CN106501203A - A kind of insulin human detection method based on aptamer - Google Patents
A kind of insulin human detection method based on aptamer Download PDFInfo
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- CN106501203A CN106501203A CN201610880554.4A CN201610880554A CN106501203A CN 106501203 A CN106501203 A CN 106501203A CN 201610880554 A CN201610880554 A CN 201610880554A CN 106501203 A CN106501203 A CN 106501203A
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- YAJCHEVQCOHZDC-QMMNLEPNSA-N actrapid Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3N=CNC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@H](C)CC)[C@H](C)CC)[C@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C(N)=O)C1=CNC=N1 YAJCHEVQCOHZDC-QMMNLEPNSA-N 0.000 title claims abstract description 68
- 229950004152 insulin human Drugs 0.000 title claims abstract description 68
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title claims abstract description 23
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 51
- 239000010931 gold Substances 0.000 claims abstract description 51
- 229910052737 gold Inorganic materials 0.000 claims abstract description 50
- 238000010521 absorption reaction Methods 0.000 claims abstract description 26
- 239000000523 sample Substances 0.000 claims abstract description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 14
- 230000002776 aggregation Effects 0.000 claims abstract description 13
- 238000004220 aggregation Methods 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 238000000825 ultraviolet detection Methods 0.000 claims abstract description 8
- 239000011780 sodium chloride Substances 0.000 claims abstract description 7
- 239000012467 final product Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 20
- 238000009835 boiling Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- 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 4
- 210000004153 islets of langerhan Anatomy 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 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 claims description 2
- NMWONDKHBZEDMY-UHFFFAOYSA-H [Au](Cl)(Cl)Cl.C(CC(O)(C(=O)[O-])CC(=O)[O-])(=O)[O-].[Na+].[Na+].[Na+] Chemical compound [Au](Cl)(Cl)Cl.C(CC(O)(C(=O)[O-])CC(=O)[O-])(=O)[O-].[Na+].[Na+].[Na+] NMWONDKHBZEDMY-UHFFFAOYSA-H 0.000 claims description 2
- 238000013019 agitation Methods 0.000 claims description 2
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims description 2
- 239000000376 reactant Substances 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims 3
- 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 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 11
- 238000012986 modification Methods 0.000 abstract description 3
- 230000004048 modification Effects 0.000 abstract description 3
- 238000004737 colorimetric analysis Methods 0.000 abstract description 2
- 239000012295 chemical reaction liquid Substances 0.000 abstract 1
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 11
- 239000008280 blood Substances 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 229940125396 insulin Drugs 0.000 description 6
- 102000004877 Insulin Human genes 0.000 description 5
- 108090001061 Insulin Proteins 0.000 description 5
- 238000003756 stirring Methods 0.000 description 4
- 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 3
- 239000008103 glucose Substances 0.000 description 3
- 210000000496 pancreas Anatomy 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 201000001421 hyperglycemia Diseases 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108091081406 G-quadruplex Proteins 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- RJHLTVSLYWWTEF-UHFFFAOYSA-K gold trichloride Chemical class Cl[Au](Cl)Cl RJHLTVSLYWWTEF-UHFFFAOYSA-K 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 206010022498 insulinoma Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 208000021255 pancreatic insulinoma Diseases 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 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
- G01N21/17—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
-
- 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
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of insulin human detection method based on aptamer, the method is first to synthesize nanometer gold, in nanometer gold surface modification insulin human aptamers, obtains probe；By a series of insulin human of variable concentrations first respectively with the probe reaction, react with NaCl solution respectively again, again by this series reaction liquid by ultraviolet detection nanometer gold state of aggregation and the uv absorption peak value of dispersed, set up the standard curve of the corresponding nanometer gold state of aggregation uv absorption peak value of insulin human concentration value and nanometer gold dispersed uv absorption peak value ratio, prepare liquid is detected and establishing criteria curve is analyzed, obtained final product the actual concentrations of insulin human in prepare liquid；The method realizes quick detection to insulin human using colorimetry, and have the advantages that good stability, low cost, simple to operate.
Description
Technical field
The present invention relates to a kind of method of detection insulin human, more particularly to a kind of based on aptamer quick detection people's pancreas
The method of island element；Belong to biological nano field.
