CN107290284A - A kind of gold nanorod chiral dimer for preparing thorn-like platinum parcel is used for the detection method of DNA damage - Google Patents
A kind of gold nanorod chiral dimer for preparing thorn-like platinum parcel is used for the detection method of DNA damage Download PDFInfo
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Abstract
A kind of gold nanorod chiral dimer for preparing thorn-like platinum parcel is used for the method that DNA damage is detected, belongs to technical field of analytical chemistry and technical field of food safety.Step of the present invention is：（1）The synthesis of gold nanorods；（2）Thorn-like type platinum coats the synthesis of gold nanorods；（3）Thorn-like type platinum coats gold nanorods surface orientation nucleic acid function；（4）Build the gold nanorod chiral dimer of thorn-like platinum parcel；（5）Advance damage of the biotoxin to DNA；（6）Detection of the gold nanorod chiral dimer of thorn-like platinum parcel to DNA damage.Plasma chirality Spectroscopy signal can be successfully applied to bio-sensing detection field.Assembling, which is carried out, using nucleic acid forms the circular dichroism signal for gold nanorods dimer generation visible ray/near infrared region that thorn-like platinum is wrapped up and then for the detection of biotoxin and its metabolin to DNA damage.
Description
Technical field
A kind of gold nanorod chiral dimer for preparing thorn-like platinum parcel is used for the detection method of DNA damage, belongs to analysis
Technical field of chemistry and technical field of food safety.
Background technology
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 are often made to the DNA in cell
Into oxidation（Base oxidation, base come off or the fracture of single, double chain）And/or base modification（Base adduct, base methylate or shape
Into pyrimidine dimer）Structural damage Deng including.The DNA of such damage easily causes gene mutation or even causes body
Canceration.Therefore, DNA damage detection pair and environmental monitoring and food security are carried out（Particularly cross-border food security）Deng all close phase
Close, be badly in need of development at present and be directed to the high specific of DNA damage product, highly sensitive new Fast Detection Technique, realize exogenous
Chemistry accurate and quantitative analysis is carried out to the degree of DNA damage with biological substance.
The generation of circular dichroism is due to that absorption of the chiral material to left and right circularly polarized light in system is different.At present,
Circular dichroism is mainly the Spectroscopy signal by measuring large biological molecule, so that pair secondly level or tertiary structure are solved
Analysis.In recent years, chiral nanometer assembling superstructure turns into study hotspot problem of concern, particularly since we were in 2009
Since successfully constructing the chiral tetrahedral structure of plasma nanometer with similar organic chiral carbon, plasma chirality optical activity
The concern of a large amount of scientific circles and industrial quarters 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 region.And the chiral circular dichroism signal of plasma is different from the circular dichroism of biomolecule
Signal, its ultraviolet region being not only often in traditional biomolecule has stronger Spectroscopy signal, and it is also can
See that light/near infrared region generates extremely strong circular dichroism signal, and what is more important, plasma chiral signal
Most sensitive radioactivity transducing signal during sensitivity will be tested and analyzed significantly larger than at present.We by study find, this grade from
The intensity and the spectroscopy position of signal and nanoscale of signal of the sub- chiral inorganic Nanoscale assemblies in visible ray/near infrared region
Element, size, configuration and its corresponding yield for assembling primitive are closely bound up, have absolutely proved the chiral Spectroscopy signal of plasma
Bio-sensing detection field can be successfully applied to.The gold nanorods dimer production that assembling forms thorn-like platinum parcel is carried out using nucleic acid
Give birth to the circular dichroism signal of visible ray/near infrared region and then for the detection of biotoxin and its metabolin to DNA damage
Still belong to blank.
The content of the invention
It is an object of the invention to provide a kind of preparation method of the gold nanorod chiral dimer of thorn-like platinum parcel, construct
Utilize the new technology of plasma circular dichroism signal detection DNA damage.
A kind of technical scheme, gold nanorod chiral dimer for preparing thorn-like platinum parcel is used for DNA damage
Detection method.The present invention main implementation steps be：（1）The synthesis of gold nanorods；（2）Thorn-like type platinum coats the conjunction of gold nanorods
Into；（3）The gold nanorods body structure surface oriented nuclei acid functionalization of thorn-like platinum parcel；（4）Build the gold nanorods of thorn-like platinum parcel
Chiral dimer；（5）Advance damage of the biotoxin to DNA；（6）The gold nanorod chiral dimer of thorn-like platinum parcel is damaged to DNA
The detection of wound.
