CN106546749B - Method of protein is detected based on nanogold DNA composites - Google Patents
Method of protein is detected based on nanogold DNA composites Download PDFInfo
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- CN106546749B CN106546749B CN201610949304.1A CN201610949304A CN106546749B CN 106546749 B CN106546749 B CN 106546749B CN 201610949304 A CN201610949304 A CN 201610949304A CN 106546749 B CN106546749 B CN 106546749B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
Abstract
The present invention provides a kind of based on nanogold DNA composites detection method of protein, including the preparation of nanogold DNA compounds, the generation of nanogold DNA protein ternary complexs, nano gold catalysis nitrophenol and sodium borohydride reaction (color development system), range estimation reaction solution become colourless time (CCT values), according to the CCT/CCT of different proteins by yellow0Value draws LDA figures, and the testing protein of concentration known is added into above-mentioned color development system, calculates the CCT/CCT of testing protein0Value, scheme with reference to LDA so as to which testing protein be identified.The present invention is established on the basis of linear discriminant analysis technology, only need to observe by the naked eye, and with the time of timer recording solution color change, without any expensive instrument, different proteins can successfully be identified, test limit reaches 30nM, and new technological means is provided for protein high flux quick detection.
Description
Technical field
The present invention relates to one kind based on nanogold-DNA composites detection method of protein.
Background technology
The early diagnosis for being detected as a variety of diseases of protein provides very valuable information, such as hypoalbuminemia
(the U.Andreasson such as disease, senile dementia, cancer, prostatitis, AIDS；E.Portelius；
M.E.Andersson,et al.,Biomarker Med.2007,1,59-78；V.U.Bai；A.Kaseb；S.Tejwani,et
al.,Proc.Natl.Acad.Sci.2007,104,2343-2348；J.Hardy；D.J.Selkoe,Science 2002,
297,353-356).Mainly include two major classes currently for the detection method of protein：One kind is high performance liquid chromatography mass spectrum connection
Usage and two-dimensional gel electrophoresis method.The shortcomings that needing professional's operation, expensive equipment, time-consuming be present in this method.It is another
It is the immunization method based on Ag-Ab specific recognition biomarker.Not only complex operation, workload are big, anti-for this method
Former and antibody has to come from biological living, causes financial cost high, and their limitednumber.Both approaches are difficult
To realize the high-throughout quick detection of protein.
The content of the invention
It is an object of the invention to provide one kind based on nanogold-DNA composites detection method of protein.
It is of the invention that method of protein, bag are detected based on nanogold-DNA composites in order to realize the object of the invention
Include following steps：
S1, nanogold particle preparation：It is made by reduction of sodium citrate gold chloride, chlorauric acid solution concentration is 50mM,
The mol ratio of gold chloride and sodium citrate is 3.5-3.8:1 (preferably 3.5:1), in resulting solution nanogold particle average grain diameter
About 13-18nm (preferably 13nm)；
S2, nanogold-DNA compounds preparation：It is 1 that concentration is added into the above-mentioned μ L of solution 50 containing nanogold particle
μM to be modified with DNA solution 10 the μ L, the DNA of sulfydryl be poly (A) for one end15, obtain nanogold-poly (A)15Compound；
S3, nanogold-DNA- protein ternary complexs generation：To the above-mentioned solution containing nanogold-DNA compounds
Each 50 μ L of 11 kinds of protein solutions of M1 concentration are separately added into 60 μ L, 30min is incubated in 37 DEG C；Simultaneously with ddH2O is blank
Control, i.e., add ddH into the above-mentioned μ L of solution 60 containing nanogold-DNA compounds2The μ L of O 50,30min is incubated in 37 DEG C；
Wherein, 11 kinds of protein include trypsase (Try), bovine hemoglobin (Hem), bovine serum albumin (BSA),
Human albumin (HSA), pepsin (Pep), papain (Pap), HRPO (HRP), soft iron albumen
(TRF), cytochromes-C (Cyt-C), fiber original albumen (Fib) and canavaline (Con)；
S4, nano gold catalysis nitrophenol and sodium borohydride reaction generation amino-phenol：Add into the μ L of S3 resulting solutions 110
Enter the μ L of nitrophenol solution 200 that concentration is 5mM and the μ L of sodium borohydride solution 200 that concentration is 0.6M, utilize exposed nanometer
Its reaction of golden watch surface catalysis；