Background technology
Insulin is a kind of proteohormone of islet cells secrete in pancreas, and the glucose in blood can be promoted to enter
The histiocytes such as liver, muscle and fat, synthesis glycogen or are transformed into other nutrient substance and are stored up in the cell；Can promote again
Glucose oxidation Decomposition releases energy, and utilizes for body, is the major hormone for maintaining blood glucose in normal level.Insulin secretion is not
When foot or shortage, hyperglycemia, even diabetes can be caused.Insulin human is also the mark as insulinoma and wound simultaneously
Thing.Accordingly, it is capable in no Accurate Determining blood insulin concentration, to early diagnosiss of hyperglycemia and diabetes, clinical and basic
Research has important value.Insulin human detection method includes immunoassay, chromatography, electrochemical methods and fluorescence point
Analysis method, this several method are cumbersome, take, high cost, it is difficult to be applied to live detection immediately.Therefore it is quick that a kind of energy is invented
The content of insulin in detection human blood, and susceptiveness is high, it is easy to operate, and the method for energy specific recognition insulin human just shows
Obtain particularly important.
Content of the invention
For the defect that prior art is present, the purpose of the present invention is to be that offer is a kind of simple to operate, quick, inexpensive
The method by colorimetric determination insulin human.
In order to realize above-mentioned technical purpose, the invention provides a kind of insulin human detection method based on aptamer,
The method is comprised the following steps：
1) first pass through reduction of sodium citrate gold chloride and prepare nanometer gold；
2) again insulin human aptamers are modified in the nanometer gold surface, obtains probe；
3) by a series of insulin human titer of variable concentrations first respectively with the probe reaction, then molten with NaCl respectively
Liquid reacts, and each gained reactant liquor by ultraviolet detection nanometer gold state of aggregation and the uv absorption peak value of dispersed, sets up people respectively
The corresponding nanometer gold state of aggregation uv absorption peak value of insulin concentration value and the standard of nanometer gold dispersed uv absorption peak value ratio
Curve；
4) prepare liquid containing insulin human is passed through ultraviolet detection by 3) step, and establishing criteria curve is analyzed, i.e.,
Obtain the concentration of insulin human in prepare liquid.
Technical scheme, be mainly based upon nanometer gold in dispersity when, have its uniqueness in 518nm or so
Absworption peak, and when nanometer gold is assembled, there is meeting red shift in the absworption peak of 518nm or so in which, and can occur in 685nm or so new
Absworption peak, the size at its peak represents the degree of aggregation, while metachromasia can occur, (solution colour also can therewith by claret
Become au bleu, discernable by eye can be passed through).And technical scheme, insulin human aptamers are adsorbed on nanometer gold
Probe is constituted, in the presence of insulin human, G- tetrades knot can be formed with the fit combination of the insulin human of absorption on nanometer gold
Structure so that the salt tolerance of nanometer gold reduces, assembles nanometer gold, therefore can pass through nanometer gold aggregation in ultraviolet determination prepare liquid
The ratio of state 685nm or so absorption peak and nanometer gold 518nm or so absorption peak, can be with the concentration of real-time characterization mark.
Preferred scheme, nanometer gold size are 13nm～15nm.
More preferably scheme, the preparation process of described nanometer gold is：The preparation process of the nanometer gold is：By concentration it is
After 0.005～0.015wt% chlorauric acid solutions are heated to boiling, the sodium citrate solution that concentration is 1.5～2.5wt% is added to enter
Row reaction, solution are changed into claret again from the colourless purple that is changed into, and keep boiling 15～20 minutes, stirring in the state of claret
Room temperature is cooled under the conditions of mixing, nanometer gold is obtained final product；Wherein, gold chloride is 1: 3～5 with the mass ratio of sodium citrate.By preferred
Scheme can obtain the nanometer gold of uniform particle diameter.
Preferred scheme, is reacted fit for insulin human with nanometer gold at room temperature, obtains probe.More preferably it is：By people
Insulin is fit with nanometer gold in the ratio of 50～80nM: 2～5nM, reacts 10～15 minutes, obtain at a temperature of 25 DEG C～40 DEG C
Arrive probe.In preferred technical scheme, the aptamer modified amount of the insulin human of detecting probe surface is easy to regulate and control.Simple adjustment can be passed through
Insulin human adaptation bulk concentration during incubation, and fit and nanometer gold reaction ratio, realize fit in nanometer golden watch
The binding capacity in face, it is easy to make the fit binding capacity in nanometer gold surface reach maximization.
