CN105223347B - A kind of semi-quantitative visual enzyme-linked immune analytic method - Google Patents
A kind of semi-quantitative visual enzyme-linked immune analytic method Download PDFInfo
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- CN105223347B CN105223347B CN201510604707.8A CN201510604707A CN105223347B CN 105223347 B CN105223347 B CN 105223347B CN 201510604707 A CN201510604707 A CN 201510604707A CN 105223347 B CN105223347 B CN 105223347B
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Abstract
A kind of semi-quantitative visual enzyme immunoassay new method of disclosure of the invention, comprises the following steps：（1）The preparation of colour developing nanometer gold（2）Enzyme-linked Immunosorbent Assay and chromogenic reaction.Raw material of the present invention is easy to get, low cost, need not any complex detection equipment, can determine whether testing result by naked eyes, and method is simple to operate, repeatability, good stability, can be directly used for sxemiquantitative quick detection various clinical marker albumen, are expected to be used widely in the field such as life sciences and clinical medicine detection.
Description
Technical field
The invention belongs to analytical chemistry field is and in particular to a kind of semi-quantitative visual enzyme-linked immune analytic method.
Background technology
Enzyme-linked immunosorbent assay（Enzyme Linked ImmunoSorbent Assay）, abbreviation ELISA, adopt
Determinand is connected with enzyme by antigen with the specific reaction of antibody, then produces color reaction by enzyme-to-substrate, to tested substance
Carry out a kind of detection method of qualitative or quantitative analysis.Due to its have the advantages that quick, sensitive, easy, be easy to standardization, make
It is rapidly developed and is extensively applied.It based on immunological response, by antibody, the specific reaction of antigen and enzyme pair
A kind of experimental technique with high specific and hypersensitivity that the efficient catalytic effect of substrate combines.
The testing result of enzyme-linked immunosorbent assay is the color reaction after adding substrate.Traditional enzyme linked immunological is inhaled
It is color signal or chemiluminescence signal that the substrate of enzyme is typically passed through chemical transformation by attached method, the enzyme linked immunological of this mode
Absorption method has very big advantage for the mensure of the object of high concentration.But the object for low concentration, remain
Problem, such as：In low concentration, the color signal of generation is very little, so that it cannot distinguish whether create color signal；
And, in actual object is analyzed, it is also possible to produce because of non-specific adsorption in the presence of there is no object
Raw weak color signal.It is thus desirable to a kind of color is easier the signal producing method of identification, such as produce different colors.
In recent years, with surface plasma resonance technology（SPR）Development with ripe, traditional for introducing surface plasma resonance
Enzyme linked immunosorbent assay also increasingly arouses people's interest.The feature of this technology is the surface of precious metal material used
Plasma resonance signal is extremely related to the structural parameters of this metal.With the naked eye detect the detection of copper ion using this technology
Limit can reach nanomole rank.The hypersensitivity of surface plasma body resonant vibration property based on noble metal and the rich of color so that
Plasma enzyme linked immunosorbent assay becomes the very potential detection technique of one kind.
Although SPR detection technique has extraordinary application prospect in ELISA field, but compared with traditional ELISA, base
Exploitation in the enzyme linked immunosorbent assay detection technique of surface plasma body resonant vibration is but relatively lagged behind with application, does not also have so far
There is the commercialization instrument report of correlation.
Content of the invention
It is an object of the invention to provide a kind of semi-quantitative visual enzyme-linked immune analytic method.The method has operation letter
List, low cost, high repeatability and other advantages, workable.Can be directly used for detecting various biological samples using the method, have
Hope and be used widely in the field such as life sciences and clinical medicine detection, there is significant economic benefit.
For achieving the above object, the present invention adopts the following technical scheme that：
（1）The preparation of colour developing nanometer gold：Gold according to required for the synthesis of reduction of sodium citrate method plants colloidal sol, its particle diameter first
For 15 ± 5 nm；Then growth method is planted according to gold, synthesize cetyl trimethylammonium bromide（CTAB）The nanometer gold of parcel
（CTAB@Au）, its particle diameter is 45 ± 15 nm.
（2）The visualization immunoassay of object：First according to classic ELISA, capture antibody antigen detection antibody
There is specific reaction, then by the horseradish peroxidase (HRP) of streptavidin and biotinylated detection antibody phase
In conjunction with thus HRP is linked on antibody；After adding substrate, reaction a period of time, hydrochloric acid is added to terminate enzymatic reaction；Simultaneously
Generate the TMB of yellow2+.Now add the nanometer gold that the CTAB preparing in advance wraps up, TMB2+Will occur instead with nanometer gold
Should, thus producing obvious color change, highly sensitive detection antigen protein.