S5, visual observations solution become colourless reaction time, i.e. CCT values (Color Change Time) by yellow, point
CCT values corresponding to different proteins, and blank control ddH are not write down2CCT corresponding to O0Value, and different proteins are calculated respectively
Corresponding CCT/CCT0Value, draw form；
S6, the DNA is replaced with to poly (C)15, repeat S1~S5；
S7, the DNA is replaced with to poly (T)15, repeat S1~S5；
S8, the reaction different proteins CCT/CCT that will be drawn for above-mentioned three kinds of DNA0The form Input Software IBM of value
SPSS Statistics 19 (parameter is arranged to give tacit consent to), scheme so as to obtain LDA corresponding to different proteins；
S9, according to S1 and S2 the nanogold-DNA complex solutions for above-mentioned three kinds of DNA are prepared respectively, respectively to three kinds
The μ L of testing protein solution 50 of M1 concentration are added in the μ L of nanogold-DNA complex solutions 60,30min is incubated in 37 DEG C；Simultaneously
With ddH2O is blank control；
S10, the μ L of nitrophenol solution 200 and concentration that into the μ L of S9 resulting solutions 110, addition concentration is 5mM are 0.6M's
The μ L of sodium borohydride solution 200, are reacted；Visual observations solution becomes the colourless reaction time by yellow, and calculates CCT/
CCT0Value, substitute into software described in S8, result is shown according to LDA figures, testing protein is identified.
Foregoing method, every kind of protein does 5~6 repetitions in S3.
Foregoing method, in S3 and S9,300nM≤M1≤2000 μM.Preferably, M1 is 300nM or 500nM (i.e. ternarys
The final concentration of 30nM or 50nM of protein in complex solution).
Foregoing method, the dosage and the same S3 of reaction condition of each reaction reagent in S9；The concentration of each reaction reagent in S10,
Dosage and the same S4 of reaction condition.
The principle schematic of above-mentioned detection method is shown in Fig. 1.
The present invention also provides a kind of kit for being used to detect protein, and the kit can detect and distinguish 11 hatching eggs
White matter Try, Hem, BSA, HSA, Pep, Pap, HRP, TRP, CytC, Fib and Con；
The kit includes following reagent：Nanogold-poly (A)15Complex solution, nanogold-poly (C)15It is compound
Thing solution, nanogold-poly (T)15Complex solution, nitrophenol, sodium borohydride and ddH2O etc..
Nanogold-the poly (A)15The preparation method of complex solution is as follows：
1. the preparation of nanogold particle：It is made by reduction of sodium citrate gold chloride, chlorauric acid solution concentration is 50mM, chlorine
The mol ratio of auric acid and sodium citrate is 3.5-3.8:1 (preferably 3.5:1), the average grain diameter of nanogold particle is in resulting solution
13-18nm (preferably 13nm)；
2. nanogold-poly (A)15The preparation of complex solution：Add into the above-mentioned μ L of solution 50 containing nanogold particle
It is that DNA solution 10 μ L, the DNA that 1 μM one end is modified with sulfydryl are poly (A) to enter concentration15, obtain nanogold-poly
(A)15Complex solution；
Nanogold-the poly (C)15Complex solution and nanogold-poly (T)15The preparation method of complex solution is same
On.
The present invention also provides application of the kit in protein high flux quick detection.
The present invention further provides application of the kit in the protein mixture detection of different component.
The present invention is established on the basis of linear discriminant analysis (Linear Discriminant Analysis) technology, can
Effectively solve traditional sensors between recognition element and object there is " key pattern ", i.e., one-to-one specific bond,
And every kind of composition in complex mixture is designed with single-minded selective specific sensing unit, it is time-consuming and unrealistic
The problem of.Compared with multi-dimension array sensor before this, present invention, avoiding their existing deficiencies：Preparation process is numerous
Trivial, complex operation；Experimental cost expense is added using fluorescence molecule marker DNA；Need large-scale instrument and specialty
Operating personnel.The present invention only needs to observe by the naked eye, and with the time of timer recording solution color change, without any
Expensive instrument, you can successfully identify 11 kinds of protein, test limit reaches 30nM.This method can be to the protein of different component
Mixture is identified (differentiation rate is 100%), it may also be used for the protein identification in blood serum sample.
Brief description of the drawings
Fig. 1 is the schematic diagram that the present invention detects method of protein based on nanogold-DNA composites.
