CN104597021B - A kind of method of the gold nano cluster fluorescence probe quick detection DNA concentration based on glutathione functionalization - Google Patents
A kind of method of the gold nano cluster fluorescence probe quick detection DNA concentration based on glutathione functionalization Download PDFInfo
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- 239000010931 gold Substances 0.000 title claims abstract description 43
- 239000000523 sample Substances 0.000 title claims abstract description 35
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 title claims abstract description 34
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 229910052737 gold Inorganic materials 0.000 title claims abstract description 29
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 229960003180 glutathione Drugs 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 16
- 238000007306 functionalization reaction Methods 0.000 title claims abstract description 15
- 108010024636 Glutathione Proteins 0.000 title claims abstract description 14
- 229960004169 mitoxantrone hydrochloride Drugs 0.000 claims abstract description 12
- ZAHQPTJLOCWVPG-UHFFFAOYSA-N mitoxantrone dihydrochloride Chemical compound Cl.Cl.O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO ZAHQPTJLOCWVPG-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 20
- 238000010791 quenching Methods 0.000 claims description 9
- 230000000171 quenching effect Effects 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 244000309466 calf Species 0.000 claims description 3
- 230000005284 excitation Effects 0.000 claims description 3
- 210000001541 thymus gland Anatomy 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- 239000007853 buffer solution Substances 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 10
- 229940079593 drug Drugs 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 5
- 238000011275 oncology therapy Methods 0.000 abstract description 5
- 150000004056 anthraquinones Chemical class 0.000 abstract description 3
- 230000003993 interaction Effects 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 238000001917 fluorescence detection Methods 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- 235000003969 glutathione Nutrition 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 description 2
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Chemical compound C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000000695 excitation spectrum Methods 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 229910004042 HAuCl4 Inorganic materials 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 241000894007 species Species 0.000 description 1
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- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of method based on gold nano cluster fluorescence probe quick detection DNA concentration, comprise the following steps：Prepare the gold nano cluster of glutathione functionalization（GSH‑Au NCs）Fluorescent detection probe, the fluorescence probe can quantitative determination Anthraquinones cancer therapy drug mitoxantrone hydrochloride, the quantitative determination to DNA is then realized using the interaction of DNA and mitoxantrone hydrochloride.The fluorescent detection probe that the present invention is provided is easily prepared and preserves；Agents useful for same has no toxic side effect；Fluorescence detection method sensitivity of the invention is high, detection limit is low, the range of linearity is wide, it is easy, easy to operate the features such as.
Description
Technical field
It is fast based on gold nano cluster fluorescence probe the invention belongs to technical field of nano material application, more particularly to one kind
Speed, the new method of Sensitive Detection DNA concentration.
Background technology
In recent years, fluorescence metal nanocluster, especially gold nano cluster (Au NCs), visit as a kind of new fluorescence
Pin is extensively concerned.This has the characteristics such as small size, good water solubility, good biocompatibility, good light stability mainly due to it.So far
Untill the present, using the work that glutathione is templated synthesis fluorescence nano gold cluster fluoroscopic examination DNA, there is not been reported.
Detection DNA concentration, helps to study small-molecule substance（Particularly cancer therapy drug）Interaction between DNA,
Understanding genetic transcription, DNA replication dna, some diseases origin, illustrate the mechanism of action of cancer therapy drug, effective design dna
There is very important effect in targeted drug.At present, the method for existing detection DNA cuts both ways.Wherein, using wider
Two methods be based on to the electrochemical process of guanine/oxidation of drug signal [referring to：Rauf, S.; Gooding, J. J.;
Akhtar, K.; Ghauri, M. A.; Rahman, M.; Anwar,M. A.; Khalid,A. M. J. Pharm.
Biomed. Anal. 2005,37,205-217.] and fluorescence signal change based on organic dyestuff fluorescence method [referring to：Ni,
Y.; Lin, D.; Kokot, S. Talanta. 2005,65,1295–1302.].However, both approaches are still present one
A little limitation, such as electrochemical research is generally carried out in acid condition, and organic dyestuff has quick photobleaching phenomenon.
