CN103197080A - Preparation method for nanogold-protein composite material with enzyme catalysis performance - Google Patents
Preparation method for nanogold-protein composite material with enzyme catalysis performance Download PDFInfo
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- CN103197080A CN103197080A CN2013101306999A CN201310130699A CN103197080A CN 103197080 A CN103197080 A CN 103197080A CN 2013101306999 A CN2013101306999 A CN 2013101306999A CN 201310130699 A CN201310130699 A CN 201310130699A CN 103197080 A CN103197080 A CN 103197080A
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Abstract
The invention provides a preparation method for a nanogold-protein composite material with an enzyme catalysis performance. The preparation method comprises the steps of: firstly dispersing nanogold sol into phosphoric acid buffer solution containing Tween 20 after degassing the nanogold sol by nitrogen purging, mixing the solution with mercaptoundecanoic acid, and standing and reacting at the normal temperature; carrying out centrifuging, supernatant discarding and gravity suspension on the mixed solution until supernatant does not contain the mercaptoundecanoic acid; centrifuging and collecting nanogold protected by MUA (mercaptoundecanoic acid), and dispersing the nanogold into the phosphoric acid buffer solution to be standby; mixing the nanogold protected by the MUA with EDAC (Carbodiimide Hydrochloride), NHS (N-Hydroxysuccinimide) and incision enzyme, adding the mixture into MES (Morpholineethanesulfonic acid) buffer solution, and standing and reacting at the normal temperature; and finally repeatedly carrying out the centrifuging, the supernatant discarding and the gravity suspension, and dispersing the obtained composite materials into the phosphoric acid buffer solution. According to the preparation method provided by the invention, the complex chemical modification to enzyme is not involved, and the activity of the enzyme is not influenced; the reaction is carried out at the room temperature, and the reaction conditions are mild; and the prepared functional nanogold-protein composite material is hardly influenced by solution media and has excellent stability.
Description
Technical field
The present invention relates to a kind of preparation method of functional form nano composite material, particularly a kind of preparation method with nm of gold-protein compound substance of enzymatic performance.
Background technology
Nm of gold generally refers to gold chloride (HAuCl
4) a certain size the gold grain that under the reductive agent effect, aggregates into, these particles are because electrostatic interaction becomes stable colloidal state, so be called collaurum again.Nm of gold also has excellent biological compatibility simultaneously except the surface effect with general nano particle, quantum size effect.Because the nano Au particle of biomacromolecule and functionalization is in nanometer dimension together, when adding the nanogold particle of functionalization in corresponding living things system, it has directed adsorption effect and enrichment effect etc.Both can make not only stable but also have a biology/nano composite material of certain quantitative relation by chemistry or physisorption.Nm of gold has the characteristic of high electron density, at the visible pitchy particle of microscopically, when these particles are assembled in a large number, naked eyes red color visible or pink spot, thus with biomacromolecule (as protein) in conjunction with and functionalization the nm of gold compound substance can be used in the qualitative or semiquantitative method for quick.
People last for many years for the applied research of the nm of gold of biomacromolecule functionalization, obtained great achievement, as nm of gold being applied to the microscope spike, flow cytometer, immunity marking technology, biology sensor, biochip, the fast detecting that agricultural and veterinary chemicals is residual, pathogenic microorganisms detects etc., therefore nano gold mark technology (Nanogold Labelling Techique) is described as one of modern four big labelling techniques, has in biomarker analysis fields such as biomedicine, molecular biology extensively and important use.
The mechanism that nm of gold is combined with biomolecule is generally considered to be the positive charge group of the electronegative and protein of nm of gold surface institute, because Electrostatic Absorption forms non-covalent absorption.PH value and the salinity of this absorption and solution are closely related, change these character of solution, cause the gathering of colloid gold particle or coming off of biomolecule easily.Therefore, seeking functionalized nano gold-biomacromolecule compound substance that a kind of simple and practical method is come synthesizing stable, is the market demand that is increasing at present, has broad application prospects.
Summary of the invention
The object of the present invention is to provide a kind of nm of gold-protein composite material and preparation method thereof with enzymatic performance.In the inventive method, the amido in conjunction with on employing carboxyl and the protein of protein and nm of gold carries out cross-linking reaction, produce firm covalent bond, stability of nano gold is subjected to solution medium to influence bigger shortcoming in the classic method thereby overcome, and is a kind of preparation method of functionalized nano gold more reliably.