Fig. 2 is to detect the result distinguished to not homologous protein in the embodiment of the present invention 1；Wherein, A：With 11 kinds of protein
The CCT/CCT of (final concentration 30nM)0The block diagram that is done by ordinate of ratio；B, C and D is respectively final concentration 10nM, 30nM
With the identification shot chart of 50nM protein；CCT represents the solution colour transformation period containing protein.CCT0Represent no albumen
The solution changes color time existing for matter.
Fig. 3 is the detection differentiation result to mixing not homologous protein in the embodiment of the present invention 2；Wherein, A：Mixing is dense eventually
Spend the identification shot chart for 30nM bovine serum albumins (BSA) and human albumin (HSA)；B：Mix final concentration of 30nM cow's serums
The identification shot chart of albumen (BSA) and canavaline (Con).
Fig. 4 is the detection recognition result to mixing not homologous protein in blood serum sample in the embodiment of the present invention 3；Wherein,
A, B is respectively to be based on CCT/CCT0Fingerprint image and protein identification shot chart.
In Fig. 2-Fig. 4, factor 1 and factor 2 represent typical discriminator 1 and typical discriminator 2 respectively.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
Embodiment 1 is based on nanogold-DNA composites detection method of protein
What the present embodiment provided detects method of protein based on nanogold-DNA composites, comprises the following steps：
1st, the preparation of nanogold particle：It is made by reduction of sodium citrate gold chloride, chlorauric acid solution concentration is 50mM, chlorine
The mol ratio of auric acid and sodium citrate is 3.5:1, the average grain diameter of nanogold particle is about 13nm in resulting solution；
2nd, the preparation of nanogold-DNA compounds：It is 1 μ that concentration is added into the above-mentioned μ L of solution 50 containing nanogold particle
DNA solution 10 the μ L, the DNA that M one end is modified with sulfydryl are poly (A)15, obtain nanogold-poly (A)15Compound；
3rd, to be separately added into 300nM (100nM or 500nM) into the above-mentioned μ L of solution 60 containing nanogold-DNA compounds dense
Each 50 μ L of 11 kinds of protein solutions of degree, 30min is incubated in 37 DEG C；Every kind of protein at least does 5 repetitions；Simultaneously with ddH2O
For blank control, i.e., add ddH into the above-mentioned μ L of solution 60 containing nanogold-DNA compounds2The μ L of O 50, in 37 DEG C of incubations
30min；
Wherein, 11 kinds of protein includes Try, Hem, BSA, HSA, Pep, Pap, HRP, TRP, CytC, Fib and Con；
4th, nano gold catalysis nitrophenol and sodium borohydride reaction generation amino-phenol：Add into the μ L of S3 resulting solutions 110
Enter the μ L of nitrophenol solution 200 that concentration is 5mM and the μ L of sodium borohydride solution 200 that concentration is 0.6M, utilize exposed nanometer
Its reaction of golden watch surface catalysis；
5th, visual observations solution becomes colourless reaction time, i.e. CCT values by yellow, and it is corresponding to write down different proteins respectively
CCT values, and blank control ddH2CCT corresponding to O0Value, and CCT/CCT corresponding to different proteins is calculated respectively0Value, is painted
Tabulation lattice；
6th, the DNA is replaced with into poly (C)15, repeat S1~S5；
7th, the DNA is replaced with into poly (T)15, repeat S1~S5；
8th, the reaction different proteins CCT/CCT that will be drawn for above-mentioned three kinds of DNA0The form Input Software IBM of value
SPSS Statistics 19 (parameter is arranged to give tacit consent to), scheme so as to obtain LDA corresponding to different proteins；
9th, the nanogold-DNA complex solutions for above-mentioned three kinds of DNA are prepared respectively according to step 1 and 2, respectively to three
Each 50 μ of testing protein solution of 300nM (100nM or 500nM) concentration is added in the μ L of kind nanogold-DNA complex solutions 60
L, 30min is incubated in 37 DEG C；Simultaneously with ddH2O is blank control；
10th, the μ L of nitrophenol solution 200 and concentration that addition concentration is 5mM into the μ L of step 9 resulting solution 110 are 0.6M
The μ L of sodium borohydride solution 200, reacted；Visual observations solution becomes the colourless reaction time by yellow, and calculates CCT/
CCT0Value, substitute into software described in step 8, result is shown according to LDA figures, testing protein is identified.
The result that homologous protein detection is not distinguished is shown in Fig. 2.
