CN105925963A - Method for preparing gold substrate through self-assembly of dry mediate gold nanoparticles and application of method - Google Patents
Method for preparing gold substrate through self-assembly of dry mediate gold nanoparticles and application of method Download PDFInfo
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- CN105925963A CN105925963A CN201610347942.6A CN201610347942A CN105925963A CN 105925963 A CN105925963 A CN 105925963A CN 201610347942 A CN201610347942 A CN 201610347942A CN 105925963 A CN105925963 A CN 105925963A
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- gold
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- nano grain
- mpeg
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- C—CHEMISTRY; METALLURGY
- C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
- C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
- C23C18/00—Chemical coating by decomposition of either liquid compounds or solutions of the coating forming compounds, without leaving reaction products of surface material in the coating; Contact plating
- C23C18/16—Chemical coating by decomposition of either liquid compounds or solutions of the coating forming compounds, without leaving reaction products of surface material in the coating; Contact plating by reduction or substitution, e.g. electroless plating
- C23C18/31—Coating with metals
- C23C18/42—Coating with noble metals
- C23C18/44—Coating with noble metals using reducing agents
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- C—CHEMISTRY; METALLURGY
- C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
- C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
- C23C18/00—Chemical coating by decomposition of either liquid compounds or solutions of the coating forming compounds, without leaving reaction products of surface material in the coating; Contact plating
- C23C18/16—Chemical coating by decomposition of either liquid compounds or solutions of the coating forming compounds, without leaving reaction products of surface material in the coating; Contact plating by reduction or substitution, e.g. electroless plating
- C23C18/1601—Process or apparatus
- C23C18/1633—Process of electroless plating
- C23C18/1646—Characteristics of the product obtained
- C23C18/165—Multilayered product
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
-
- 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
Abstract
The invention discloses a method for preparing a gold substrate through self-assembly of dry mediate gold nanoparticles and application of the method. Gold nanoparticles are modified through mPEG-SH; then the gold nanoparticles modified through the high-temperature dry mediate of mPEG-SH are assembled on a 96-pore plate substrate; and gold atoms are deposited on the substrate on which the gold nanoparticles are assembled, and the uniform gold substrate is formed. The method can be used for ELISA detection reactions and has the beneficial effects of being simple, fast and little in agent dosage; non-specific adsorption in the detection process can be effectively avoided, and a new platform is provided for environment monitoring and diseases diagnosis.
Description
Technical field
The invention belongs to technical field of analysis and detection, be specifically related to a kind of dry mediation gold nano grain self assembly and prepare the side of gold substrate
Method, and the application in ELISA detection technique.
Background technology
Enzyme-linked immunosorbent assay (Enzyme Linked Immunosorbent Assay, ELISA) is a kind of based on antigen-antibody
The detection reaction that specificity interacts.ELISA detection target is utilized mainly to be carried out, first by the method for double-antibody sandwich
Fixed trapped antibody in substrate, then utilizes the specificity of antigen-antibody to interact, capture antibody on successively immobilized antigen and
Detection antibody, finally by the enzyme Journal of Molecular Catalysis substrate colour developing of labelling on detection antibody, luminous or aerogenesis mensuration target concentration.Additionally
Also has dual-antigen sandwich method etc..At present, ELISA has been widely used in the targets such as protein, little molecule, cell, antibacterial
Detection, occupy critical role in fields such as environmental monitoring, food safety and medicals diagnosis on disease.Utilize ELISA method to carry out detection to have
The advantage that specificity is good, stable and highly sensitive, but, when ELISA detects target, need to hatch 12h at 4 DEG C and complete
Capture antibody in substrate is coated process, the most quite time-consuming；Further, the 96 orifice plate bases commonly used with ELISA detection due to antibody
End affinity is weak, and during coated antibody, reagent consumption is big.Different from 96 orifice plate substrates, gold substrate is strong with affinity of antibody, combination
Speed is fast, utilizes gold substrate to carry out ELISA detection and can effectively reduce the consumption of reagent and shorten antibody and be coated the time used.So
And, mostly the method for preparation gold substrate is that, based on plating or the method for vacuum evaporation, the method needs to use large-scale instrument, cost at present
Height and reagent consumption are big.
