CN105911047A - Method for detecting cholesterol based on gold-silver core-shell nanoparticle colourimetry - Google Patents
Method for detecting cholesterol based on gold-silver core-shell nanoparticle colourimetry Download PDFInfo
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- CN105911047A CN105911047A CN201610200535.2A CN201610200535A CN105911047A CN 105911047 A CN105911047 A CN 105911047A CN 201610200535 A CN201610200535 A CN 201610200535A CN 105911047 A CN105911047 A CN 105911047A
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- cholesterol
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 title claims abstract description 132
- 235000012000 cholesterol Nutrition 0.000 title claims abstract description 63
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 38
- 239000011258 core-shell material Substances 0.000 title abstract description 5
- PQTCMBYFWMFIGM-UHFFFAOYSA-N gold silver Chemical group [Ag].[Au] PQTCMBYFWMFIGM-UHFFFAOYSA-N 0.000 title abstract description 5
- 238000004737 colorimetric analysis Methods 0.000 title abstract 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000006243 chemical reaction Methods 0.000 claims abstract description 19
- 108010089254 Cholesterol oxidase Proteins 0.000 claims abstract description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000001301 oxygen Substances 0.000 claims abstract description 10
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 10
- 239000003054 catalyst Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 44
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 36
- 239000010931 gold Substances 0.000 claims description 36
- 229910052737 gold Inorganic materials 0.000 claims description 36
- 239000002245 particle Substances 0.000 claims description 33
- PLIKAWJENQZMHA-UHFFFAOYSA-N 4-aminophenol Chemical compound NC1=CC=C(O)C=C1 PLIKAWJENQZMHA-UHFFFAOYSA-N 0.000 claims description 26
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(I) nitrate Inorganic materials [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 19
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 13
- 229910052709 silver Inorganic materials 0.000 claims description 11
- 239000004332 silver Substances 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 8
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 8
- 239000012498 ultrapure water Substances 0.000 claims description 8
- 238000009835 boiling Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000001509 sodium citrate Substances 0.000 claims description 6
- 108090000854 Oxidoreductases Proteins 0.000 claims description 5
- 102000004316 Oxidoreductases Human genes 0.000 claims description 5
- 239000002202 Polyethylene glycol Substances 0.000 claims description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 5
- 229920001223 polyethylene glycol Polymers 0.000 claims description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- NICDRCVJGXLKSF-UHFFFAOYSA-N nitric acid;trihydrochloride Chemical compound Cl.Cl.Cl.O[N+]([O-])=O NICDRCVJGXLKSF-UHFFFAOYSA-N 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 238000002371 ultraviolet--visible spectrum Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 19
- 108090000790 Enzymes Proteins 0.000 abstract description 10
- 102000004190 Enzymes Human genes 0.000 abstract description 10
- 239000002086 nanomaterial Substances 0.000 abstract description 8
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000005054 agglomeration Methods 0.000 abstract 1
- 230000002776 aggregation Effects 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 5
- 101710134784 Agnoprotein Proteins 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 3
- 210000000232 gallbladder Anatomy 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 235000005979 Citrus limon Nutrition 0.000 description 2
- 244000131522 Citrus pyriformis Species 0.000 description 2
- 229910004042 HAuCl4 Inorganic materials 0.000 description 2
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 150000002431 hydrogen Chemical class 0.000 description 2
- 206010025482 malaise Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 229930193551 sterin Natural products 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- CDAWCLOXVUBKRW-UHFFFAOYSA-N 2-aminophenol Chemical compound NC1=CC=CC=C1O CDAWCLOXVUBKRW-UHFFFAOYSA-N 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 229930003316 Vitamin D Natural products 0.000 description 1
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002343 gold Chemical group 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000002082 metal nanoparticle Substances 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 235000019166 vitamin D Nutrition 0.000 description 1
- 239000011710 vitamin D Substances 0.000 description 1
- 150000003710 vitamin D derivatives Chemical class 0.000 description 1
- 229940046008 vitamin d Drugs 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a method for detecting cholesterol based on gold-silver core-shell nanoparticle colourimetry. The method comprises the following steps: with cholesterol oxidase as a catalyst, reacting cholesterol with oxygen to produce hydrogen peroxide; reacting the produced hydrogen peroxide with a sliver nanometer shell layer on the surface of a gold-silver core-shell nanoparticle so as to etch the nanometer shell layer; and carrying out detection by using an ultraviolet-visible spectroscopic instrument. The method provided by the invention utilizes the characteristic that the surface plasma resonance (SPR) absorption peak intensity and color of the gold-silver core-shell nanoparticle substantially change before and after the reaction, so an extra developing agent is not needed, detection can be carried out without usage of a precise instrument, and complex and time-consuming enzyme immobilization is not needed; and thus, the defect of possible agglomeration of the nanomaterial in enzyme immobilization is avoided and the detection method is simplified. The method provided by the invention has the advantages of low cost, rapidness, easy, sensitivity and good specificity.