Preferred scheme, is 50pM, 250pM, 500pM, 1nM, 10nM, 50nM, 100nM, 150nM and 200nM by concentration
Insulin human titer respectively with 15～20min of probe reaction, then respectively with the NaCl solution of 50～100mM reaction 10～
After 15min, the uv absorption peak value of nanometer gold state of aggregation and dispersed in ultraviolet detection insulin human standard solution is respectively adopted,
And set up the corresponding nanometer gold state of aggregation uv absorption peak value of insulin human concentration value and nanometer gold dispersed uv absorption peak value ratio
The standard curve of rate.
Preferred scheme, insulin human aptamers are the aptamers of insulin human specific binding in blood.
The method of the present invention acts on corresponding insulin human using insulin human aptamers, with higher specificity
And discernment, the sensitivity of detection is higher.
The insulin human aptamers adopted in the present invention program can be obtained by existing conventional method screening technique, or
Person directly passes through to be commercially available, such as raw work biological engineering Shanghai (share) company limited.
The insulin human aptamers adopted in the present invention program are mainly incorporated into system by electrostatic force absorption without any modification
Standby nanometer gold surface.
Hinge structure, the Advantageous Effects that technical solution of the present invention is brought：
1) by naked eyes, change of the technical scheme according to color, directly can substantially judge that insulin human contains
Amount number, insulin human concentration can accurately be determined using colorimetry, reach the purpose of quick detection insulin human.
2) probe that technical scheme builds uses the corresponding aptamers of insulin human in blood, not dry
Material is disturbed, test limit is low, with specific recognition, detection sensitivity is higher.
3) preparation of technical scheme probe and, operation simple, reproducible for insulin human detection process
Simply.Probe is only with a kind of nucleic acid molecules chain, and without ornamental equivalent, there is the modification such as sulfydryl, signaling molecule relative to using
Fit for, cost is very low.
4) probe of structure of the invention is the complex of nanometer gold and aptamers, fit direct seizure object, therefore
Can be used directly to detect mark.
5) technical scheme acts on insulin human with insulin human aptamers (aptamer), while basis
The ultraviolet curve of solution analyzing the content of insulin human in blood, with sensitivity high, detection limit is low, high specificity spy
Point.
6) the fit sequence that technical solution of the present invention is adopted is variable, can be generalized to detection corresponding after fit replacing sequence
Other marks, such as polypeptide, DNA, RNA so that sensor can be more widely used, with certain universality.
Description of the drawings
【Fig. 1】Principle schematic for present invention detection insulin human；
【Fig. 2】For detecting the ultraviolet figure of variable concentrations mark；
【Fig. 3】It is that the corresponding nanometer gold state of aggregation purple of insulin human concentration value is set up by ultraviolet detection insulin human titer
Outer absorption peak and nanometer gold dispersed uv absorption peak value ratio (A685/A518) standard curve.
Specific embodiment
Following examples are intended to further illustrate present invention, rather than limit the protection model of the claims in the present invention
Enclose.
Embodiment 1
(1) prepared by nanometer gold, and its nanometer gold prepares probe with fit combination, and step is as follows：
First by 0.01% gold chlorides of 100mL under agitation, it is heated to seething with excitement, then adds in the rapid solution toward boiling
Enter 2% sodium citrate solutions of 2mL, continue stirring and be allowed to mix homogeneously, now stirring should not be excessively violent, in case mixing is uneven
Even, while maintenance fluidized state, when color from colourless be changed into purple and be changed into claret again when, start timing, continue heating
15min, then from oil bath pan takes out solution, places stirring on magnetic stirring apparatuss and is cooled to room temperature, then using ultraviolet point
Light photometer and particle size analyzer carry out the sign of nano-Au solution particle size and degree of scatter.