Protein mixture of the embodiment 2 based on nanogold-DNA composites detection different component
Comprise the following steps：
1st, the preparation of nanogold particle：It is made by reduction of sodium citrate gold chloride, chlorauric acid solution concentration is 50mM, chlorine
The mol ratio of auric acid and sodium citrate is 3.5:1, the average grain diameter of nanogold particle is about 13 in resulting solution；
2nd, the preparation of nanogold-DNA compounds：It is 1 μ that concentration is added into the above-mentioned μ L of solution 50 containing nanogold particle
DNA solution 10 the μ L, the DNA that M one end is modified with sulfydryl are poly (A)15, obtain nanogold-poly (A)15Compound；
3rd, two kinds of protein of 300nM concentration are separately added into the above-mentioned μ L of solution 60 containing nanogold-DNA compounds
Solution (BSA and HSA mixed solutions or BSA and Con mixed solutions) each 50 μ L of mixing, 30min is incubated in 37 DEG C；Per hatching egg
White matter at least does 5 repetitions；Simultaneously with ddH2O is blank control, i.e., to the above-mentioned μ of solution 60 containing nanogold-DNA compounds
DdH is added in L2The μ L of O 50,30min is incubated in 37 DEG C；
4th, nano gold catalysis nitrophenol and sodium borohydride reaction generation amino-phenol：Add into the μ L of S3 resulting solutions 110
Enter the μ L of nitrophenol solution 200 that concentration is 5mM and the μ L of sodium borohydride solution 200 that concentration is 0.6M, utilize exposed nanometer
Its reaction of golden watch surface catalysis；
5th, visual observations solution becomes colourless reaction time, i.e. CCT values by yellow, and it is corresponding to write down different proteins respectively
CCT values, and blank control ddH2CCT corresponding to O0Value, and CCT/CCT corresponding to different proteins is calculated respectively0Value, is painted
Tabulation lattice；
6th, the DNA is replaced with into poly (C)15, repeat S1~S5；
7th, the DNA is replaced with into poly (T)15, repeat S1~S5；
8th, the reaction different proteins CCT/CCT that will be drawn for above-mentioned three kinds of DNA0The form Input Software IBM of value
SPSS Statistics 19 (parameter is arranged to give tacit consent to), scheme so as to obtain LDA corresponding to different proteins；
9th, the nanogold-DNA complex solutions for above-mentioned three kinds of DNA are prepared respectively according to step 1 and 2, respectively to three
The μ L of testing protein solution 50 of 300nM concentration are added in the μ L of kind nanogold-DNA complex solutions 60, in 37 DEG C of incubations
30min；Simultaneously with ddH2O is blank control；
10th, the μ L of nitrophenol solution 200 and concentration that addition concentration is 5mM into the μ L of step 9 resulting solution 110 are 0.6M
The μ L of sodium borohydride solution 200, reacted；Visual observations solution becomes the colourless reaction time by yellow, and calculates CCT/
CCT0Value, substitute into software described in step 8, result is shown according to LDA figures, testing protein is identified.
The detection differentiation result for mixing not homologous protein is shown in Fig. 3.
In addition, for other protein species (including Try, Hem, BSA, HSA, Pep, Pap, HRP, TRF, Cyt-C, Fib
And Con) mixing situation and 300nM concentrations above albumen mixing situation, it is necessary to experimenter for testing protein solution kind
The number of class, standard LDA figures are repainted according to above-mentioned steps 1-8, solution to be measured is repeated the above steps 9-10.