Its concrete grammar step is：
（1）Before the synthesis the required glass drying oven used all is soaked with chloroazotic acid, then rinsed with substantial amounts of water,
Use ultra-pure water rinse clean afterwards；
（2）Synthesis particle diameter is in the nanometer gold of 15 ± 5nm：Using the synthesis of reduction of sodium citrate method, with vigorous stirring by 100
mL、2.5×10-4M gold chloride is placed in 120 DEG C of oil bath pans 30 min that flow back；Then it is added thereto to the lemon of 10 mL 1wt%
Lemon acid sodium solution, reacts 20 min under stirring, is then shut off heating, continues to be stirred at reflux to room temperature.
（3）Synthesis particle diameter is in the nanometer gold of 45 ± 15nm：Growth method synthesis is planted using gold, prepares growth-promoting media 60 mL first,
Wherein contain gold chloride 2.5 × 10-4M, cetyl trimethylammonium bromide（CTAB）0.01 M, is subsequently adding the anti-of 0.1 M
Bad hematic acid（Vc）3 mL, stir 2 min, be eventually adding above-mentioned 15 nm about nanometer gold 30 mL, at 30 DEG C after mix homogeneously
Water-bath in place 6-8h.
（4）Above-mentioned nanometer gold is centrifuged under the rotating speed of 10000rpm 10min, abandoning supernatant, the 0.1 of precipitation equivalent
The CTAB of M disperses again.
（5）Test kit is taken out from refrigerator, and recovers to room temperature；According to traditional ELISA mode by antibody antigen and
Enzyme links together, and is then added thereto to corresponding TMB developer, adds the HCl terminating reaction of 2 M after incubation 30min, and
Generate the TMB of respective amount2+.
（6）Finally will be added in the hole of ELISA Plate with 0.1M again scattered nanometer gold, different amounts of TMB2+And nanometer
Gold will occur different etching effects, thus different colors.
Beneficial effects of the present invention：
（1）Conventional ELISA method only produces a kind of color change（Yellow is from shallow to deep）, and visualization of the present invention half
Quantitative immunoassay method can produce two kinds of color changes, thus considerably increase this method for naked eyes detect accuracy.
（2）Visualization sxemiquantitative immunoassay of the present invention is higher compared to conventional ELISA method colour developing sensitivity,
Detection limit is lower.
（3）Colour developing nanometer gold preparation is simple, low cost, and repeatability is high.
（4）Semi-quantitative visual enzyme-linked immune analytic method of the present invention is by traditional ELISA kits and colour developing nanometer
Gold composition, raw material is easy to get, and easily realizes merchandized handling.
Brief description
Fig. 1 is the schematic diagram of plasma ELISA of the present invention.Wherein left-half is to pass
The ELISA schematic diagram of system, right half part senses schematic diagram for plasma.
Fig. 2 is the transmission electron microscope picture of colour developing nanometer gold of the present invention.
Fig. 3 is the detection that application plasma enzyme linked immunosorbent assay carries out antigen（Prostate specific antigen, Prostate
Specific Antigen）Ratio chromatic graph and spectrogram.
Specific embodiment
Embodiment 1
Following examples combine accompanying drawing to illustrate to apply the micro-fluidic chip of present invention preparation to carry out actual sample analysis
Operating process：
(1) before the synthesis the required glass drying oven used all is soaked with chloroazotic acid, then rinsed with substantial amounts of water,
Use ultra-pure water rinse clean afterwards；
(2) synthesis particle diameter is in the nanometer gold of 15 ± 5nm：Using the synthesis of reduction of sodium citrate method, with vigorous stirring by 100
mL、2.5×10-4M gold chloride is placed in 120 DEG C of oil bath pans 30 min that flow back；Then it is added thereto to the lemon of 10 mL 1wt%
Lemon acid sodium solution, reacts 20 min under stirring, is then shut off heating, continues to be stirred at reflux to room temperature.
(3) synthesis particle diameter is in the nanometer gold of 45 ± 15 nm：Growth method synthesis is planted using gold, prepares growth-promoting media 60 first
ML, wherein contains gold chloride 2.5 × 10-4M, cetyl trimethylammonium bromide（CTAB）0.01 M, is subsequently adding 0.1 M's
Ascorbic acid（Vc）3 mL, stir 2 min, be eventually adding above-mentioned 15 nm about nanometer gold 30 mL, 30 after mix homogeneously
DEG C water-bath in place 6 h.
(4) above-mentioned nanometer gold is centrifuged under the rotating speed of 10000 rpm 10 min, abandoning supernatant, precipitation equivalent
The CTAB of 0.1 M disperses again.
Prepare first particle diameter be 45 nm about nanometer gold, and nanometer gold is disperseed again with the CTAB of 0.1 M, accompanying drawing 2
Transmission electron microscope picture for the nanometer gold of this particle diameter.