Description
Technical field
The invention belongs to biosensor technique field, be specifically related to a kind of based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination gallbladder
The method of sterin.
Background technology
Cholesterol plays an important role in cell.It is not only the important composition composition of cell membrane, is also synthesis bile
The raw material of acid, vitamin D and steroid hormone.Cholesterol is one of index of biochemistry detection.Cholesterol level should
Maintaining in normal level, cholesterol level is higher, cancer (colon cancer, pulmonary carcinoma, breast carcinoma, leukemia, child
The brain cancer, gastric cancer, hepatocarcinoma), diabetes, coronary heart disease, hypertension, the sickness rate of diabetes also rise.
When cholesterol level is on the low side, anemia, the sickness rate of syndrome of becoming thin uprise.
Noble metal nanometer material, especially gold silver nano material absorb due to its SPR in visible near infrared spectrum region
Unique tunable optical characteristics of causing and cause substantial amounts of concern.In in the past few decades, people have developed many
The method of nano material based on one-component in order to detect various analysis material such as glucose, cholesterol, uric acid, DNA,
Various enzymes and metal ion etc..Compared with the metal nanoparticle of one-component, containing double gold of two kinds of different metal elements
Belonging to nanoparticle can be by controlling the size of core, and the thickness of shell, chemical composition, the distance between particle etc. regulates and controls
The wavelength of gas ions resonance, the character such as intensity.Between additionally bimetal nano particles is because of its two kinds of different metal components
Mutually synergism and make plasma resonance character greatly strengthen.It is known that compared with golden nanometer particle, silver nanoparticle
Particle shows higher and sensitiveer SPR, but the Nano silver grain how preparing character highly stable remains one
Individual challenge, and people have been easy to prepare size and morphology controllable, stability and good biocompatibility now, and
SPR absorbs golden nanometer particle widely.In order to combine the advantage of golden nanometer particle and Nano silver grain, prepared by people
Go out gold/galactic nucleus-core/shell nanoparticles and be applied to various field.
In the method for various cholesterol detection, the macroscopic advantage that color detection method has, it is not required to costly
Instrument and the professional and technical personnel of specific training, in numerous analyzing detecting methods, there is competitive advantage.Major part
The method of existing color detection cholesterol utilizes the catalytic performance of nano material mostly.Specific as follows: first by cholesterol
Oxidase is fixed on the surface of nano material, then utilizes cholesterol oxidation enzyme catalysis cholesterol to generate peroxide with oxygen reaction
Change hydrogen, nano material catalyzing hydrogen peroxide oxidized color developing agent and there is chromogenic reaction.Whole detection process contain complexity and
Time-consuming enzyme fixation procedure, and enzyme fixation procedure may result in the reunion of nano material.Need in the process simultaneously
Add extra developer such as TMB, ABTS2-Deng, add the complexity of system undoubtedly.Therefore these shortcomings limit
The application of these methods.