Therefore need that a kind of energy is quick, sensitive effective detection DNA concentration new method badly.
The content of the invention
The purpose of the present invention is directed to the shortcoming of prior art presence, there is provided a kind of quick, sensitive quantitative determination DNA concentration
New method.
To achieve these goals, technical scheme is as follows：
A kind of method based on gold nano cluster fluorescence probe quick detection DNA concentration, comprises the following steps：
Step one：By the gold nano cluster of glutathione functionalization（GSH-Au NCs）The aqueous solution and the phosphorus that pH values are 7.0
The isometric ratio of phthalate buffer is well mixed, and takes mixed solution in suprasil cuvette；
Step 2：A series of mitoxantrone hydrochloride solution of various concentrations is added, various concentrations hydrochloric acid rice support is detected respectively
Influence of the anthraquinone to gold nano cluster fluorescence probe signal, draws mitoxantrone hydrochloride concentration linear with the fluorescence intensity of probe
Relation curve；
Step 3：Add a series of DNA of various concentrations molten in AuNCs-MTX systems after step 2 fluorescent quenching
Liquid, influences of the detection various concentrations DNA to AuNCs-MTX system fluorescence probe signals, draws the glimmering of AuNCs-MTX system probes
Luminous intensity with DNA concentration increased linear relationship curve；
Step 4：In the AuNCs-MTX systems that DNA solution to be measured is added to after fluorescent quenching, detect it to fluorescence
The influence value of the AuNCs-MTX system fluorescence intensities after quenching, the linear relationship curve that correspondence step 3 is obtained, calculates to be measured
DNA solution concentration.
In 2.0 ~ 18 μM of concentration ranges, hydrochloric acid rice holds in the palm anthracene to the gold nano cluster of the step 2 GSH-PX activity functionalization
Quinone concentration shows good linear relationship with the fluorescence intensity of probe.
Fluorometric investigation condition in the step 2 is：The slit width of XRF is set as 20nm, and setting is excited
The a length of 392nm of light wave, the emission peak fluorescence intensity at detection 570nm wavelength.
DNA concentration is in 2.0 ~ 32 μ g mL in the step 3-1In the range of, DNA concentration shows with the fluorescence intensity of probe
Good linear relationship.
Interaction based on Anthraquinones cancer therapy drug Yu DNA, this experiment also can be used to detect other species DNA concentrations,
DNA includes calf thymus DNA and milt DNA.
Principle of the invention is：Based on energy of photoelectron transfer（PIET）Mechanism, MTX absorption makes it on nanocluster surface
Fluorescent quenching, DNA is combined with MTX makes it depart from from nanocluster surface, and fluorescence recovers, using MTX as a kind of model drug,
Because it can effectively be combined with DNA, for detecting DNA concentration.
Advantageous Effects acquired by the present invention are：Fluorescent detection probe is easily prepared and preserves；The equal nothing of agents useful for same
Poison.
Brief description of the drawings
The fluorescence gold nano cluster of Fig. 1 glutathione functionalization（GSH-Au NCs）Excitation and emission spectra figure.
Influence figures of the MTX of Fig. 2 various concentrations to the fluorescence intensity of GSH-Au NCs.
The linear relationship chart of Fig. 3 GSH-Au NCs fluorescence intensities and MTX concentration.
Influence figures of the DNA of Fig. 4 various concentrations to the fluorescence intensity of Au NCs-MTX.
The linear relationship chart of Fig. 5 Au NCs-MTX systems fluorescence intensities and DNA concentration.
Specific embodiment
The synthesizing glutathion of embodiment 1.（GSH）The fluorescence gold nano cluster probe of functionalization, fluoroscopic examination DNA concentration.
