma chiral signal
Sensitivity will be significantly larger than most sensitive radioactivity transducing signal in detection and analysis at present.We pass through the study found that this it is equal from
Sub- chiral inorganic Nanoscale assemblies are in the intensity of visible light/near infrared region signal and the spectroscopy position of signal and nanoscale
Element, size, configuration and its corresponding yield for assembling primitive are closely bound up, have absolutely proved plasma chirality Spectroscopy signal
It can be successfully applied to bio-sensing detection field.The gold nanorods dimer production that assembling forms thorn-like platinum package is carried out using nucleic acid
The circular dichroism signal of raw visible light/near infrared region is used for the detection of biotoxin and its metabolin to DNA damage in turn
Still belong to blank.
Summary of the invention
The object of the present invention is to provide a kind of preparation methods of the gold nanorod chiral dimer of thorn-like platinum package, construct
Utilize the new technology of plasma circular dichroism signal detection DNA damage.
Technical solution of the present invention, a kind of gold nanorod chiral dimer preparing thorn-like platinum package is for DNA damage
Detection method.Main implementation steps of the invention are as follows: (1) synthesis of gold nanorods；(2) conjunction of thorn-like type platinum cladding gold nanorods
At；(3) the gold nanorods body structure surface oriented nuclei acid functionalization of thorn-like platinum package；(4) gold nanorods of building thorn-like platinum package
Chiral dimer；(5) advance damage of the biotoxin to DNA；(6) the gold nanorod chiral dimer of thorn-like platinum package damages DNA
The detection of wound.
Specific steps are as follows:
(1) synthesis of gold nanorods
According to a kind of patent " preparation method of the self-assembled material with the surface reinforced Raman active " (patent No.: ZL
201010605799.9) method synthesis draw ratio be about 3.0 gold nanorods.It is simple that steps are as follows:
1. crystal seed synthesizes: taking the three of preconfigured 2 g/L of 0.1 mL to be hydrated tetra chlorauric acid at 28.0 DEG C and be added to 1
In the cetyl trimethylammonium bromide solution of 0.2 M of mL, at this point, solution colour becomes yellowish-brown by colourless.Then up
It states and the 0.01 M sodium borohydride solution newly prepared of 0.12 mL is added in mixed system quickly stirs 2 minutes.Solution colour is
Light brown is become from yellowish-brown.The crystal seed of above-mentioned synthesis is put into 28.0 DEG C of water-bath and stands reaction 30 minutes, made not complete
The full sodium borohydride for participating in crystal seed synthesis is depleted, (first is that the gold not reacted completely in the further reduction system of sodium borohydride
Ion and be consumed, second is that stand reaction process in sodium borohydride due to hydrolysis cause its content to further decrease), preparation
Crystal seed will carry out next step reaction in post synthesis in 4 hours.
2. gold nanorods growth course: after crystal seed synthesis is completed, carrying out the growth of gold nanorods.2 g/L's of 5 mL
The ultrapure water of 4 mL is added in three hydration tetra chlorauric acids after being added in the cetyl trimethylammonium bromide of 5 mL, 0.2 M, mix
It is even.The 0.01 M silver nitrate solution of 0.125 mL is added in above-mentioned mixed system, is mixed, then by 0.1 M of 65 μ L
Above-mentioned system solution is added in ascorbic acid solution, reaction 2 minutes is vigorously stirred at 28.0 DEG C, solution becomes colourless by brown.Most
Afterwards, the seed-solution gentle agitation that 0.05 mL is added mixes 20 seconds.28.0 DEG C reaction 2 hours after solution color be it is drabon
Color.The gold nanorods solution of synthesis is centrifuged 10 minutes by 10000 rpms, supernatant is abandoned, using the hexadecane of 0.005 M
Base trimethylammonium bromide solution is dispersed precipitating again, and cleaning is primary, finally by gold nanorods solution be dispersed in it is initial etc.
In the solution of volume, it is stored at room temperature preservation, to carry out the characterization and experiment demand of next step.