Concretely comprise the following steps：
（1）The synthesis of gold nanorods
According to patent《A kind of preparation method of the self-assembled material with surface reinforced Raman active》（The patent No.：ZL
201010605799.9）Method synthesis draw ratio be about 3.0 gold nanorods.Simple step is as follows：
1. crystal seed is synthesized：0.1 mL 2 g/L being pre-configured with three hydration tetra chlorauric acids are taken to be added to 1 mL at 28.0 DEG C
0.2 M cetyl trimethylammonium bromide solution in, now, solution colour becomes yellowish-brown by colourless.State then up
The 0.01 M sodium borohydride solutions that 0.12 mL new preparation is added in mixed system are quickly stirred 2 minutes.Solution colour i.e. by
Yellowish-brown is changed into light brown.The crystal seed of above-mentioned synthesis is put into 28.0 DEG C of water-bath and stands reaction 30 minutes, is made incomplete
The sodium borohydride for participating in crystal seed synthesis is depleted,（One be the gold that does not react completely in the further reduction system of sodium borohydride from
Son and be consumed, two be stand course of reaction in sodium borohydride due to hydrolysis cause its content further to reduce）, the crystalline substance of preparation
Next step reaction will be carried out in post synthesis by planting in 4 hours.
2. gold nanorods growth course：After crystal seed synthesis is completed, the growth of gold nanorods is carried out.5 mL 2 g/L's
Three hydration tetra chlorauric acids add 4 mL ultra-pure water after being added in the M of 5 mL 0.2 cetyl trimethylammonium bromide, mix
It is even.0.125 mL 0.01 M silver nitrate solutiones are added in above-mentioned mixed system, are mixed, then by 65 μ L 0.1 M
Ascorbic acid solution, which is added, is stirred vigorously reaction 2 minutes at above-mentioned system solution, 28.0 DEG C, solution becomes colourless by brown.Most
Afterwards, the seed-solution gentle agitation for adding 0.05 mL is mixed 20 seconds.The color of solution is drabon after 28.0 DEG C of reactions 2 hours
Color.The gold nanorods solution of synthesis is centrifuged 10 minutes by 10000 rpms, supernatant is abandoned, using 0.005 M hexadecane
Base trimethylammonium bromide solution by precipitate is disperseed again, cleaning once, most at last gold nanorods solution be dispersed in initially etc.
In the solution of volume, preservation is stored at room temperature, to carry out the sign and experiment demand of next step.
（2）Thorn-like type platinum coats the synthetic method of gold nanorods
Into 5mL gold nanorods solution, 50 μM of sodium iodide, the rear 0.05M for adding 10mL cetyl trimethyl are added
After ammonium bromide, mix, be subsequently added 500 μ L 0.2 mM silver nitrate solution, mix, add 500 μ L 0.1M it is anti-
Bad hematic acid solution, is mixed, and 70 DEG C are incubated after 1 h, 480 μ L 0.1M hydrochloric acid and 440 μ L 2mM chloroplatinic acid are added, 70
After DEG C incubation reaction 4h, 6500rpm centrifuges 10 min, abandons supernatant, using 5 mM cetyl trimethylammonium bromide solution point
Dissipate, it is stand-by；
（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：Above-mentioned 10 nM prepared thorn-like type platinum is taken to coat
The mL of gold nanorods 2, adds 30 μ L 1 mM dithiothreitol (DTT) solution, mixes, shaking reaction 4h, is centrifuged using 6000rpm
5min, abandons supernatant, disperses answering for gold nanorods-dithiothreitol (DTT) with 2 mL 5 mM cetyl trimethylammonium bromide solution
Compound, it is 4 DEG C of preservations, standby；
2. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 2：Take above-mentioned treated thorn-like type platinum cladding gold nano
The μ L of solution 300 of the compound of rod-dithiothreitol (DTT) add the DNA2 of 3 μ L 10 μM of sulfydryl modification, mix, 25 DEG C, shake
Reaction 8h, 6000rpm centrifugation 5min is shaken, supernatant is removed, is disperseed with 300 μ L 5 mM cetyl trimethylammonium bromide solution
Thorn-like type platinum coats gold nanorods-dithiothreitol (DTT)-DNA2 compound, and using 5 mM cetyl trimethyl bromination
Ammonium salt solution is cleaned once, 4 DEG C of preservations, standby；
3. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 3：Take above-mentioned treated thorn-like type platinum cladding gold nano