Therefore develop a kind of simple, quickly, accurately, the new method of detection by quantitative cholesterol there is in terms of Clinical detection weight
Want meaning！
Summary of the invention
Goal of the invention: the problem existed for above-mentioned prior art, invention provides one and receives based on gold/silver core-shell structure copolymer
The method of rice corpuscles colorimetric determination cholesterol.The present invention acts on, for cholesterol oxidase, the peroxide that cholesterol produces
Change hydrogen reacts with the silver nanoparticle shell in gold/galactic nucleus-core/shell nanoparticles and makes it etch, and makes optical property significantly change,
Thus set up colorimetric determination cholesterol, it is simple that it has method, highly sensitive, accuracy good, is applicable to the body of complexity
The advantages such as system, and experimental result the most just can directly observe and without valuable large-scale instrument.
Technical scheme is as follows:
The invention provides a kind of method based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol, its feature exists
In, the method includes: make cholesterol generate hydrogen peroxide with oxygen reaction with cholesterol oxidase for catalyst；To generate
The silver nanoparticle shell on hydrogen peroxide and gold/galactic nucleus-core/shell nanoparticles surface react, etch silver nanoparticle shell；Utilize ultraviolet
-visible spectrophotometer detects.
A kind of based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol method the most excellent that the present invention proposes
The scheme is selected to be:
The synthesis step of described gold/galactic nucleus-core/shell nanoparticles includes: repaiied by sulfhydrylation methyl Polyethylene Glycol (SH-PEG)
The golden nanometer particle of decorations is dispersed in buffer solution, and adds AgNO3After mix homogeneously, add para-aminophenol
(p-AP) 10～40min, and are at room temperature reacted；Described buffer solution is DEA buffer solution；Wherein DEA
Buffer solution is containing 0.2-3mM MgSO4The DEA buffer solution that the concentration that pH is 8-11 is 550-650mM；Its
In, the concentration of described AgNO3 is 0.1～0.5M；The concentration of described para-aminophenol (p-AP) is 0.1～1mM.
The preparation process of the golden nanometer particle that described SH-PEG modifies includes: add SH-PEG in golden nanometer particle,
Being stirred continuously down and react 1～3h at room temperature, final solution, 12200～13200rpm, is centrifuged 20～30 at 2-10 DEG C
Min, redissolves in ultra-pure water after being centrifuged, and 2-10 DEG C of preservation obtains the golden nanometer particle that SH-PEG modifies.
The preparation process of described golden nanometer particle includes: soaked glass drying oven chloroazotic acid used in experiment, then with ultrapure
Water is rinsed well, dries stand-by；It is heated to the HAuCl4 solution that 30-70mL concentration is 0.5-3mM seething with excitement, and
Under fluidized state continuously stirred, add 1-10mL concentration be the sodium citrate solution of 30-50mM, continue keep boiling
10-30min；Remove thermal source, stir 10-30min, be cooled to room temperature, prepare golden nanometer particle.
A diameter of 5-30nm of described golden nanometer particle, wherein the diameter of golden nanometer particle is preferably 13nm.
The described step making cholesterol and oxygen reaction generate hydrogen peroxide for catalyst with cholesterol oxidase includes: to not
It is 1～2mg mL with the cholesterol solution of concentration adds concentration-1Cholesterol oxidase, incubation 0.3 at 30-45 DEG C
～1h, the volume ratio of described cholesterol solution and cholesterol oxidase solution is 3～1.
The described silver nanoparticle shell by the hydrogen peroxide of generation with gold/galactic nucleus-core/shell nanoparticles surface reacts and includes: by gold/
Galactic nucleus-core/shell nanoparticles solution joins and makes cholesterol generate peroxidating with oxygen reaction with cholesterol oxidase for catalyst
In the solution of hydrogen, and reaction 0.5～1h at room temperature.
The present invention reacts generation hydrogen peroxide, the mistake of generation by cholesterol and oxygen under cholesterol oxidation enzyme catalysis
Hydrogen oxide reacts with the silver nanoparticle shell on gold/galactic nucleus-core/shell nanoparticles surface, thus etches silver nanoparticle shell, causes SPR
Absorption peak strength is remarkably decreased, the character of the notable change of simultaneous solution colour, colorimetric determination cholesterol.