(2) synthetic method of thorn-like type platinum cladding gold nanorods
Into the gold nanorods solution of 5mL, 50 μM of sodium iodide, the cetyl three of the rear 0.05M that 10mL is added is added
It after methyl bromide ammonium, mixes, the silver nitrate solution of 0.2 mM of 500 μ L is then added, mix, add the 0.1M of 500 μ L
Ascorbic acid solution, mix, after 70 DEG C of 1 h of incubations, the hydrochloric acid of the 0.1M of 480 μ L of addition and the chloroplatinic acid of 440 μ L 2mM,
After 70 DEG C of incubation reaction 4h, 6500rpm is centrifuged 10 min, abandons supernatant, and the cetyl trimethylammonium bromide using 5 mM is molten
Liquid dispersion, for use；
(3) thorn-like type platinum coats gold nanorods surface orientation nucleic acid function
1. thorn-like type platinum cladding gold nanorods carry out end face closing to it: taking the thorn-like type platinum of above-mentioned 10 nM prepared
2 mL of gold nanorods is coated, the dithiothreitol (DTT) solution of 1 mM of 30 μ L is added, is mixed, shaking reaction 4h, using 6000rpm
It is centrifuged 5min, abandons supernatant, disperses gold nanorods-dithiothreitol (DTT) with the cetyl trimethylammonium bromide solution of 5 mM of 2 mL
Compound, it is 4 DEG C of preservations, spare；
2. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 2: taking above-mentioned processed thorn-like type platinum cladding gold
10 μM of the mercapto-modified DNA2 of 3 μ L is added in the 300 μ L of solution of nanometer rods-dithiothreitol (DTT) compound, mixes, 25
DEG C, shaking reaction 8h, 6000rpm be centrifuged 5min, supernatant is removed, with the cetyl trimethylammonium bromide solution of 5 mM of 300 μ L
Disperse thorn-like type platinum and coat gold nanorods-dithiothreitol (DTT)-DNA2 compound, and using the cetyl trimethyl of 5 mM
Ammonium bromide solution cleaning is primary, 4 DEG C of preservations, spare；
3. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 3: taking above-mentioned processed thorn-like type platinum cladding Jenner
10 μM of the mercapto-modified DNA3 of 3 μ L is added in the 300 μ L of solution of rice stick-dithiothreitol (DTT) compound, mixes, 25 DEG C,
Shaking reaction 8h, 6000rpm centrifugation 5min, removes supernatant, with the cetyl trimethylammonium bromide solution pair of 5 mM of 300 μ L
Thorn-like type platinum cladding gold nanorods-dithiothreitol (DTT)-DNA3 compound is dispersed, and using the cetyl three of 5 mM
The cleaning of methyl bromide ammonium salt solution is primary, 4 DEG C of preservations, spare；
(4) preparation of the gold nanorod chiral dimer of the thorn-like platinum package of nucleic acid function
The thorn-like gold nanorods for the platinum package for taking DNA2 the and DNA3 lateral orientation of the 50 above-mentioned preparations of μ L to modify are mixed
It closes, whirlpool mixing, 10 μM of DNA1, the whirlpool mixing of 0.5 μ L is added, after being incubated at room temperature 8h, hybridizes instead by nucleic acid specificity
It answers, so that the gold nanorods for the thorn-like platinum package that DNA2 and DNA3 are modified respectively form side assembling dimer；
One DNA damage of table detection base sequence used
| Number | Sequence (5 ' -3 ') | Length |
| DNA 1 | ACCGGAGGCC CATCCTCACC | 20 |
| DNA2 | SH-GGCCTCCGGT | 10 |
| DNA 3 | GGTGAGGATG-SH | 10 |
(5) advance damage of the biotoxin to DNA
The aflatoxin B1 of μm ol/L of 10 μM of DNA1 and 10,5,1,0.1,0.01 is mixed, room temperature reaction
The DNA1 of advance damage is placed on 4 DEG C of refrigerators by 2.5 h, spare；Meanwhile for the metabolite aspergillus flavus of aflatoxin B1
Toxin M1 and aflatoxin M 2, will according to the step of aflatoxin B1 and identical concentration and DNA1 carry out being incubated for 2.5 h
DNA1 after advance damage is placed on 4 DEG C of refrigerators, spare；
(6) detection of the gold nanorod chiral dimer of thorn-like platinum package to DNA damage
Using circular dichroism signal to aflatoxin B1 and its metabolite Aflatoxins M1 and aspergillus flavus poison