1st, the gold nano cluster of synthesizing glutathion functionalization（GSH-Au NCs）Fluorescence probe：
The gold nano cluster of glutathione functionalization（GSH-Au NCs）It is by glutathione（GSH）Reduction gold chloride
（HAuCl4•4H20）Synthesis.The glutathione solution and chlorauric acid solution of 5mM are respectively configured, are kept in dark place at 4 DEG C standby.
It is stirred vigorously down, by configured chlorauric acid solution（10mL, 5mM）It is added to glutathione solution（10mL, 5mM）In, this is mixed
Close liquid successive reaction 4 days under room temperature, illumination condition.Solution colour is changed into dark-brown from colourless, eventually becomes light yellow.Should
Reaction solution is centrifuged（15000r/min）20 minutes, take supernatant liquor.Obtain the gold nano cluster of glutathione functionalization（GSH-Au
NCs）The aqueous solution, its concentration is 2.5mM (being calculated with the quantity of gold atom), and solution is saved backup under conditions of 4 DEG C.Paddy Guang
The gold nano cluster of sweet peptide functionalization（GSH-Au NCs）Excited at 392nm, maximum emission peak is obtained at 570nm.
2nd, cancer therapy drug --- mitoxantrone hydrochloride is detected（MTX）
First by the gold nano cluster of glutathione functionalization（GSH-Au NCs）The aqueous solution and phosphate buffer（pH
7.0）By 1:1 ratio is well mixed, and is configured to the solution of 1.25mM, takes 3ml solution in the quartz colorimetric utensil of four sides printing opacity.
A series of solution of concentration is subsequently adding, detects various concentrations mitoxantrone hydrochloride to gold nano cluster fluorescence probe signal respectively
Influence（Solution final concentration is respectively 2.0,4.0,6.0,8.0,10,12,14,16,18 μM）.The slit width of XRF
It is set as 20nm, a length of 392nm of excitation light wave, the emission peak fluorescence intensity at detection 570nm wavelength is set.Result display is added
After medicine, there is Fluorescence-quenching, and as drug concentration increases, fluorescence probe signal intensity weakens therewith, in 2.0 ~ 18 μ
In M concentration ranges, mitoxantrone hydrochloride concentration shows good linear relationship with the fluorescence intensity of probe, and its detection is limited to
0.1μM。
3rd, calf thymus DNA is detected（ctDNA）
The gold nano cluster of glutathione functionalization（GSH-Au NCs）Fluorescence be quenched by mitoxantrone hydrochloride after can
Recovered with DNA, therefore can be used to detect DNA.Add a series of differences dense in AuNCs-MTX systems after above-mentioned fluorescent quenching
The DNA solution of degree.Influences of the detection various concentrations DNA to AuNCs-MTX system fluorescence probe signals respectively（Solution final concentration point
Wei not 2.0,4.0,6.0,8.0,10,12,14,16,18,20,22,24,26,28,30,32 μ g mL-1）.XRF it is narrow
Seam width is set as 20nm, and setting excitation spectrum is 392nm, the emission peak fluorescence intensity at detection 570nm wavelength.Result shows
After adding DNA, AuNCs-MTX systems fluorescence probe recovers.And as DNA concentration increases, fluorescence probe signal intensity increases therewith
By force, in 2.0 ~ 32 μ g mL-1In concentration range, DNA concentration shows good linear relationship with the fluorescence intensity of probe, its inspection
Rising limit is 0.6 μ g mL-1.Test as a comparison, studied using the gold nano cluster of bovine serum albumin(BSA) functionalization, as a result
Show that its fluorescence intensity and MTX show good linear relationship, but fluorescence recovery effects are not obvious after adding DNA, therefore can not
For the detection to DNA.