To achieve these goals, the present invention adopts following technical scheme:
A kind of preparation method with nm of gold-protein compound substance of enzymatic performance comprises:
(1) preparation of the nm of gold of sulfydryl undecanoic acid root (MUA) protection;
The nm of gold of the sulfydryl undecanoic acid root protection that (2) step (1) is obtained is connected the described nm of gold-protein compound substance with enzymatic performance of preparation with restriction endonuclease;
Wherein, step (1) specifically comprises the steps:
(1-1) the nm of gold colloid is purged degassing 10-60 second with nitrogen;
(1-2) the nm of gold colloid after will outgasing is scattered in the phosphate buffer solution that contains polysorbas20, and stable at least 20 minutes, add sulfydryl undecanoic acid aqueous solution then, left standstill under the normal temperature 2-4 hour, obtain mixed liquor; Wherein, the mol ratio of nm of gold colloid and sulfydryl undecanoic acid is 1: (100-200);
(1-3) the mixed liquor centrifuging that step (1-2) is obtained behind the removal supernatant liquor, is dissolved in precipitation in the phosphate buffer solution ultrasonic dispersion;
(1-4) repeating step (1-3) centrifugal, go clearly, resuspended process, in supernatant liquor, do not contain the sulfydryl undecanoic acid, the nm of gold of the sulfydryl undecanoic acid root protection of collecting centrifugal is scattered in the phosphate buffer solution standby;
Step (2) specifically comprises the steps:
(2-1) phosphate buffer solution and 1-ethyl-3-(3-dimethyl amine propyl group) carbodiimide hydrochloride (EDAC), N-hydroxy-succinamide (NHS) and the restriction endonuclease of the nm of gold that the sulfydryl undecanoic acid root that obtains in the step (1) is protected join in 2-(N-morpholino) ethyl sulfonic acid (MES) buffer solution, normal temperature leaves standstill reaction 2-4 hour, obtains mixed liquor;
(2-2) the mixed liquor centrifuging that step (2-1) is obtained behind the removal supernatant liquor, is dissolved in precipitation in the phosphate buffer solution ultrasonic dispersion;
(2-3) repeating step (2-2) centrifugal, go clearly, resuspended process, in supernatant liquor, do not contain EDAC, NHS, MES and restriction endonuclease;
Nm of gold-protein the compound substance with enzymatic performance is collected in (2-4) centrifuging, and the nm of gold with the enzymatic performance-protein compound substance that obtains is scattered in the phosphate buffer solution and gets final product.
Further, in the step (1-2), the concentration that is scattered in the nm of gold colloid in the phosphate buffer solution that contains polysorbas20 is 4-5nmol/L.
Further, phosphate buffer solution need add and contain polysorbas20 in the step (1-2), contains in the phosphate buffer solution of polysorbas20, and polysorbas20 concentration is 0.1-1mg/mL.
Further, the middle concentration that adds sulfydryl undecanoic acid in the sulfydryl undecanoic acid aqueous solution of step (1-2) is 1-10mmol/L.
Further, in the step (1-4), the concentration that is scattered in the nm of gold of the sulfydryl undecanoic acid root protection in the phosphate buffer solution is 4-5nmol/L.
Further, the EDAC that adds in step (2-1) mixed liquor and the mol ratio of NHS are 1: (1-5); Need in the step (2-1) with 2-(N-morpholino) ethyl sulfonic acid (MES) buffer solution, its solvent is ultrapure water (resistivity is 18m Ω cm); The concentration of 2-(N-morpholino) ethyl sulfonic acid MES in step (2-1) mixed liquor is 5-20mmol/L.MES has the stability that helps enzyme in the coupled reaction process, after reaction finishes, removes with centrifugal method and to get final product.
Further, in step (2-1) mixed liquor, the concentration of the nm of gold of sulfydryl undecanoic acid root protection is 2-3nmol/L, and the concentration of restriction endonuclease is 1000-5000U/mL.
Further, in the step (2-4), be scattered in the concentration 4-5nmol/L of the nm of gold with the enzymatic performance-protein compound substance in the phosphate buffer solution.