In this example, selected object is prostate characteristic antigen it is therefore desirable to connect corresponding antibody and enzyme
Pick up, specific implementation method is as follows：The ELISA Plate being loaded with capture antibody is fixed, is added thereto to 100 uL PSA antigens molten
Liquid, is incubated 2.5 h at room temperature with shaking table, capture antibody and antigen will be dynamically connected certainly, then be washed with cleanout fluid four times, and will
Water in hole blots；It is added thereto to the biotinylated detection antibody of 100 uL again, with being incubated 1 h under shaking table room temperature, repeat clear
Wash step；Then add 100 uL Streptavidins-horseradish peroxidase in each hole（HRP）, under room temperature, it is incubated 45min；?
All of antibody, antigen and enzyme is made to connect according to the mode of Fig. 1 left-half eventually.
It is subsequently adding 100 uL TMB developers, after incubation 30 min, add 50 uL 2 M HCl terminating reaction.Finally plus
Enter 100 uL nanometer gold（4.182 nM）React 5 min.
Fig. 3 is the ratio chromatic graph adopting this plasma enzyme-linked immunosorbent assay PSA and spectrogram, and this is than chromatic graph from a left side
PSA concentration of turning right is followed successively by 0,0.15,0.40,0.60,0.80,1.0,2.0,3.0 ng/mL, the detection of the method visual colorimetric determination
It is limited to 0.15 ng/mL.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
Claims (4)
1. a kind of semi-quantitative visual enzyme-linked immune analytic method it is characterised in that：The method comprising the steps of：
（1）The preparation of colour developing nanometer gold：First nanometer gold is synthesized according to reduction of sodium citrate method, its particle diameter is 15 ± 5 nm；So
Afterwards growth method, the nanometer gold of synthesis cetyl trimethylammonium bromide parcel are planted according to gold, its particle diameter is 45 ± 15 nm；
（2）Visual retrieval proteantigen：First according to classic ELISA method, capture antibody antigen detection antibody occurs special
Then the horseradish peroxidase of streptavidin is combined by opposite sex reaction with biotinylated detection antibody, thus will
Horseradish peroxidase is linked on antibody；After adding corresponding TMB developer, react 30min, add hydrochloric acid to terminate enzymatic anti-
Should；Generate the TMB of yellow simultaneously2+；Now add step（1）The nanometer of the cetyl trimethylammonium bromide parcel preparing
Gold, TMB2+Will react with nanometer gold, thus originally single yellow change transitions are the double-colored change of red-yellow, pass through
The change of range estimation solution colour can carry out semi-quantitative analyses to target antigen.
2. according to claim 1 semi-quantitative visual enzyme-linked immune analytic method it is characterised in that：Step（1）Middle Fructus Citri Limoniae
Sour sodium reduction concretely comprise the following steps with vigorous stirring by 100 mL, 2.5 × 10-4M gold chloride is placed in 120 DEG C of oil bath pans and returns
Flow 30 min；Then it is added thereto to the sodium citrate solution of 10 mL 1wt%, react 20 min under stirring, then
Close heating, continue to be stirred at reflux to room temperature.
3. according to claim 1 semi-quantitative visual enzyme-linked immune analytic method it is characterised in that：Described gold plants growth
Method is to prepare growth-promoting media 60 mL first, wherein contains gold chloride 2.5 × 10-4M, cetyl trimethylammonium bromide 0.01 M,
Ascorbic acid 3 mL being subsequently adding 0.1 M, as reducing agent, stirs 2 min, is eventually adding the synthesis of reduction of sodium citrate method
Nanometer gold 30 mL, places 6-8 h in 30 DEG C of water-bath after mix homogeneously, synthesis cetyl trimethylammonium bromide parcel
Nanometer gold.
4. according to claim 1 semi-quantitative visual enzyme-linked immune analytic method it is characterised in that：Step（2）Middle yellow
TMB2+Response time with the nanometer gold of cetyl trimethylammonium bromide parcel is 5 ~ 30 min, and reaction temperature is room temperature.
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| CN108181462A (en) * | 2018-01-25 | 2018-06-19 | 福州大学 | A kind of method of saxitoxin in more quick detection marine products of color visualization |
| CN111175509A (en) * | 2020-02-21 | 2020-05-19 | 福州大学 | ELISA visual detection kit constructed based on Fenton reaction and application of kit in detection of ZEN |
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Effective date of registration: 20190410 Address after: Room 301A South of Building A, No. 96 Xiangxing Road, Xiangxing Industrial Zone, Xiamen City, Fujian Province Patentee after: Jin Ruibo (Xiamen) Biological Technology Co., Ltd. Address before: 350108 new campus of Fuzhou University, No. 2, Xue Yuan Road, University Town, Minhou street, Minhou, Fujian. Patentee before: Fuzhou University |
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