Claims (5)
1. a kind of method based on gold nano cluster fluorescence probe quick detection DNA concentration, comprises the following steps：
Step one：By the gold nano cluster of glutathione functionalization（GSH-Au NCs）The aqueous solution and the phosphate that pH values are 7.0
The isometric ratio of buffer solution is well mixed, and takes mixed solution in suprasil cuvette；
Step 2：A series of mitoxantrone hydrochloride solution of various concentrations is added, various concentrations mitoxantrone hydrochloride is detected respectively
Influence to gold nano cluster fluorescence probe signal, draws the linear relationship of mitoxantrone hydrochloride concentration and the fluorescence intensity of probe
Curve；
Step 3：A series of DNA solution of various concentrations is added in AuNCs-MTX systems after step 2 fluorescent quenching, is examined
Influences of the various concentrations DNA to AuNCs-MTX system fluorescence probe signals is surveyed, the fluorescence for drawing AuNCs-MTX system probes is strong
Spend with the increased linear relationship curve of DNA concentration；
Step 4：In the AuNCs-MTX systems that DNA solution to be measured is added to after fluorescent quenching, detect it to fluorescent quenching
The influence value of AuNCs-MTX system fluorescence intensities afterwards, the linear relationship curve that correspondence step 3 is obtained, calculates to be measured
The concentration of DNA solution.
2. the method based on gold nano cluster fluorescence probe quick detection DNA concentration according to claim 1, its feature exists
Mitoxantrone hydrochloride shows good linear pass in 2.0-18 μM of concentration range with the fluorescence intensity of probe in step 2
System.
3. the method based on gold nano cluster fluorescence probe quick detection DNA concentration according to claim 1, its feature exists
Test condition in step 2 is：The slit width of XRF is set as 20nm, sets a length of 392nm of excitation light wave,
Emission peak fluorescence intensity at detection 570nm wavelength.
4. the method based on gold nano cluster fluorescence probe quick detection DNA concentration according to claim 1, its feature exists
DNA concentration is in 2.0 ~ 32 μ g mL in step 3-1In the range of, DNA concentration and the fluorescence intensity of probe show good linear
Relation.
5. according to any one of claim 1-4 claim based on gold nano cluster fluorescence probe quick detection DNA
The method of concentration, DNA includes calf thymus DNA and milt DNA.
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| CN105572087A (en) * | 2016-01-11 | 2016-05-11 | 东北师范大学 | Mercury ion colorimetric detection test paper capable of being cyclically used and application method thereof |
| CN105772740B (en) * | 2016-03-17 | 2018-03-27 | 湖北大学 | The preparation method and applications of gold nanoclusters |
| CN106010512B (en) * | 2016-05-11 | 2018-10-02 | 中国石油大学(华东) | One kind being used for Hg2+The SiO of detection2/ Au nanocomposites and preparation method thereof |
| CN107014787B (en) * | 2016-09-30 | 2020-11-10 | 盐城工学院 | Application of glutathione template gold nanocluster in detection of cysteine and lysine |
| CN112391158A (en) * | 2019-11-08 | 2021-02-23 | 重庆科技学院 | Multicolor fluorescent probe for detecting early markers of cardiac injury and preparation and use methods thereof |
| CN111269715B (en) * | 2020-02-13 | 2022-11-29 | 中国科学院合肥物质科学研究院 | Ratiometric fluorescent probe and application thereof in visual detection of glutathione |
| CN111568922B (en) * | 2020-05-09 | 2022-05-03 | 深圳深见医药科技有限公司 | Treatment of atypical antipsychotic-induced adverse reactions |
| CN113480999B (en) * | 2021-07-02 | 2023-03-10 | 澳门大学 | Fluorescent metal nanocluster and preparation method and application thereof |
| CN114235768A (en) * | 2021-12-18 | 2022-03-25 | 中国科学院新疆理化技术研究所 | Method for rapidly detecting TNT (trinitrotoluene) through fluorescence |
| CN115386367A (en) * | 2022-08-17 | 2022-11-25 | 合江县公共检验检测中心 | Fluorescent paper sensor |
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| CN103575715A (en) * | 2013-11-06 | 2014-02-12 | 盐城工学院 | Method for detecting mitoxantrone based on luminous gold nanocluster |
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