Further, step (1-2), (1-3), (1-4), (2-2) and (2-4) in used buffer solution be phosphate buffer solution, its solute concentration is 5-20mmol/L.
Further, above-mentioned restriction endonuclease is restriction enzyme, is selected from EcoRI endonuclease, Nt.AlwI nicking restriction endonuclease, EcoRV restriction endonuclease, SmaI restriction endonuclease, BamHI restriction endonuclease, HhaI restriction endonuclease, XcmI restriction endonuclease and BbvCI restriction endonuclease.Above-mentioned restriction endonuclease all can directly be purchased in market.
The present invention compared with prior art has following characteristics:
The preparation method that the present invention has a nm of gold-protein compound substance of enzymatic performance does not relate to the complicated chemical of enzyme is modified, and does not influence its activity; And be reflected at room temperature and carry out, mild condition has been avoided chemical reaction and the purge process of a lot of complexity.The final nm of gold with the enzymatic performance-protein composite finished product that obtains is not easy to be subjected to the influence of solution medium; carboxyl on the nm of gold of the amido on its zymoprotein and the protection of sulfydryl undecanoic acid root carries out cross-linking reaction; produce firm covalent bond, had good stability.
Description of drawings
Fig. 1 has the synthetic route synoptic diagram of the nm of gold-protein compound substance of enzymatic performance for the present invention's preparation;
Fig. 2 is nm of gold colloid of the present invention, the nm of gold of sulfydryl undecanoic acid root (MUA) protection and the ultraviolet-visible absorption spectroscopy figure of nm of gold-protein compound substance.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment, should be understood that these embodiment only are used for explanation the present invention and are not used in restriction protection scope of the present invention.
Nm of gold of the present invention can be prepared by well known to a person skilled in the art technology, K.C.Grabar for example, R.G.Freeman, M.B.Hommer, M.J.Natan, Anal.Chem.1995,67,735., reported specifically in the document that sodium citrate reduction gold chloride prepares the reaction that diameter is the nm of gold about 10 nanometers.
Used buffer solution, sulfydryl undecanoic acid, 1-ethyl-3-(3-dimethyl amine propyl group) carbodiimide hydrochloride, the N-hydroxy-succinamide of the present invention purchased the company in the Ao Liqi of Sigma (Sigma Aldrich), and endonuclease is all purchased the Inc in New England Biolabs.
Embodiment 1
Preparation with nm of gold-EcoRI endonuclease compound substance of enzymatic performance, its preparation route as shown in Figure 1.
1, preparation nm of gold colloid:
(1) 50mL chlorauric acid solution (1mmol/L) is heated to boiling.
(2) sodium citrate solution of 5mL prepared fresh (sodium citrate concentration is 1wt%) joins rapidly in the chlorauric acid solution, waits solution colour to become redness, continues reflux solution and obtains the nm of gold colloid in 20 minutes, and the diameter of nm of gold is about 10 nanometers.
2, the nm of gold of preparation sulfydryl undecanoic acid root (MUA) protection:
(2-1) the nm of gold colloid that step (1) is obtained purges the degassing 30 seconds with nitrogen.
(2-2) the nm of gold colloid of getting after 450 μ L outgas is scattered in the phosphate buffer solution that contains polysorbas20 of 450 μ L, and stable at least 20 minutes, add sulfydryl undecanoic acid solution 100 μ L then, left standstill under the normal temperature 4 hours, obtain mixed liquor; Wherein, the concentration that contains polysorbas20 in the phosphate buffer solution of polysorbas20 is 0.5mg/mL, and the solute concentration in the phosphate buffer solution is 10mM; The concentration of sulfydryl undecanoic acid is 5mM in the sulfydryl undecanoic acid solution.
(2-3) mixed liquor centrifugal (16000rpm) that step (2-2) is obtained is removed the upper strata after the stillness of night, precipitation is dissolved in the phosphate buffer solution of 10mM ultrasonic dispersion.
(2-4) repeating step (2-3) centrifugal, go clearly, resuspended process, do not contain the sulfydryl undecanoic acid in the stillness of night until the upper strata, the nm of gold of the sulfydryl undecanoic acid root protection of collecting centrifugal is scattered in the phosphate buffer solution (10mM) of 1mL standby.