Beneficial effect: the present invention, relative to prior art, has the advantage that
(1) system of the present invention is simply not required to add extra developer.
(2) present invention utilizes gold/galactic nucleus core/shell nanoparticles rear notable variation characteristic of color before the reaction, it is not necessary to by
Precision instrument detects, and simplifies detection method, therefore can use colorimetric determination cholesterol.
(3) present invention need not complicated and time-consuming enzyme fixation procedure in whole detection process, it is to avoid enzyme fixation procedure
And cause the probability of the reunion of nano material.
(4) present invention has the advantage that simplicity, sensitivity and specificity are good.
(5) present invention is successfully used in the detection of actual sample, and accuracy is high.
Accompanying drawing explanation
Fig. 1 shows schematic diagram based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol.
Fig. 2 shows cholesterol detection experimental principle proof diagram.A line: the ultra-violet absorption spectrum of golden nanometer particle；B line:
AgNO is added in golden nanometer particle3The ultra-violet absorption spectrum of rear solution；C line: after adding p-AP in golden nanometer particle
The ultra-violet absorption spectrum of solution；D line: add AgNO in golden nanometer particle3With the ultra-violet absorption spectrum of solution after p-AP；
E line: add AgNO in golden nanometer particle3After p-AP, after reaction a period of time, then by this solution and cholesterol and
The ultra-violet absorption spectrum of solution after the mixing of cholesterol oxidation enzyme reaction solution；
Fig. 3 shows the ultraviolet spectrogram of detection by quantitative cholesterol.Fig. 3 A: in the presence of the cholesterol of variable concentrations,
The ultraviolet spectrogram (concentration of cholesterol: 0,0.3,5,25,50,100,200,300,400,600,800 μM) arrived；Figure
3B: uv absorption intensity and the concentration matched curve of cholesterol；The illustration of Fig. 3 B: between absorbance and cholesterol concentration
Linear relationship.
Detailed description of the invention
Below in conjunction with the accompanying drawings the present invention is further described.
The reagent used in this experiment and instrument:
Gold chloride (HAuCl4·4H2O), sodium citrate (Na3C6H5O7·2H2O), silver nitrate (AgNO3), right
Amino-phenol (p-AP), cholesterol oxidase (ChOx), cholesterol, diethanolamine (DEA), ultraviolet spectrometry light
Degree meter (Cary 100, Agilent, Singapore), transmission electron microscope (JEM-2010, Hitachi, Japan), mix whirlpool
Rotation instrument (IKA German), centrifuge (Eppendorf German), digital camera (DSC-W730, Sony, Japan).
Embodiment 1:
Method based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol, comprises the following steps:
The synthesis step of the golden nanometer particle that SH-PEG modifies: used all glass drying oven chloroazotic acid are soaked, then uses
Ultrapure water is clean, dries stand-by；By 30-70mL, such as the HAuCl that concentration is 1mM of 50mL4Molten
Liquid is heated to (the wherein HAuCl that seethes with excitement4The concentration range of solution also can solve the application skill to be solved at 0.5-3mM
Art problem), and continuously stirred under fluidized state, and addition 1-10mL, preferably 5mL concentration are the lemon of 38.8mM
Lemon acid sodium solution (in the inventive solutions, the concentration range of sodium citrate solution can be 30-50mM)；Continue
Keep boiling 10-30min, be preferably 15min；Remove thermal source, stirring 10-30min, preferably 15min；Cooling
To room temperature, prepare golden nanometer particle；Take solution of gold nanoparticles 1mL prepared, and be added thereto to 6mL,
The SH-PEG of 25mM, reacts 2h under being stirred continuously at room temperature, and final solution is at 13200rpm, 2-10 DEG C
Under centrifugal 20-30min (preferably 25min) under (preferably 4 DEG C), redissolve in ultra-pure water after centrifugal three times, 2-10 DEG C
Preserve (preferably 4 DEG C), obtain the golden nanometer particle that SH-PEG modifies.