Embodiment 3 identifies the protein in blood serum sample based on nanogold-DNA composites
Comprise the following steps：
1st, the preparation of nanogold particle：It is made by reduction of sodium citrate gold chloride, chlorauric acid solution concentration is 50mM, chlorine
The mol ratio of auric acid and sodium citrate is 3.5:1, the average grain diameter of nanogold particle is about 13 in resulting solution；
2nd, the preparation of nanogold-DNA compounds：It is 1 μ that concentration is added into the above-mentioned μ L of solution 50 containing nanogold particle
DNA solution 10 the μ L, the DNA that M one end is modified with sulfydryl are poly (A)15, obtain nanogold-poly (A)15Compound；
3rd, 11 kinds of protein that 3 μM of concentration is separately added into the above-mentioned μ L of solution 60 containing nanogold-DNA compounds are molten
Each 50 μ L of liquid, 30min is incubated in 37 DEG C；Every kind of protein at least does 5 repetitions；Simultaneously with ddH2O is blank control, i.e., upwards
State in the μ L of solution 60 containing nanogold-DNA compounds and add ddH2The μ L of O 50,30min is incubated in 37 DEG C；
Wherein, 11 kinds of protein includes Try, Hem, BSA, HSA, Pep, Pap, HRP, TRP, CytC, Fib and Con；
4th, nano gold catalysis nitrophenol and sodium borohydride reaction generation amino-phenol：Add into the μ L of S3 resulting solutions 110
Enter the μ L of nitrophenol solution 200 that concentration is 5mM and the μ L of sodium borohydride solution 200 that concentration is 0.6M, utilize exposed nanometer
Its reaction of golden watch surface catalysis；
5th, visual observations solution becomes colourless reaction time, i.e. CCT values by yellow, and it is corresponding to write down different proteins respectively
CCT values, and blank control ddH2CCT corresponding to O0Value, and CCT/CCT corresponding to different proteins is calculated respectively0Value, is painted
Tabulation lattice；
6th, the DNA is replaced with into poly (C)15, repeat S1~S5；
7th, the DNA is replaced with into poly (T)15, repeat S1~S5；
8th, the reaction different proteins CCT/CCT that will be drawn for above-mentioned three kinds of DNA0The form Input Software IBM of value
SPSS Statistics 19 (parameter is arranged to give tacit consent to), scheme so as to obtain LDA corresponding to different proteins；
9th, the nanogold-DNA complex solutions for above-mentioned three kinds of DNA are prepared respectively according to step 1 and 2, respectively to three
The μ L of testing protein solution 50 of 30nM concentration are added in the μ L of kind nanogold-DNA complex solutions 60,30min are incubated in 37 DEG C；
Simultaneously with ddH2O is blank control；
10th, the μ L of nitrophenol solution 200 and concentration that addition concentration is 5mM into the μ L of step 9 resulting solution 110 are 0.6M
The μ L of sodium borohydride solution 200, reacted；Visual observations solution becomes the colourless reaction time by yellow, and calculates CCT/
CCT0Value, substitute into software described in step 8, result is shown according to LDA figures, testing protein is identified.
The detection recognition result that not homologous protein is mixed in blood serum sample is shown in Fig. 4.
In addition, it is necessary to which experimenter is according to testing protein solution in the case of other concentrating proteins are in blood serum sample
Concentration, repaint standard LDA figures according to above-mentioned steps 1-8, solution to be measured repeated the above steps 9-10.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
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Claims (8)
1. based on nanogold-DNA composites detection method of protein, it is characterised in that comprise the following steps：
S1, nanogold particle preparation：It is made by reduction of sodium citrate gold chloride, chlorauric acid solution concentration is 50mM, and chlorine is golden
The mol ratio of acid and sodium citrate is 3.5-3.8:1, the average grain diameter of nanogold particle is 13-18nm in resulting solution；
S2, nanogold-DNA compounds preparation：It is 1 μM that concentration is added into the above-mentioned μ L of solution 50 containing nanogold particle
DNA solution 10 the μ L, the DNA that one end is modified with sulfydryl are poly (A)15, obtain nanogold-poly (A)15Compound；
S3,11 kinds of protein solutions for being separately added into the above-mentioned μ L of solution 60 containing nanogold-DNA compounds M1 concentration are each
50 μ L, 10min is incubated in 37 DEG C；Simultaneously with ddH2O is blank control, i.e., to the above-mentioned solution containing nanogold-DNA compounds
DdH is added in 60 μ L2The μ L of O 50,30min is incubated in 37 DEG C；
Wherein, 11 kinds of protein includes trypsase, bovine hemoglobin, bovine serum albumin, human albumin, stomach cardia
Enzyme, papain, HRPO, soft iron albumen, cytochromes-C, fiber original albumen and canavaline；
S4, nano gold catalysis nitrophenol and sodium borohydride reaction generation amino-phenol：Added into the μ L of S3 resulting solutions 110 dense