3, prepare the nm of gold-EcoRI endonuclease compound substance with enzymatic performance:
(3-1) (MES concentration is 0.1M in the MES buffer solution for the nm of gold that the sulfydryl undecanoic acid root that obtains in the 500 μ L steps (2) is protected and 2-(N-morpholino) the ethyl sulfonic acid buffer solution of 100 μ L; pH=5.3), 1-ethyl-3-of 5 μ L (3-dimethyl amine propyl group) carbodiimide hydrochloride (EDAC; 0.1M); N-hydroxy-succinamide (the NHS of 10 μ L; 0.1M), the EcoRI endonuclease (100000U/mL) of 50 μ L and 335 μ L ultrapure waters (its resistivity is 18m Ω cm) mix; leave standstill reaction 2 hours under the normal temperature, obtain mixed liquor.
The upper strata stillness of night is removed in (3-2) the mixed liquor centrifuging that step (2-1) is obtained, and precipitation is dissolved in the phosphate buffer solution ultrasonic dispersion;
(3-3) repeating step (3-2) centrifugal, go clearly, resuspended process, in supernatant liquor, do not contain EDAC, NHS, MES and enzyme.
Nm of gold-protein the compound substance with enzymatic performance is collected in (3-4) centrifuging, and the nm of gold with enzymatic performance-protein compound substance of collecting is scattered in 500 μ L phosphate buffer solutions, and (10mM gets final product in pH=7).
The nm of gold of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root (MUA) protection and the ultraviolet-visible absorption spectroscopy of nm of gold-EcoRI endonuclease compound substance are as shown in Figure 2.Modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the EcoRI as can be seen from Figure 2, illustrate that the character of collaurum is not affected.
The nm of gold of present embodiment-EcoRI endonuclease compound substance identifies that through λ DNA its EcoRI endonuclease activity is not affected.
Embodiment 2
Have the preparation of the nm of gold-Nt.AlwI nicking restriction endonuclease compound substance of enzymatic performance, dezymotize to outside the Nt.AlwI restriction endonuclease, other steps are identical with embodiment 1.
Nm of gold and the nm of gold-Nt.AlwI nicking restriction endonuclease compound substance of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root MUA protection obtain ultraviolet-visible absorption spectroscopy figure after testing; from this collection of illustrative plates, modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the Nt.AlwI as can be known, illustrate that the character of collaurum is not affected.
The nm of gold of present embodiment-Nt.AlwI nicking restriction endonuclease compound substance identifies that through supercoil pUC101DNA (dam-/dcm-) its Nt.AlwI nicking endonuclease activity is not affected.
Embodiment 3
Have the preparation of the nm of gold-EcoRV restriction enzyme compound substance of enzymatic performance, dezymotize to outside the EcoRV restriction endonuclease, other steps are identical with embodiment 1.
Nm of gold and the nm of gold-EcoRV restriction enzyme compound substance of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root MUA protection obtain ultraviolet-visible absorption spectroscopy figure after testing; from this collection of illustrative plates, modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the EcoRV as can be known, illustrate that the character of collaurum is not affected.
The nm of gold of present embodiment-EcoRV restriction enzyme compound substance identifies that through λ DNA its EcoRV restriction enzyme enzymatic activity is not affected.
Embodiment 4
Have the preparation of the nm of gold-SmaI restriction enzyme compound substance of enzymatic performance, dezymotize to outside the SmaI restriction endonuclease, other steps are identical with embodiment 1.
Nm of gold and the nm of gold-SmaI restriction enzyme compound substance of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root MUA protection obtain ultraviolet-visible absorption spectroscopy figure after testing; from this collection of illustrative plates, modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the SmaI as can be known, illustrate that the character of collaurum is not affected.
The nm of gold of present embodiment-SmaI restriction enzyme compound substance identifies that through λ DNA (HindIII digestion) its SmaI restriction enzyme enzymatic activity is not affected.
Embodiment 5
Have the preparation of the nm of gold-BamHI restriction enzyme compound substance of enzymatic performance, dezymotize to outside the BamHI restriction endonuclease, other steps are identical with embodiment 1.