The μ L of solution 300 of the compound of rod-dithiothreitol (DTT) add the DNA3 of 3 μ L 10 μM of sulfydryl modification, mix, 25 DEG C, shake
Reaction 8h, 6000rpm centrifugation 5min is shaken, supernatant is removed, with 300 μ L 5 mM cetyl trimethylammonium bromide solution to thorn
Shape type platinum cladding gold nanorods-dithiothreitol (DTT)-DNA3 compound is disperseed, and using 5 mM cetyl front three
Base ammonium bromide solution is cleaned once, 4 DEG C of preservations, standby；
（4）The preparation of the gold nanorod chiral dimer of the thorn-like platinum parcel of nucleic acid function
The thorn-like gold nanorods that the platinum for taking DNA2 the and DNA3 lateral orientations of the 50 above-mentioned preparations of μ L to modify is wrapped up are mixed, whirlpool
Whirlpool is mixed, and is added 0.5 μ L 10 μM of DNA1, whirlpool and is mixed, after incubation at room temperature 8h, by nucleic acid specificity hybridization reaction, makes
Obtain the gold nanorods formation side assembling dimer for the thorn-like platinum parcel that DNA2 and DNA3 are modified respectively；
The DNA damage of table one detection base sequence used
| Numbering | 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
μm ol/L of 10 μM of DNA1 and 10,5,1,0.1,0.01 aflatoxin B1 is mixed, and reacts at room temperature 2.5 h,
The DNA1 of advance damage is placed on 4 DEG C of refrigerators, it is standby；Simultaneously for the metabolite Aflatoxins M1 of aflatoxin B1
And aflatoxin M 2 is according to the step of aflatoxin B1 and identical concentration, and DNA1 carries out being incubated 2.5 h, by advance damage
DNA1 afterwards is placed on 4 DEG C of refrigerators, standby；
（6）Detection of the gold nanorod chiral dimer of thorn-like platinum parcel to DNA damage
Using circular dichroism signal to aflatoxin B1 and its metabolite Aflatoxins M1 and aflatoxin M 2
Detected, draw molar ellipticity~aflatoxin B1, M1 and M2 concentration standard curve：Take the 50 above-mentioned preparations of μ L
The thorn-like gold nanorods of the platinum parcel of DNA2 and DNA3 lateral orientations modification are mixed, whirlpool is mixed, and are added by step 5 place
The DNA1 for the advance damage managed（0.5 μL）, whirlpool is mixed, after incubation at room temperature 8h, by nucleic acid specificity hybridization reaction so that
The thorn-like gold nanorods formation side assembling structure for the platinum parcel that DNA2 and DNA3 are modified respectively；
Due to the aflatoxin B1 of addition or the difference of its metabolite M1 and M2 concentration, aflatoxin and corresponding
The adduct of guanine formation aflatoxin-guanine in DNA1, and then DNA2 and DNA3 can not be carried out with DNA1
Specific nucleic acid recognizing hybridization, the yield for the dimer that the gold nanorods for causing thorn-like platinum to wrap up are formed is different and then causes
Ion circular dichroism signal produces difference, and the assembling determined using circular dichroism instrument under different aflatoxin concentration is produced
The molar ellipticity of thing, according to the molar ellipticity of the different aflatoxin concentration determined under 855 nm wavelength, draws yellow bent
The standard curve of mould toxin concentration.
Beneficial effects of the present invention：We by study find, the plasma chiral inorganic Nanoscale assemblies visible ray/
The intensity of the signal of near infrared region and the spectroscopy position of signal assembled with nanoscale the element of primitive, size, configuration and its
Corresponding yield is closely bound up, has absolutely proved that the chiral Spectroscopy signal of plasma can be successfully applied to bio-sensing detection neck
Domain.The circle two that assembling forms gold nanorods dimer generation visible ray/near infrared region of thorn-like platinum parcel is carried out using nucleic acid
Coloured light spectrum signal and then still belong to blank for the detection of biotoxin and its metabolin to DNA damage.
Brief description of the drawings
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-likes platinum parcel.
The circular dichroism figure of the gold nanorods dimer of Fig. 4 thorn-likes platinum parcel.
The standard curve of the plasma circular dichroism detection method of Fig. 5 aflatoxin B1s.