Gold/galactic nucleus-core/shell nanoparticles synthesis step: the golden nanometer particle that SH-PEG modifies is dispersed in 550-650mM
(pH=9.8, wherein containing 1mM MgSO in 600mM DEA for (preferably 600mM) DEA buffer solution4)
In (in technical scheme, DEA buffer solution pH scope be 8-11, preferably pH be 9.8；MgSO can be predicted4
Concentration range is 0.2-3mM, preferably 1mM), and to be added thereto to 2 μ L concentration be 0.25M AgNO3(this
Invention AgNO3Concentration range is 0.1～0.5M, and preferred concentration is 0.25M) after mix homogeneously, add 93.75 μ L
Concentration is the p-AP of 0.667mM (p-AP concentration range of the present invention is 0.1～1mM, and preferred concentration is 0.667mM),
Final solution volume is 350 μ L, reacts 40min at room temperature, prepares gold/galactic nucleus-core/shell nanoparticles.
Cholesterol detection step: will be 1.5mg mL containing concentration-1Cholesterol oxidase solid with variable concentrations gallbladder respectively
The solution mixing of alcohol (0,0.3,5,25,50,100,200,300,400,600,800 μM), described cholesterol solution and gallbladder
The volume ratio of sterin oxidase solution be 2 (volume ratio scope 3～1, preferred volume ratio in technical solution of the present invention
It is 2), mixed volume is 150 μ L, after both are sufficiently mixed, reacts 30min in 37 DEG C of thermostat water baths
After, after 350 μ L gold/galactic nucleus-core/shell nanoparticles is joined above cholesterol oxidation enzyme catalysis cholesterol and oxygen reaction
In solution, react 30min at room temperature, after reaction terminates, go final solution to scan ultraviolet-visible spectrum.Analyze
Obtain ultraviolet absorpting spectrum, draw cholesterol linear graph.Experimental result see Fig. 3: cholesterol at 0.3 to 300 μMs in good
Good linear relationship, detection limit is 0.15 μM.Note: reaction cumulative volume is 500 μ L.
Embodiment 2
Method based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol, comprises the following steps:
The synthesis step of the golden nanometer particle that SH-PEG modifies: used all glass drying oven chloroazotic acid are soaked, then
Clean with ultrapure water, dry stand-by；It is the HAuCl of 1mM by 50mL concentration4Solution is heated to boiling, and
Under fluidized state continuously stirred, add 5mL concentration be the sodium citrate solution of 38.8mM, continue keep boiling 15
min；Remove thermal source, stir 15min, be cooled to room temperature, prepare golden nanometer particle；Take the Jenner prepared
Rice corpuscles solution 1mL, and add 6mL, concentration is the SH-PEG of 25mM, the most anti-under being stirred continuously
Answering 2h, final solution is at 13200rpm, and at 4 DEG C, centrifugal 25min, redissolves in ultra-pure water after centrifugal three times, 4 DEG C of guarantors
Deposit, obtain the golden nanometer particle that SH-PEG modifies.
Gold/galactic nucleus-core/shell nanoparticles synthesis step: the golden nanometer particle that SH-PEG modifies is dispersed in 600mM
(pH=9.8, wherein containing 1mM MgSO in the DEA of 600mM for DEA buffer solution4In), and add wherein
Enter 2 μ L, the AgNO of 0.25M3After mix homogeneously, add 31.25 μ L, the p-AP of 0.667mM, final solution
Volume is 350 μ L, reacts 40min at room temperature, can be prepared by gold/galactic nucleus-core/shell nanoparticles.
Cholesterol detection step: will be containing 1.5mg mL-1Cholesterol oxidase and variable concentrations cholesterol (0,0.09,0.5,
5,25,50,100,150,200,300 μMs) solution mixing, described cholesterol solution and the body of cholesterol oxidase solution
Long-pending ratio is 2, and mixed volume is 150 μ L, after being sufficiently mixed, reacts 30min in 37 DEG C of thermostat water baths
After, 350 μ L gold/galactic nucleus-core/shell nanoparticles is joined in above 150 μ L reactant liquors, reacts 30min at room temperature,
After reaction terminates, go final solution to scan ultraviolet-visible spectrum.Analysis obtains ultraviolet absorpting spectrum, draws cholesterol
Linear graph.Cholesterol is at 0.09 to 150 μMs in good linear relationship, and detection limit is 0.04 μM.Note: reaction is total
Volume is 500 μ L.