Nm of gold and the nm of gold-BamHI restriction enzyme compound substance of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root MUA protection obtain ultraviolet-visible absorption spectroscopy figure after testing; from this collection of illustrative plates, modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the BamHI as can be known, illustrate that the character of collaurum is not affected.
The nm of gold of present embodiment-BamHI restriction enzyme compound substance identifies that through λ DNA its BamHI restriction enzyme enzymatic activity is not affected.
Embodiment 6
Have the preparation of the nm of gold-HhaI restriction enzyme compound substance of enzymatic performance, dezymotize to outside the HhaI restriction endonuclease, other steps are identical with embodiment 1.
Nm of gold and the nm of gold-HhaI restriction enzyme compound substance of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root MUA protection obtain ultraviolet-visible absorption spectroscopy figure after testing; from this collection of illustrative plates, modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the HhaI as can be known, illustrate that the character of collaurum is not affected.
The nm of gold of present embodiment-HhaI restriction enzyme compound substance identifies that through λ DNA its HhaI restriction enzyme enzymatic activity is not affected.
Embodiment 7
Have the preparation of the nm of gold-XcmI restriction enzyme compound substance of enzymatic performance, dezymotize to outside the XcmI restriction endonuclease, other steps are identical with embodiment 1.
Nm of gold and the nm of gold-XcmI restriction enzyme compound substance of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root MUA protection obtain ultraviolet-visible absorption spectroscopy figure after testing; from this collection of illustrative plates, modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the XcmI as can be known, illustrate that the character of collaurum is not affected.
The nm of gold of present embodiment-XcmI restriction enzyme compound substance identifies that through λ DNA its XcmI restriction enzyme enzymatic activity is not affected.
Embodiment 8
Have the preparation of the nm of gold-BbvCI restriction enzyme compound substance of enzymatic performance, dezymotize to outside the BbvCI restriction endonuclease, other steps are identical with embodiment 1.
Nm of gold and the nm of gold-BbvCI restriction enzyme compound substance of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root MUA protection obtain ultraviolet-visible absorption spectroscopy figure after testing; from this collection of illustrative plates, modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the BbvCI as can be known, illustrate that the character of collaurum is not affected.
The nm of gold of present embodiment-BbvCI restriction enzyme compound substance identifies that through λ DNA its BbvCI restriction enzyme enzymatic activity is not affected.
Embodiment 9
Preparation with nm of gold-EcoRI endonuclease compound substance of enzymatic performance:
1, preparation nm of gold colloid, its preparation method is identical with embodiment 1:
2, the nm of gold of preparation sulfydryl undecanoic acid root (MUA) protection:
(2-1) the nm of gold colloid that step (1) is obtained purges the degassing 60 seconds with nitrogen.
(2-2) the nm of gold colloid after will outgasing is scattered in the phosphate buffer solution that contains polysorbas20, the concentration that is scattered in the nm of gold colloid in the phosphate buffer solution that contains polysorbas20 is 5nmol/L, stable at least 20 minutes, add sulfydryl undecanoic acid aqueous solution then, wherein the mol ratio of nm of gold colloid and sulfydryl undecanoic acid is 1: 150, left standstill under the normal temperature 3 hours, and obtained mixed liquor; Wherein, the concentration that contains polysorbas20 in the phosphate buffer solution of polysorbas20 is 1mg/mL, and the solute concentration in the phosphate buffer solution is 10mM; The concentration of sulfydryl undecanoic acid is 8mM in the sulfydryl undecanoic acid solution.
(2-3) mixed liquor centrifugal (16000rpm) that step (2-2) is obtained is removed the upper strata after the stillness of night, precipitation is dissolved in the phosphate buffer solution of 10mM ultrasonic dispersion.
(2-4) repeating step (2-3) centrifugal, go clearly, resuspended process; do not contain the sulfydryl undecanoic acid in the stillness of night until the upper strata; standby in the phosphate buffer solution (its solute concentration 10mM) that the nm of gold that the centrifugal sulfydryl undecanoic acid root of collecting is protected is scattered in, the concentration that is scattered in the nm of gold of the sulfydryl undecanoic acid root protection in the phosphate buffer solution is 4nmmol/L.