The μ L of sodium borohydride solution 200 for being 0.6M for 5mM the μ L of nitrophenol solution 200 and concentration are spent, utilize exposed nanometer golden watch
Its reaction of surface catalysis；
S5, visual observations solution become colourless reaction time, i.e. CCT values by yellow, write down respectively corresponding to different proteins
CCT values, and blank control ddH2CCT corresponding to O0Value, and CCT/CCT corresponding to different proteins is calculated respectively0Value, draw
Form；
S6, the DNA is replaced with to poly (C)15, repeat S1~S5；
S7, the DNA is replaced with to poly (T)15, repeat S1~S5；
S8, the reaction different proteins CCT/CCT that will be drawn for above-mentioned three kinds of DNA0The form Input Software IBM SPSS of value
Statistics 19, scheme so as to obtain LDA corresponding to different proteins；
S9, according to S1 and S2 the nanogold-DNA complex solutions for above-mentioned three kinds of DNA are prepared respectively, respectively to three kinds of nanometers
The μ L of testing protein solution 50 of M1 concentration are added in the μ L of gold-DNA complex solutions 60,30min is incubated in 37 DEG C；While with
ddH2O is blank control；
S10, the μ L of nitrophenol solution 200 that concentration is 5mM and the boron hydrogen that concentration is 0.6M are added into the μ L of S9 resulting solutions 110
Change the μ L of sodium solution 200, reacted；Visual observations solution becomes the colourless reaction time by yellow, and calculates CCT/CCT0Value,
Substitute into software described in S8, result is shown according to LDA figures, testing protein is identified；
In step S3 and S9,300nM≤M1≤2000 μM；
The poly (A)15The DNA fragmentation being made up of 15 base A, the poly (C)15The DNA being made up of 15 base C
Fragment, the poly (T)15The DNA fragmentation being made up of 15 base T.
2. according to the method for claim 1, it is characterised in that every kind of protein does 5~6 repetitions in S3.
3. according to the method for claim 1, it is characterised in that M1 is 300nM or 500nM.
4. according to the method described in claim any one of 1-3, it is characterised in that the dosage of each reaction reagent and reaction bar in S9
The same S3 of part；The concentration of each reaction reagent, dosage and the same S4 of reaction condition in S10.
5. a kind of kit for being used to detect protein, it is characterised in that the kit can detect and distinguish 11 kinds of albumen
Matter：Trypsase, bovine hemoglobin, bovine serum albumin, human albumin, pepsin, papain, horseradish peroxidating
Enzyme, soft iron albumen, cytochromes-C, fiber original albumen and canavaline；
The kit includes following reagent：Nanogold-poly (A)15Complex solution, nanogold-poly (C)15Compound is molten
Liquid, nanogold-poly (T)15Complex solution, nitrophenol, sodium borohydride and ddH2O；
Wherein, the nanogold-poly (A)15The preparation method of compound is as follows：It is made by reduction of sodium citrate gold chloride,
Chlorauric acid solution concentration is 50mM, and the mol ratio of gold chloride and sodium citrate is 3.5-3.8:1, nanogold particle in resulting solution
Average grain diameter be 13-18nm；One end that concentration is 1 μM is added into the above-mentioned μ L of solution 50 containing nanogold particle to be modified with
DNA solution 10 the μ L, the DNA of sulfydryl are poly (A)15, obtain nanogold-poly (A)15Compound；
According to the method described above, the DNA is replaced with into poly (C)15Or poly (T)15, respectively obtain nanogold-poly (C)15It is multiple
Compound, nanogold-poly (T)15Compound；
Wherein described poly (A)15The DNA fragmentation being made up of 15 base A, the poly (C)15It is made up of 15 base C
DNA fragmentation, the poly (T)15The DNA fragmentation being made up of 15 base T.
6. kit according to claim 5, it is characterised in that the nanogold-poly (A)15The system of complex solution
Preparation Method is as follows：
1. the preparation of nanogold particle：It is made by reduction of sodium citrate gold chloride, chlorauric acid solution concentration is 50mM, gold chloride
Mol ratio with sodium citrate is 3.5-3.8:1, the average grain diameter of nanogold particle is 13-18nm in resulting solution；
2. nanogold-poly (A)15The preparation of complex solution：Added into the above-mentioned μ L of solution 50 containing nanogold particle dense
DNA solution 10 the μ L, the DNA that the one end spent for 1 μM is modified with sulfydryl are poly (A)15, obtain nanogold-poly (A)15It is multiple
Polymer solution；
Nanogold-the poly (C)15Complex solution and nanogold-poly (T)15The preparation method of complex solution is same as above.
7. application of the kit of claim 5 or 6 in protein high flux quick detection.
8. application of the kit of claim 5 or 6 in the protein mixture detection of different component.
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| CN201610949304.1A CN106546749B (en) | 2016-10-26 | 2016-10-26 | Method of protein is detected based on nanogold DNA composites |
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