Embodiment
The gold nanorod chiral dimer that embodiment 1 prepares thorn-like platinum parcel is used for DNA damage check
（1）The synthesis of gold nanorods
According to patent《A kind of preparation method of the self-assembled material with surface reinforced Raman active》（The patent No.：ZL
201010605799.9）Method synthesis draw ratio be about 3.0 gold nanorods.Simple step is as follows：
1. crystal seed is synthesized：0.1 mL 2 g/L being pre-configured with three hydration tetra chlorauric acids are taken to be added to 1 mL at 28.0 DEG C
0.2 M cetyl trimethylammonium bromide solution in, now, solution colour becomes yellowish-brown by colourless.State then up
The 0.01 M sodium borohydride solutions that 0.12 mL new preparation is added in mixed system quickly stir 2min.Solution colour i.e. by
Yellowish-brown is changed into light brown.The crystal seed of above-mentioned synthesis is put into 28.0 DEG C of water-bath and stands reaction 30min, makes completely not join
The sodium borohydride synthesized with crystal seed is depleted,（One is the gold ion not reacted completely in the further reduction system of sodium borohydride
And be consumed, two be that sodium borohydride causes its content further to reduce due to hydrolyzing in course of reaction is stood）, the crystal seed of preparation
Will the interior progress next step reactions of 4h in post synthesis.
2. gold nanorods growth course：After crystal seed synthesis is completed, the growth of gold nanorods is carried out.5 mL 2 g/L's
Three hydration tetra chlorauric acids add 4 mL ultra-pure water after being added in the M of 5 mL 0.2 cetyl trimethylammonium bromide, mix
It is even.0.125 mL 0.01 M silver nitrate solutiones are added in above-mentioned mixed system, are mixed, then by 65 μ L 0.1 M
Ascorbic acid solution, which is added, is stirred vigorously reaction 2min at above-mentioned system solution, 28.0 DEG C, solution becomes colourless by brown.Most
Afterwards, the seed-solution gentle agitation for adding 0.05 mL mixes 20s.The color of solution is drabon color after 28.0 DEG C of reaction 2h.Close
Into gold nanorods solution by 10000rpm centrifuge 10min, supernatant is abandoned, using 0.005 M cetyl trimethyl bromination
Ammonium salt solution will be precipitated to be disperseed again, cleaning once, most at last gold nanorods solution be dispersed in in initially isometric solution,
Preservation is stored at room temperature, to carry out the sign and experiment demand of next step.
（2）Thorn-like type platinum coats the synthetic method of gold nanorods
Into 5mL gold nanorods solution, 50 μM of sodium iodide, the rear 0.05M for adding 10mL cetyl trimethyl are added
After ammonium bromide, mix, be subsequently added 500 μ L 0.2 mM silver nitrate solution, mix, add 500 μ L 0.1M it is anti-
Bad hematic acid solution, is mixed, and 70 DEG C are incubated after 1 h, 480 μ L 0.1M hydrochloric acid and 440 μ L 2mM chloroplatinic acid are added, 70
After DEG C incubation reaction 4h, 6500rpm centrifuges 10 min, abandons supernatant, using 5 mM cetyl trimethylammonium bromide solution point
Dissipate, it is stand-by.
（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：Above-mentioned 10 nM prepared thorn-like type platinum is taken to coat
The mL of gold nanorods 2, add 30 μ L 1 mM dithiothreitol (DTT) solution, mix, shaking reaction 4 hours, using 6000rpm from
Heart 5min, abandons supernatant, disperses gold nanorods-dithiothreitol (DTT) with 2 mL 5 mM cetyl trimethylammonium bromide solution
Compound, it is 4 DEG C of preservations, standby.
2. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 2：Take above-mentioned treated thorn-like type platinum cladding gold
The μ L of solution 300 of the compound of nanometer rods-dithiothreitol (DTT) add the DNA2 of 3 μ L 10 μM of sulfydryl modification, mix, 25
DEG C, shaking reaction 8h, 6000rpm centrifugation 5min, supernatant is removed, with 300 μ L 5 mM cetyl trimethylammonium bromide solution
Scattered thorn-like type platinum coats gold nanorods-dithiothreitol (DTT)-DNA2 compound, and using 5 mM cetyl trimethyl
Ammonium bromide solution is cleaned once, 4 DEG C of preservations, standby.
3. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 3：Take above-mentioned treated thorn-like type platinum cladding Jenner
The μ L of solution 300 of the compound of rice rod and dithiothreitol (DTT) add the DNA3 of 3 μ L 10 μM of sulfydryl modification, mix, 25
℃.Shaking reaction 8h, 6000rpm centrifugation 5min, removes supernatant, with 300 μ L 5 mM cetyl trimethylammonium bromide solution
The compound that thorn-like type platinum coats gold nanorods-dithiothreitol (DTT)-DNA3 is disperseed, and using 5 mM cetyl
Trimethylammonium bromide solution is cleaned once, 4 DEG C of preservations, standby.