Therefore, development is simple, quick, reagent consumes little and without large-scale instrument method and prepares gold substrate, for shortening ELISA
Used by coated antibody, time, reduction reagent dosage are significant.
Summary of the invention
In place of it is an object of the invention to overcome the deficiencies in the prior art, it is provided that prepared by a kind of dry mediation gold nano grain self assembly
The method of gold substrate and application thereof, have simple, quick, cheap advantage, also solve in existing elisa technique and be coated capture
The deficiencies such as antibody time-consumingly length, reagent dosage are big.
One of the technical solution adopted for the present invention to solve the technical problems is:
A kind of method that gold substrate is prepared in dry mediation gold nano grain self assembly, including:
1) by the gold nano grain concentration of particle diameter 5～30nm be 0.2～5 μM, molecular weight be 0.75～20kDa mPEG-SH
(Polyethylene Glycol of methoxyl group sulfydryl modification) is modified, and obtains the gold nano grain that mPEG-SH modifies；
2) it is dried at 65～95 DEG C, to mediate the gold nano grain of the above-mentioned mPEG-SH modification that concentration is 1～25nM at enzyme
It is self-assembly of in the substrate that the macromolecular materials such as the polyethylene of target, polypropylene, polrvinyl chloride, polystyrene or cellulose are made
Uniform gold nano grain layer；MPEG-SH in modification can with gold nano grain completely, be adequately bonded, and can
In self assembling process, promote that gold nano grain is uniformly distributed；It is dried at 65～95 DEG C and can promote while rapidly removing moisture
Solution flows, thus ensures further while adding fast speed and form uniform gold nano grain layer；
3) reducing agent also original content is utilized to be not less than the method for gold chloride of 0.31mM in step 2) the uniform gold nano that obtains
Deposit gold atom on granular layer, thus in ELISA Plate, form homogeneous gold substrate.Described reducing agent can be oxammonium hydrochloride., anti-bad
Hematic acid, hydroquinone or sodium citrate etc., preferably oxammonium hydrochloride..The auri of generation can be regulated and controled by the amount of reducing agent Yu gold chloride
The thickness at the end.
In one embodiment: described step 1) in, the particle diameter of gold nano grain is 13～20nm.
In one embodiment: described step 1) in, the molecular weight of mPEG-SH is 5kDa.
In one embodiment: described step 1) in, final concentration of 0.5～1 μM of mPEG-SH.
In one embodiment: described step 2) in, temperature is 75～85 DEG C
In one embodiment: described step 2) in, the gold nano grain concentration that mPEG-SH modifies is 2.5～10nM.
In one embodiment: described step 3) in, described reducing agent is oxammonium hydrochloride., and the reaction density of oxammonium hydrochloride. is 2～50mM.
In one embodiment: the reaction density of described oxammonium hydrochloride. is 5～20mM.
In one embodiment: described step 3) in, the concentration of described gold chloride is 10～20mM.
The two of the technical solution adopted for the present invention to solve the technical problems are:
Said method application in ELISA detection technique, the target of described ELISA detection can be cell, protein and little
Molecules etc., may be used for direct method, indirect method, double antibody sandwich method, dual-antigen sandwich method, competition law etc..
The technical program is compared with background technology, and it has the advantage that
First, the gold nano grain making mPEG-SH modify by the method for dry mediation self assembly is adsorbed in 96 orifice plate substrates, this
Method ADSORPTION OF GOLD nano-particle homogeneity is good；Secondly, utilize oxammonium hydrochloride. reduction gold chloride to deposit gold atom in substrate, simple, honest and clean
It is controlled that valency, reagent consume thickness that is little and that form gold substrate；Again, utilize the strong adsorption of gold substrate and antibody, carry out ELISA
Detection can reduce reagent dosage when being coated capture antibody, shortening is coated the time used；Finally, the gold substrate that prepared by the method is fitted
Detect for the ELISA of the multiple targets such as protein, little molecule, cell and antibacterial.Have simple, quick, honest and clean based on the method
Valency and the few advantage of reagent consumption, will further facilitate the development of ELISA detection method.