(2) Criterion curve：Variable concentrations (0.05,0.25,0.5,1,10,50,100,150,200nM) are prepared respectively
Insulin human, be sequentially added in the probe solution of the nanometer gold and fit composition for building above, due to probe solution
In aptamers be corresponding insulin human aptamer, it can form stable G- tetrad structures, meeting with insulin human
Compete fit for the insulin human adsorbed on nanometer gold.Amount due to adding insulin human is different, the G- tetrades of formation
Amount also different, will be also different for fit for the insulin human adsorbed on the nanometer gold amount for competing, when add 70mM NaCl when,
Nanometer gold can be made certain coagulation, so as to whole solution can assume different colors, quickly people's pancreas can be judged by naked eyes
The substantially content of island element, then ultraviolet absorption curve is surveyed, the ratio of absorption peak strength at the absorption peak strength of 685nm and 518nm
Can be different, such that it is able to accurate quantitative analysis insulin human.
(3) the quantitative analyses step to insulin human is as follows：When for detecting actual sample, can be by using actual sample
Replace standard solution, carry out above-mentioned same operation step, calculate uv absorption peak ratio, can be obtained according to linear standard curve
Go out the corresponding concentration of sample.
As can be seen from Figure 2 as the amount of the insulin human for adding increases, absorb at absorption peak and 518nm at 685nm
The ratio of peak value gradually increases.It is also seen that states of the Au-aptamer-insulin under 50～100mM NaCl, corresponding
The size of absworption peak in ultraviolet-visible absorption spectroscopy.
Insulin human concentration is bigger in certain concentration range as can be seen from Figure 3, its A685With A518Ratio more next
Bigger, can tend to balance after reaching a certain concentration, can be seen that from illustration：In low strength range, there is good its linearly to close
System.
Claims (6)
1. a kind of insulin human detection method based on aptamer, it is characterised in that：Comprise the following steps：
1) first pass through reduction of sodium citrate gold chloride and prepare nanometer gold；
2) again insulin human aptamers are modified in the nanometer gold surface, obtains probe；
3) by a series of insulin human titer of variable concentrations first respectively with the probe reaction, then anti-with NaCl solution respectively
Should, each gained reactant liquor by ultraviolet detection nanometer gold state of aggregation and the uv absorption peak value of dispersed, sets up people's islets of langerhans respectively
The corresponding nanometer gold state of aggregation uv absorption peak value of plain concentration value and the standard curve of nanometer gold dispersed uv absorption peak value ratio；
4) prepare liquid containing insulin human is passed through ultraviolet detection by 3) step, and establishing criteria curve is analyzed, and obtains final product and treats
Survey the concentration of insulin human in liquid.
2. the insulin human detection method based on aptamer according to claim 1, it is characterised in that：Described nanometer
Golden size is 13nm～15nm.
3. the insulin human detection method based on aptamer according to claim 1 and 2, it is characterised in that：Described receive
The preparation process of meter Jin is：Concentration is heated to after boiling for 0.005～0.015wt% chlorauric acid solutions, it is 1.5 to add concentration
The sodium citrate solution of～2.5wt% is reacted, and solution is changed into claret again from the colourless purple that is changed into, in the state of claret
Lower holding boiling 15～20 minutes, then it is cooled to room temperature under agitation, obtain final product nanometer gold；Wherein, gold chloride and citric acid
The mass ratio of sodium is 1:3～5.
4. the insulin human detection method based on aptamer according to claim 1, it is characterised in that：By insulin human
Fit reacted with nanometer gold at room temperature, obtain probe.
5. the insulin human detection method based on aptamer according to claim 4, it is characterised in that：Insulin human is fitted
Body is 50～80nM with the reaction ratio of nanometer gold:2～5nM.
6. the insulin human detection method based on aptamer according to claim 1, it is characterised in that：By concentration it is
The insulin human titer of 50pM, 250pM, 500pM, 1nM, 10nM, 50nM, 100nM, 150nM and 200nM is anti-with probe respectively
15～20min is answered, then is reacted after 10～15min with the NaCl solution of 50～100mM respectively, ultraviolet detection people's islets of langerhans is respectively adopted
The uv absorption peak value of nanometer gold state of aggregation and dispersed in plain standard solution, and set up the corresponding nanometer gold of insulin human concentration value
State of aggregation uv absorption peak value and the standard curve of nanometer gold dispersed uv absorption peak value ratio.
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