（4）The preparation of the gold nanorod chiral dimer of the thorn-like platinum parcel of nucleic acid function
The thorn-like gold nanorods that the platinum for taking DNA2 the and DNA3 lateral orientations of the 50 above-mentioned preparations of μ L to modify is wrapped up are mixed, whirlpool
Whirlpool is mixed, and is added 0.5 μ L 10 μM of DNA1, whirlpool and is mixed, after incubation at room temperature 8h, by nucleic acid specificity hybridization reaction, makes
Obtain the gold nanorods formation side assembling dimer for the thorn-like platinum parcel that DNA2 and DNA3 are modified respectively.
The DNA damage of table one detection base sequence used
| Numbering | Sequence (5 ' -3 ') | Length |
| DNA1 | ACCGGAGGCC CATCCTCACC | 20 |
| DNA2 | SH-GGCCTCCGGT | 10 |
| DNA3 | GGTGAGGATG-SH | 10 |
（5）Advance damage of the biotoxin to DNA
μm ol/L of 10 μM of DNA1 and 10,5,1,0.1,0.01 aflatoxin B1 is mixed, room temperature reaction 2.5
H, 4 DEG C of refrigerators are placed on by the DNA of advance damage, standby.Simultaneously for the metabolite Aflatoxins M1 of aflatoxin B1
And aflatoxin M 2 is according to the step of aflatoxin B1 and identical concentration, and DNA1 carries out being incubated 2.5 h, by advance damage
DNA afterwards is placed on 4 DEG C of refrigerators, standby.
（6）Detection of the thorn-like gold nanorod chiral dimer of platinum parcel to DNA damage；
Using circular dichroism signal to aflatoxin B1 and its metabolite Aflatoxins M1 and aflatoxin M 2
Detected, draw molar ellipticity~aflatoxin B1, M1 and M2 concentration standard curve：Take the 50 above-mentioned preparations of μ L
The thorn-like gold nanorods of the platinum parcel of DNA2 and DNA3 lateral orientations modification are mixed, whirlpool is mixed, and are added by step 5 place
The DNA1 for the advance damage managed（0.5 μL）, whirlpool mix, incubation at room temperature 8h after, by nucleic acid specificity hybridization reaction so that
The thorn-like gold nanorods formation side assembling structure for the platinum parcel that DNA2 and DNA3 are modified respectively.
Due to the aflatoxin B1 of addition or the difference of its metabolite M1 and M2 concentration, aflatoxin and phase
The adduct of guanine formation aflatoxin-guanine in the DNA1 answered, and then make it that DNA2 and DNA3 can not be with DNA1
Specific nucleic acid recognizing hybridization 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
Play plasma circular dichroism signal and produce difference.The group under different aflatoxin concentration is determined using circular dichroism instrument
The molar ellipticity of product is filled, according to the molar ellipticity of the different aflatoxin concentration determined under 855 nm wavelength, is drawn
The standard curve of aflatoxin concentration.