Accompanying drawing explanation
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Fig. 1 is that the present invention is dried mediation gold nano grain self assembly and prepares gold substrate for schematic diagram that ELISA detects.
Fig. 2 is that in embodiment 1, variable concentrations different molecular weight mPEG-SH modifies gold nano grain in 96 orifice plate substrates assembling preparations
(A) of gold substrate photo in kind and (B) are in the absorption of 405nm.
Fig. 3 is that in embodiment 1, variable concentrations gold chloride prepares (A) photo in kind of gold substrate with (B) substrate 405nm's
Absorb.
Fig. 4 is to be dried mediation gold nano grain self assembly in embodiment 1 to prepare the scanning electron microscope (SEM) photograph of gold substrate.
Fig. 5 is that in embodiment 2,1 μ g/mL (A) HRP and (B) SA-HRP adsorbs in gold substrate and 96 orifice plate substrates
Dynamic process.
Fig. 6 is that in embodiment 2, variable concentrations (A) HRP and (B) SA-HRP hatches 1h in gold substrate and 96 orifice plate substrates
The amount that can adsorb.
Fig. 7 is the knot utilizing (A) gold substrate and (B) 96 orifice plate to carry out ELISA detection variable concentrations H5N1 in embodiment 3
Really schematic diagram.
Detailed description of the invention
Present disclosure is illustrated below by embodiment:
Embodiment 1: be dried mediation gold nano grain self assembly and prepare gold substrate
1) synthesis 16nm gold nano grain (AuNPs): add 100mL 0.01wt% gold chloride in round-bottomed flask, continuously
The lower boiling reflux of stirring, at the uniform velocity adds 1mL 3wt% sodium citrate, continues stirring and boils 30min, and synthesis particle diameter is 16nm's
AuNPs.The concentration characterizing the method synthesis gold nano grain according to ultravioletvisible absorption is 2.5nM.
2) in 96 hole ELISA Plate, add 100 μ L above-mentioned 2.5nM 16nm AuNPs and 5 μ L variable concentrations different molecular weights
MPEG-SH, mix homogeneously, obtain the gold nano grain that mPEG-SH modifies；
3) it being dried at 80 DEG C, with mediating steps 2) gold nano grain modified of the mPEG-SH that obtains is in 96 hole ELISA Plate
Uniform gold nano grain layer it is self-assembly of in substrate；
4) it is subsequently adding 50 μ L variable concentrations gold chlorides and 50 μ L 20mM oxammonium hydrochloride .s, 25 DEG C of reaction 30min, utilizes salt
The method of acid hydroxylamine reduction gold chloride is in step 3) deposit gold atom on the uniform gold nano grain layer that obtains, thus at 96 hole enzymes
Homogeneous gold substrate is formed in target.With milli-Q water 3 times, utilize microplate reader measure different sample 405nm absorption with
Effect under contrast different condition；Utilizing scanning electron microscope to characterize the pattern of gold substrate, result is as shown in Figure 4.
As shown in Figures 2 and 3, the molecular weight of mPEG-SH is preferred microplate reader measurement result with 5kDa, and concentration is with 0.5～1 μM
It is preferred；The concentration of gold chloride is preferred with 10mM.
Embodiment 2: the dynamic process of gold substrate adhesion protein characterizes
Embodiment 1 obtain there are 96 hole ELISA Plate of gold substrate in add 100 μ L variable concentrations HRP (peroxidase)
Or SA-HRP (Streptavidin/peroxidase), incubated at room different time, then wash 3 times with PBS.Add 100
The tmb substrate of μ L commercialization, room temperature reaction 20min, add 50 μ L 2M H2SO4Terminate reaction, utilize microplate reader to measure
Different samples are in the absorption of 450nm.Matched group uses 96 conventional hole ELISA Plate, and operation is ibid.