3, prepare the nm of gold-EcoRI endonuclease compound substance with enzymatic performance:
(3-1) nm of gold and MES, EDAC, NHS, EcoRI endonuclease (100000U/mL) and ultrapure water (its resistivity is 18m Ω cm) that the sulfydryl undecanoic acid root that obtains in the step (2) is protected) mix, leave standstill reaction 3 hours under the normal temperature, obtain mixed liquor; Wherein in the mixed liquor, the volumetric molar concentration of MES is 5mmol/L, and the mol ratio of EDAC and NHS is 1: 5, and the concentration of the nm of gold of sulfydryl undecanoic acid root protection is 2nmol/L, and the concentration of EcoRI endonuclease is 1000U/mL.
The upper strata stillness of night is removed in (3-2) the mixed liquor centrifuging that step (2-1) is obtained, and precipitation is dissolved in the phosphate buffer solution ultrasonic dispersion;
(3-3) repeating step (3-2) centrifugal, go clearly, resuspended process, in supernatant liquor, do not contain EDAC, NHS, MES and enzyme.
(3-4) centrifuging, collection has the nm of gold-protein compound substance of enzymatic performance, the nm of gold with enzymatic performance-EcoRI endonuclease compound substance of collecting is scattered in phosphate buffer solution (10mM, pH=7) get final product in, the concentration that is scattered in the nm of gold with the enzymatic performance-EcoRI endonuclease compound substance in the phosphate buffer solution is 4nmmol/L.
Nm of gold and the nm of gold-EcoRI endonuclease compound substance of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root (MUA) protection obtain ultraviolet-visible absorption spectroscopy after testing.From ultraviolet-visible absorption spectroscopy, modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the EcoRI as can be known, illustrate that the activity of collaurum is not affected.
The nm of gold of present embodiment-EcoRI endonuclease compound substance identifies that through λ DNA its EcoRI endonuclease activity is not affected.
Embodiment 10
Preparation with nm of gold-EcoRI endonuclease compound substance of enzymatic performance:
1, preparation nm of gold colloid, its preparation method is identical with embodiment 1:
2, the nm of gold of preparation sulfydryl undecanoic acid root (MUA) protection:
(2-1) the nm of gold colloid that step (1) is obtained purges the degassing 10 seconds with nitrogen.
(2-2) the nm of gold colloid after will outgasing is scattered in the phosphate buffer solution that contains polysorbas20, the concentration that is scattered in the nm of gold colloid in the phosphate buffer solution that contains polysorbas20 is 10nmol/L, stable at least 20 minutes, add sulfydryl undecanoic acid aqueous solution then, wherein the mol ratio of nm of gold colloid and sulfydryl undecanoic acid is 1: 200, left standstill under the normal temperature 2 hours, and obtained mixed liquor; Wherein, the concentration that contains polysorbas20 in the phosphate buffer solution of polysorbas20 is 0.8mg/mL, and the solute concentration in the phosphate buffer solution is 10mM; The concentration of sulfydryl undecanoic acid is 1mM in the sulfydryl undecanoic acid solution.
(2-3) mixed liquor centrifugal (16000rpm) that step (2-2) is obtained is removed the upper strata after the stillness of night, precipitation is dissolved in the phosphate buffer solution of 10mM ultrasonic dispersion.
(2-4) repeating step (2-3) centrifugal, go clearly, resuspended process; do not contain the sulfydryl undecanoic acid in the stillness of night until the upper strata; standby in the phosphate buffer solution (its solute concentration 10mM) that the nm of gold that the centrifugal sulfydryl undecanoic acid root of collecting is protected is scattered in, the concentration that is scattered in the nm of gold of the sulfydryl undecanoic acid root protection in the phosphate buffer solution is 5nmmol/L.
3, prepare the nm of gold-EcoRI endonuclease compound substance with enzymatic performance:
(3-1) nm of gold that the sulfydryl undecanoic acid root that obtains in the step (2) is protected is mixed with MES, EDAC, NHS, EcoRI endonuclease (100000U/mL) and ultrapure water (its resistivity is 18m Ω cm), leave standstill reaction 4 hours under the normal temperature, obtain mixed liquor; Wherein in the mixed liquor, the volumetric molar concentration of MES is 20mmol/L, and the mol ratio of EDAC and NHS is 1: 1, and the concentration of the nm of gold of sulfydryl undecanoic acid root protection is 3nmol/L, and the concentration of EcoRI endonuclease is 2500U/mL.