Above are only the preferred embodiment of the invention, be not restricted to the present invention.To those of ordinary skill in the art,
Change or the variation of other multi-forms can also be made on the basis of the above description.Here without also cannot be to all of
Embodiment illustrates.And the obvious change that thus scheme is extended out or variation are still in the protection of the present invention
Scope.
Claims (9)
1. a method based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol, it is characterised in that the method
Including: make cholesterol generate hydrogen peroxide with oxygen reaction with cholesterol oxidase for catalyst；The hydrogen peroxide that will generate
React with the silver nanoparticle shell on gold/galactic nucleus-core/shell nanoparticles surface, etch silver nanoparticle shell；Utilize ultraviolet-visible spectrum
Instrument detects.
A kind of method based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol the most according to claim 1,
It is characterized in that, the preparation method of described gold/galactic nucleus-core/shell nanoparticles includes: by polyethyleneglycol modified for sulfhydrylation methyl
Golden nanometer particle is dispersed in buffer solution, and adds AgNO3After mix homogeneously, add para-aminophenol, and in
10～40min are reacted under room temperature.
A kind of method based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol the most according to claim 2,
It is characterized in that, described buffer solution includes containing 0.2-3mM MgSO4The concentration that pH is 8-11 be
The DEA buffer of 550-650mM.
A kind of method based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol the most according to claim 2,
It is characterized in that, described AgNO3Concentration be 0.1～0.5M；The concentration of described para-aminophenol is 0.1～1mM.
5. according to the arbitrary described one of claim 2-4 based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol
Method, it is characterised in that the preparation method of the golden nanometer particle that described sulfhydrylation methyl is polyethyleneglycol modified includes: to
Adding sulfhydrylation methyl Polyethylene Glycol in golden nanometer particle, react 1～3h under being stirred continuously at room temperature, reaction terminates
After 12200～13200rpm, at 2-10 DEG C centrifugal 20～30min, centrifugal after redissolve in ultra-pure water, 2-10 DEG C of guarantor
Deposit, obtain the golden nanometer particle that sulfhydrylation methyl is polyethyleneglycol modified.
A kind of method based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol the most according to claim 5,
It is characterized in that, the preparation method of described golden nanometer particle includes: is soaked by glass drying oven chloroazotic acid, then rushes with ultra-pure water
Wash clean, dries stand-by；It is the HAuCl of 0.5-3mM by 30-70mL concentration4Solution is heated to boiling, and in boiling
Under state continuously stirred, add 1-10mL concentration be the sodium citrate solution of 30-50mM, continue keep boiling
10-30min；Remove thermal source, stir 10-30min, be cooled to room temperature, prepare golden nanometer particle.
A kind of method based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol the most according to claim 6,
It is characterized in that, a diameter of 5-30nm of described golden nanometer particle.
A kind of method based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol the most according to claim 1,
It is characterized in that, the described preparation step making cholesterol and oxygen reaction generate hydrogen peroxide for catalyst with cholesterol oxidase
Suddenly include: being separately added into concentration in the cholesterol solution of variable concentrations is 1～2mg mL-1Cholesterol oxidase, in
Incubation 0.3～1h at 30-45 DEG C；Described cholesterol solution is 3～1 with the volume ratio of cholesterol oxidase solution.
9. according to a kind of based on gold/galactic nucleus-core/shell nanoparticles colorimetric determination cholesterol the side described in claim 1 or 8
Method, it is characterised in that the described silver nanoparticle shell by the hydrogen peroxide generated with gold/galactic nucleus-core/shell nanoparticles surface reacts
Step include: gold/galactic nucleus-core/shell nanoparticles solution is joined and described makes cholesterol with cholesterol oxidase for catalyst
With in the solution that oxygen reaction generates hydrogen peroxide, and reaction 0.5～1h hour at room temperature.
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