Plain M2 is detected, and is drawn the concentration standard curve of molar ellipticity~aflatoxin B1, M1 and M2: being taken the 50 above-mentioned systems of μ L
The thorn-like gold nanorods of the platinum package of standby DNA2 and DNA3 lateral orientation modification are mixed, whirlpool mixes, and are added by step
The DNA1(0.5 μ L of rapid 5 processed advance damage), whirlpool mixes, after being incubated at room temperature 8h, by nucleic acid specificity hybridization reaction,
So that the thorn-like gold nanorods for the platinum package that DNA2 and DNA3 are modified respectively form side assembling structure；
Due to the aflatoxin B1 of addition or the difference of the concentration of its metabolite M1 and M2, aflatoxin and phase
Guanine in the DNA1 answered forms aflatoxin-guanine adduct, so that DNA2 and DNA3 can not be with DNA1
The nucleic acid recognizing hybridization of specificity is carried out, the yield for the dimer that the gold nanorods for causing thorn-like platinum to wrap up are formed is different and then draws
It plays plasma circular dichroism signal and generates difference, measure the group under different aflatoxin concentration using circular dichroism instrument
The molar ellipticity for filling product is drawn according to the molar ellipticity of the different aflatoxin concentration measured under 855 nm wavelength
The standard curve of aflatoxin concentration.
Beneficial effects of the present invention: we pass through the study found that the plasma chiral inorganic Nanoscale assemblies visible light/
The element of the spectroscopy position and nanoscale assembling primitive of the intensity of the signal of near infrared region and signal, size, configuration and its
Corresponding yield is closely bound up, has absolutely proved that plasma chirality Spectroscopy signal can be successfully applied to bio-sensing detection neck
Domain.Gold nanorods dimer generation visible light/near infrared region circle two that assembling forms thorn-like platinum package is carried out using nucleic acid
Be used for biotoxin and its metabolin still belongs to blank to the detection of DNA damage to coloured light spectrum signal in turn.
Detailed description of the invention
The electromicroscopic photograph of the gold nanorods of Fig. 1 synthesis.
The ultraviolet/visible light spectrogram of the gold nanorods of Fig. 2 synthesis.
The electromicroscopic photograph of the gold nanorods dimer of Fig. 3 thorn-like platinum package.
The circular dichroism figure of the gold nanorods dimer of Fig. 4 thorn-like platinum package.
The standard curve of the plasma circular dichroism detection method of Fig. 5 aflatoxin B1.
Specific embodiment
The gold nanorod chiral dimer that embodiment 1 prepares thorn-like platinum package is used for the damage check of DNA
(1) synthesis of gold nanorods
According to a kind of patent " preparation method of the self-assembled material with the surface reinforced Raman active " (patent No.: ZL
201010605799.9) method synthesis draw ratio be about 3.0 gold nanorods.It is simple that steps are as follows:
1. crystal seed synthesizes: taking the three of preconfigured 2 g/L of 0.1 mL to be hydrated tetra chlorauric acid at 28.0 DEG C and be added to 1
In the cetyl trimethylammonium bromide solution of 0.2 M of mL, at this point, solution colour becomes yellowish-brown by colourless.Then up
It states and the 0.01 M sodium borohydride solution newly prepared of 0.12 mL is added in mixed system quickly stirs 2min.Solution colour is
Light brown is become from yellowish-brown.The crystal seed of above-mentioned synthesis is put into 28.0 DEG C of water-bath and stands reaction 30min, made not completely
Participate in crystal seed synthesis sodium borohydride it is depleted, (first is that do not reacted completely in the further reduction system of sodium borohydride gold from
Son and be consumed, second is that stand reaction process in sodium borohydride due to hydrolysis cause its content to further decrease), the crystalline substance of preparation
Kind will the interior progress next step reaction of 4h in post synthesis.