Claims (1)
1. a kind of gold nanorod chiral dimer for preparing thorn-like platinum parcel is used for the method that DNA damage is detected, it is characterised in that
Step is：Thorn-like type platinum coats the synthesis of gold nanorods；The gold nanorods body structure surface oriented nuclei acid functionalization of thorn-like platinum parcel；
Build the gold nanorod chiral dimer of thorn-like platinum parcel；Advance damage of the biotoxin to DNA；The gold nanorods of thorn-like platinum parcel
Detection of the chiral dimer to DNA damage, concrete technology is：
（1）Thorn-like type platinum coats the synthetic method of gold nanorods
Into 5mL gold nanorods solution, 50 μM of sodium iodide, the rear 0.05M for adding 10mL cetyl trimethyl are added
After ammonium bromide, mix, be subsequently added 500 μ L 0.2 mM silver nitrate solution, mix, add 500 μ L 0.1M it is anti-
Bad hematic acid solution, is mixed, and 70 DEG C are incubated after 1 h, 480 μ L 0.1M hydrochloric acid and 440 μ L 2mM chloroplatinic acid are added, 70
After DEG C incubation reaction 4h, 6500rpm centrifuges 10 min, abandons supernatant, using 5 mM cetyl trimethylammonium bromide solution point
Dissipate, it is stand-by；
（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：Above-mentioned 10 nM prepared thorn-like type platinum is taken to coat
The mL of gold nanorods 2, adds 30 μ L 1 mM dithiothreitol (DTT) solution, mixes, shaking reaction 4h, is centrifuged using 6000rpm
5min, abandons supernatant, disperses answering for gold nanorods-dithiothreitol (DTT) with 2 mL 5 mM cetyl trimethylammonium bromide solution
Compound, it is 4 DEG C of preservations, standby；
2. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 2：Take above-mentioned treated thorn-like type platinum cladding gold nano
The μ L of solution 300 of the compound of rod-dithiothreitol (DTT) add the DNA2 of 3 μ L 10 μM of sulfydryl modification, mix, 25 DEG C, shake
Reaction 8h, 6000rpm centrifugation 5min is shaken, supernatant is removed, is disperseed with 300 μ L 5 mM cetyl trimethylammonium bromide solution
Thorn-like type platinum coats gold nanorods-dithiothreitol (DTT)-DNA2 compound, and using 5 mM cetyl trimethyl bromination
Ammonium salt solution is cleaned once, 4 DEG C of preservations, standby；
3. thorn-like type platinum coats gold nanorods lateral orientation modifying DNA 3：Take above-mentioned treated thorn-like type platinum cladding gold nano
The μ L of solution 300 of the compound of rod-dithiothreitol (DTT) add the DNA3 of 3 μ L 10 μM of sulfydryl modification, mix, 25 DEG C, shake
Reaction 8h, 6000rpm centrifugation 5min is shaken, supernatant is removed, with 300 μ L 5 mM cetyl trimethylammonium bromide solution to thorn
Shape type platinum cladding gold nanorods-dithiothreitol (DTT)-DNA3 compound is disperseed, and using 5 mM cetyl front three
Base ammonium bromide solution is cleaned once, 4 DEG C of preservations, standby；
（3）The preparation of the gold nanorod chiral dimer of the thorn-like platinum parcel of nucleic acid function
The gold nanorods that the thorn-like platinum for taking DNA2 the and DNA3 lateral orientations of the 50 above-mentioned preparations of μ L to modify is wrapped up are mixed, whirlpool
Whirlpool is mixed, and is added 0.5 μ L 10 μM of DNA1, whirlpool and is mixed, after incubation at room temperature 8h, by nucleic acid specificity hybridization reaction, makes
Obtain the gold nanorods formation side assembling dimer for the thorn-like platinum parcel that DNA2 and DNA3 are modified respectively；
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
μm ol/L of 10 μM of DNA1 and 10,5,1,0.1,0.01 aflatoxin B1 is mixed, and reacts at room temperature 2.5 h,
The DNA1 of advance damage is placed on 4 DEG C of refrigerators, it is standby；Simultaneously for the metabolite Aflatoxins M1 of aflatoxin B1
And aflatoxin M 2 is according to the step of aflatoxin B1 and identical concentration, and DNA1 carries out being incubated 2.5 h, by advance damage
DNA1 afterwards is placed on 4 DEG C of refrigerators, standby；
（5）Detection of the gold nanorod chiral dimer of thorn-like platinum parcel to DNA damage
Using circular dichroism signal to aflatoxin B1 and its metabolite Aflatoxins M1 and aflatoxin M 2
Detected, draw molar ellipticity~aflatoxin B1, M1 and M2 concentration standard curve：Take the 50 above-mentioned preparations of μ L
The gold nanorods of the thorn-like platinum parcel of DNA2 and DNA3 lateral orientations modification are mixed, whirlpool is mixed, and are added by step 4 place
The μ L of DNA1 0.5 for the advance damage managed, whirlpool is mixed, after incubation at room temperature 8h, by nucleic acid specificity hybridization reaction so that
The gold nanorods formation side assembling structure for the thorn-like platinum parcel that DNA2 and DNA3 are modified respectively；
Due to the aflatoxin B1 of addition or the difference of its metabolite M1 and M2 concentration, aflatoxin and corresponding
DNA1 in guanine formation aflatoxin-guanine adduct, and then cause DNA2 and DNA3 can not enter with DNA1
The specific nucleic acid recognizing hybridization of row, 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 produces difference, and the assembling under different aflatoxin concentration is determined using circular dichroism instrument
The molar ellipticity of product, according to the molar ellipticity of the different aflatoxin concentration determined under 855 nm wavelength, draws yellow
The standard curve of aspertoxin concentration.
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