As shown in Figure 5 and Figure 6, the speed of gold substrate adhesion protein and adsorbance are far longer than conventional 96 hole ELISA Plate to result, profit
With gold substrate carry out ELISA detection can effectively reduce reagent consumption and shorten antibody be coated the time used.
Embodiment 3: present invention application in ELISA detection technique
Utilize the present invention method detection H5N1:
Embodiment 1 obtain there are 96 hole ELISA Plate of gold substrate in add 100 μ L 2 μ g/mL capture antibody, hatch 30 for 4 DEG C
Min, 300 μ L Wash Buffer (containing 0.1wt%Tween 20PBS solution) wash three times.Subsequently, 300 μ L 2wt% are added
BSA/PBS solution, hatches 1h for 37 DEG C, and 300 μ L Wash Buffer wash three times.Add 100 μ L testing samples, incubate for 25 DEG C
Educating 1h, 300 μ L Wash Buffer wash three times.It is subsequently adding 100 μ L 0.5 μ g/mL biotin labeled detection antibody, 25 DEG C
Hatching 1h, 300 μ L Wash Buffer wash twice.Be subsequently adding 100 μ L 1:200 dilution commercialization SA-HRP, 25 DEG C
Hatching 1h, 300 μ L Wash Buffer wash twice.Add the tmb substrate of 100 μ L commercializations, room temperature reaction 20min,
Add 50 μ L 2M H2SO4Terminate reaction, utilize microplate reader to measure the absorption at 450nm of the different sample.Matched group uses routine
96 hole ELISA Plate, operation ibid.
Result is as it is shown in fig. 7, the ELISA Plate after using the present invention to assemble gold substrate detects, with 96 conventional hole ELISA Plate inspections
Survey is compared, and the two is linearly good, and result there was no significant difference.
The above, only present pre-ferred embodiments, therefore the scope that the present invention implements can not be limited according to this, i.e. according to the present invention
The equivalence change that the scope of the claims and description are made with modify, all should still belong in the range of the present invention contains.
Claims (10)
1. one kind is dried the method that gold substrate is prepared in mediation gold nano grain self assembly, it is characterised in that: including:
1) by the gold nano grain concentration of particle diameter 5～30nm be 0.2～5 μM, molecular weight be 0.75～20kDa mPEG-SH modify, obtain the gold nano grain that mPEG-SH modifies；
2) being dried at 65～95 DEG C, the gold nano grain modified to mediate the above-mentioned mPEG-SH that concentration is 1～25nM is self-assembly of uniform gold nano grain layer in ELISA Plate substrate；
3) reducing agent also original content is utilized to be not less than the method for gold chloride of 0.31mM in step 2) deposit gold atom on the uniform gold nano grain layer that obtains, thus in ELISA Plate, form homogeneous gold substrate.
Method the most according to claim 1, it is characterised in that: described step 1) in, the particle diameter of gold nano grain is 13～20nm.
Method the most according to claim 1, it is characterised in that: described step 1) in, the molecular weight of mPEG-SH is 5kDa.
Method the most according to claim 1, it is characterised in that: described step 1) in, final concentration of 0.5～1 μM of mPEG-SH.
Method the most according to claim 1, it is characterised in that: described step 2) in, temperature is 75～85 DEG C.
Method the most according to claim 1, it is characterised in that: described step 2) in, the gold nano grain concentration that mPEG-SH modifies is 2.5～10nM.
Method the most according to claim 1, it is characterised in that: described step 3) in, described reducing agent is oxammonium hydrochloride., and the reaction density of oxammonium hydrochloride. is 2～50mM.
Method the most according to claim 7, it is characterised in that: the reaction density of described oxammonium hydrochloride. is 5～20mM.
Method the most according to claim 1, it is characterised in that: described step 3) in, the concentration of described gold chloride is 10～20mM.
The method the most according to any one of claim 1 to 9 application in ELISA detection technique.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109855526A (en) * | 2019-02-28 | 2019-06-07 | 吉林大学 | A kind of resistance-type flexibility strain transducer and preparation method thereof based on dry mediation self assembly |
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