2. gold nanorods growth course: after crystal seed synthesis is completed, carrying out the growth of gold nanorods.2 g/L's of 5 mL
The ultrapure water of 4 mL is added in three hydration tetra chlorauric acids after being added in the cetyl trimethylammonium bromide of 5 mL, 0.2 M, mix
It is even.The 0.01 M silver nitrate solution of 0.125 mL is added in above-mentioned mixed system, is mixed, then by 0.1 M of 65 μ L
Above-mentioned system solution is added in ascorbic acid solution, and reaction 2min is vigorously stirred at 28.0 DEG C, and solution becomes colourless by brown.Most
Afterwards, the seed-solution gentle agitation that 0.05 mL is added mixes 20s.The color of solution is drabon color after 28.0 DEG C of reaction 2h.It closes
At gold nanorods solution by 10000rpm be centrifuged 10min, abandon supernatant, using the cetyl trimethyl bromination of 0.005 M
Ammonium salt solution is dispersed precipitating again, and cleaning is primary, finally by gold nanorods solution be dispersed in in initial isometric solution,
It is stored at room temperature preservation, to carry out the characterization and experiment demand of next step.
(2) synthetic method of thorn-like type platinum cladding gold nanorods
Into the gold nanorods solution of 5mL, 50 μM of sodium iodide, the cetyl three of the rear 0.05M that 10mL is added is added
It after methyl bromide ammonium, mixes, the silver nitrate solution of 0.2 mM of 500 μ L is then added, mix, add the 0.1M of 500 μ L
Ascorbic acid solution, mix, after 70 DEG C of 1 h of incubations, the hydrochloric acid of the 0.1M of 480 μ L of addition and the chloroplatinic acid of 440 μ L 2mM,
After 70 DEG C of incubation reaction 4h, 6500rpm is centrifuged 10 min, abandons supernatant, and the cetyl trimethylammonium bromide using 5 mM is molten
Liquid dispersion, for use.
(3) thorn-like type platinum coats gold nanorods surface orientation nucleic acid function
1. thorn-like type platinum cladding gold nanorods carry out end face closing to it: taking the thorn-like type platinum of above-mentioned 10 nM prepared
2 mL of gold nanorods is coated, the dithiothreitol (DTT) solution of 1 mM of 30 μ L is added, is mixed, shaking reaction 4 hours uses
6000rpm is centrifuged 5min, abandons supernatant, disperses gold nanorods-two with the cetyl trimethylammonium bromide solution of 5 mM of 2 mL
The compound of sulphur threitol, it is 4 DEG C of preservations, spare.
2. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 2: taking above-mentioned processed thorn-like type platinum cladding gold
10 μM of the mercapto-modified DNA2 of 3 μ L is added in the 300 μ L of solution of nanometer rods-dithiothreitol (DTT) compound, mixes, 25
DEG C, shaking reaction 8h, 6000rpm be centrifuged 5min, supernatant is removed, with the cetyl trimethylammonium bromide solution of 5 mM of 300 μ L
Disperse thorn-like type platinum and coat gold nanorods-dithiothreitol (DTT)-DNA2 compound, and using the cetyl trimethyl of 5 mM
Ammonium bromide solution cleaning is primary, 4 DEG C of preservations, spare.
3. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 3: taking above-mentioned processed thorn-like type platinum cladding Jenner
10 μM of the mercapto-modified DNA3 of 3 μ L is added in the 300 μ L of solution of the compound of rice stick and dithiothreitol (DTT), mixes, 25
℃.Shaking reaction 8h, 6000rpm centrifugation 5min, removes supernatant, with the cetyl trimethylammonium bromide solution of 5 mM of 300 μ L
Thorn-like type platinum cladding gold nanorods-dithiothreitol (DTT)-DNA3 compound is dispersed, and using the cetyl of 5 mM
The cleaning of trimethylammonium bromide solution is primary, 4 DEG C of preservations, spare.
(4) preparation of the gold nanorod chiral dimer of the thorn-like platinum package of nucleic acid function
The thorn-like gold nanorods for the platinum package for taking DNA2 the and DNA3 lateral orientation of the 50 above-mentioned preparations of μ L to modify are mixed
It closes, whirlpool mixing, 10 μM of DNA1, the whirlpool mixing of 0.5 μ L is added, after being incubated at room temperature 8h, hybridizes instead by nucleic acid specificity
It answers, so that the gold nanorods for the thorn-like platinum package that DNA2 and DNA3 are modified respectively form side assembling dimer.
One DNA damage of table detection base sequence used
| Number | Sequence (5 ' -3 ') | Length |
| DNA1 | ACCGGAGGCC CATCCTCACC | 20 |
| DNA2 | SH-GGCCTCCGGT | 10 |
| DNA3 | GGTGAGGATG-SH | 10 |
(5) advance damage of the biotoxin to DNA
The aflatoxin B1 of μm ol/L of 10 μM of DNA1 and 10,5,1,0.1,0.01 is mixed, room temperature reaction
The DNA of advance damage is placed on 4 DEG C of refrigerators by 2.5 h, spare.Meanwhile for the metabolite aspergillus flavus poison of aflatoxin B1
Plain M1 and aflatoxin M 2, will be pre- according to the step of aflatoxin B1 and identical concentration and DNA1 carry out being incubated for 2.5 h
DNA after damage is placed on 4 DEG C of refrigerators, spare.
(6) detection of the thorn-like gold nanorod chiral dimer of platinum package to DNA damage；
Using circular dichroism signal to aflatoxin B1 and its metabolite Aflatoxins M1 and aspergillus flavus poison
Plain M2 is detected, and is drawn the concentration standard curve of molar ellipticity~aflatoxin B1, M1 and M2: being taken the 50 above-mentioned systems of μ L
The thorn-like gold nanorods of the platinum package of standby DNA2 and DNA3 lateral orientation modification are mixed, whirlpool mixes, and are added by step
The DNA1(0.5 μ L of rapid 5 processed advance damage), whirlpool mix, after being incubated at room temperature 8h, by nucleic acid specificity hybridization reaction,
So that the thorn-like gold nanorods for the platinum package that DNA2 and DNA3 are modified respectively form side assembling structure.
Due to the aflatoxin B1 of addition or the difference of the concentration of its metabolite M1 and M2, aflatoxin and phase
Guanine in the DNA1 answered forms aflatoxin-guanine adduct, so that DNA2 and DNA3 can not be with DNA1
The nucleic acid recognizing hybridization of specificity is carried out, the yield for the dimer that the thorn-like gold nanorods for causing platinum to wrap up are formed is different and then draws
It plays plasma circular dichroism signal and generates difference.The group under different aflatoxin concentration is measured using circular dichroism instrument
The molar ellipticity for filling product is drawn according to the molar ellipticity of the different aflatoxin concentration measured under 855 nm wavelength
The standard curve of aflatoxin concentration.
Claims (1)
1. a kind of method that the gold nanorod chiral dimer for preparing thorn-like platinum package is used for DNA damage detection, it is characterised in that
Step are as follows: the synthesis of thorn-like type platinum cladding gold nanorods；The gold nanorods body structure surface oriented nuclei acid functionalization of thorn-like platinum package；
Construct the gold nanorod chiral dimer of thorn-like platinum package；Advance damage of the biotoxin to DNA；The gold nanorods of thorn-like platinum package
Detection of the chiral dimer to DNA damage, specifically comprises the processes of:
(1) synthetic method of thorn-like type platinum cladding gold nanorods
Into the gold nanorods solution of 5mL, 50 μM of sodium iodide, the cetyl trimethyl of the rear 0.05M that 10mL is added is added
It after ammonium bromide, mixes, the silver nitrate solution of 0.2 mM of 500 μ L is then added, mix, add the anti-of the 0.1M of 500 μ L
Bad hematic acid solution mixes, and after 70 DEG C of 1 h of incubation, the hydrochloric acid of the 0.1M of 480 μ L and the chloroplatinic acid of 440 μ L 2mM is added, 70
After DEG C incubation reaction 4h, 6500rpm is centrifuged 10 min, abandons supernatant, using the cetyl trimethylammonium bromide solution point of 5 mM
It dissipates, for use；
(2) thorn-like type platinum coats gold nanorods surface orientation nucleic acid function
1. thorn-like type platinum cladding gold nanorods carry out end face closing to it: the thorn-like type platinum of above-mentioned 10 nM prepared being taken to coat
The dithiothreitol (DTT) solution of 1 mM of 30 μ L is added in 2 mL of gold nanorods, mixes, and shaking reaction 4h is centrifuged using 6000rpm
5min abandons supernatant, is answered with cetyl trimethylammonium bromide solution dispersion gold nanorods-dithiothreitol (DTT) of 5 mM of 2 mL
Object is closed, it is 4 DEG C of preservations, spare；
2. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 2: taking above-mentioned processed thorn-like type platinum cladding gold nano
10 μM of the mercapto-modified DNA2 of 3 μ L is added in the 300 μ L of solution of stick-dithiothreitol (DTT) compound, mixes, 25 DEG C, vibration
Reaction 8h is shaken, 6000rpm is centrifuged 5min, removes supernatant, is dispersed with the cetyl trimethylammonium bromide solution of 5 mM of 300 μ L
Thorn-like type platinum coats gold nanorods-dithiothreitol (DTT)-DNA2 compound, and using the cetyl trimethyl bromination of 5 mM
Ammonium salt solution cleaning is primary, 4 DEG C of preservations, spare；
3. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 3: taking above-mentioned processed thorn-like type platinum cladding gold nano
10 μM of the mercapto-modified DNA3 of 3 μ L is added in the 300 μ L of solution of stick-dithiothreitol (DTT) compound, mixes, 25 DEG C, vibration
Reaction 8h is shaken, 6000rpm is centrifuged 5min, removes supernatant, with the cetyl trimethylammonium bromide solution of 5 mM of 300 μ L to thorn
Shape type platinum cladding gold nanorods-dithiothreitol (DTT)-DNA3 compound is dispersed, and using the cetyl front three of 5 mM
The cleaning of base ammonium bromide solution is primary, 4 DEG C of preservations, spare；
(3) preparation of the gold nanorod chiral dimer of the thorn-like platinum package of nucleic acid function
Take DNA2 the and DNA3 lateral orientation of the 50 above-mentioned preparations of μ L to modify thorn-like platinum package gold nanorods mixed, whirlpool
Whirlpool mixes, and 10 μM of DNA1 of 0.5 μ L is added, whirlpool mixing, by nucleic acid specificity hybridization reaction, makes after being incubated at room temperature 8h
The gold nanorods for obtaining the thorn-like platinum package that DNA2 and DNA3 are modified respectively form side assembling dimer；
DNA damage detection base sequence used:
DNA 1:5 '-ACCGGAGGCC CATCCTCACC -3 ',
DNA 2:5 '-SH-GGCCTCCGGT-3 ',
DNA 3:5 '-GGTGAGGATG-SH -3 '；
(4) advance damage of the biotoxin to DNA
The aflatoxin B1 of μm ol/L of 10 μM of DNA1 and 10,5,1,0.1,0.01 is mixed, room temperature reaction 2.5
The DNA1 of advance damage is placed on 4 DEG C of refrigerators by h, spare；Meanwhile for the metabolite aflatoxin of aflatoxin B1
M1 and aflatoxin M 2 will damage in advance according to the step of aflatoxin B1 and identical concentration and DNA1 carry out being incubated for 2.5 h
DNA1 after wound is placed on 4 DEG C of refrigerators, spare；
(5) detection of the gold nanorod chiral dimer of thorn-like platinum package to DNA damage
Using circular dichroism signal to aflatoxin B1 and its metabolite Aflatoxins M1 and aflatoxin M 2
It is detected, draws the concentration standard curve of molar ellipticity~aflatoxin B1, M1 and M2: taking the 50 above-mentioned preparations of μ L
The gold nanorods of the thorn-like platinum package of DNA2 and DNA3 lateral orientation modification are mixed, whirlpool mixes, and are added and are passed through step 4 place
The 0.5 μ L of DNA1 for the advance damage managed, whirlpool mix, after being incubated at room temperature 8h, by nucleic acid specificity hybridization reaction, so that
The gold nanorods for the thorn-like platinum package that DNA2 and DNA3 are modified respectively form side assembling structure；
Due to the aflatoxin B1 of addition or the difference of the concentration of its metabolite M1 and M2, aflatoxin and corresponding
DNA1 in guanine formed aflatoxin-guanine adduct so that DNA2 and DNA3 can not with DNA1 into
The nucleic acid recognizing hybridization of row specificity, the yield for the dimer that the gold nanorods for causing thorn-like platinum to wrap up are formed is different and then causes
Plasma circular dichroism signal generates difference, measures the assembling under different aflatoxin concentration using circular dichroism instrument
The molar ellipticity of product is drawn yellow according to the molar ellipticity of the different aflatoxin concentration measured under 855 nm wavelength
The standard curve of aspertoxin concentration.
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