The upper strata stillness of night is removed in (3-2) the mixed liquor centrifuging that step (2-1) is obtained, and precipitation is dissolved in the phosphate buffer solution ultrasonic dispersion;
(3-3) repeating step (3-2) centrifugal, go clearly, resuspended process, in supernatant liquor, do not contain EDAC, NHS, MES and enzyme.
(3-4) centrifuging, collection has the nm of gold-protein compound substance of enzymatic performance, the nm of gold with enzymatic performance-protein compound substance of collecting is scattered in phosphate buffer solution (10mM, pH=7) get final product in, the concentration that is scattered in the nm of gold with the enzymatic performance-EcoRI endonuclease compound substance in the phosphate buffer solution is 5nmmol/L.
Nm of gold and the nm of gold-EcoRI endonuclease compound substance of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root (MUA) protection obtain ultraviolet-visible absorption spectroscopy after testing.From ultraviolet-visible absorption spectroscopy, modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the EcoRI as can be known, illustrate that the activity of collaurum is not affected.
The nm of gold of present embodiment-EcoRI endonuclease compound substance identifies that through λ DNA its EcoRI endonuclease activity is not affected.
Embodiment 11
Preparation with nm of gold-EcoRI endonuclease compound substance of enzymatic performance:
1, preparation nm of gold colloid, its preparation method is identical with embodiment 1:
2, the nm of gold of preparation sulfydryl undecanoic acid root (MUA) protection:
(2-1) the nm of gold colloid that step (1) is obtained purges the degassing 40 seconds with nitrogen.
(2-2) the nm of gold colloid after will outgasing is scattered in the phosphate buffer solution that contains polysorbas20, the concentration that is scattered in the nm of gold colloid in the phosphate buffer solution that contains polysorbas20 is 8nmol/L, stable at least 20 minutes, add sulfydryl undecanoic acid aqueous solution then, wherein the mol ratio of nm of gold colloid and sulfydryl undecanoic acid is 1: 100, left standstill under the normal temperature 4 hours, and obtained mixed liquor; Wherein, the concentration that contains polysorbas20 in the phosphate buffer solution of polysorbas20 is 0.1mg/mL, and the solute concentration in the phosphate buffer solution is 10mM; The concentration of sulfydryl undecanoic acid is 10mM in the sulfydryl undecanoic acid solution.
(2-3) mixed liquor centrifugal (16000rpm) that step (2-2) is obtained is removed the upper strata after the stillness of night, precipitation is dissolved in the phosphate buffer solution of 10mM ultrasonic dispersion.
(2-4) repeating step (2-3) centrifugal, go clearly, resuspended process; do not contain the sulfydryl undecanoic acid in the stillness of night until the upper strata; standby in the phosphate buffer solution (its solute concentration 10mM) that the nm of gold that the centrifugal sulfydryl undecanoic acid root of collecting is protected is scattered in, the concentration that is scattered in the nm of gold of the sulfydryl undecanoic acid root protection in the phosphate buffer solution is 4.5nmmol/L.
3, prepare the nm of gold-EcoRI endonuclease compound substance with enzymatic performance:
(3-1) nm of gold that the sulfydryl undecanoic acid root that obtains in the step (2) is protected is mixed with MES, EDAC, NHS, EcoRI endonuclease (100000U/mL) and ultrapure water (its resistivity is 18m Ω cm), leave standstill reaction 2 hours under the normal temperature, obtain mixed liquor; Wherein in the mixed liquor, the volumetric molar concentration of MES is 10mmol/L, and the mol ratio of EDAC and NHS is 1: 3, and the concentration of the nm of gold of sulfydryl undecanoic acid root protection is 2.5nmol/L, and the concentration of EcoRI endonuclease is 5000U/mL.
The upper strata stillness of night is removed in (3-2) the mixed liquor centrifuging that step (2-1) is obtained, and precipitation is dissolved in the phosphate buffer solution ultrasonic dispersion;
(3-3) repeating step (3-2) centrifugal, go clearly, resuspended process, in supernatant liquor, do not contain EDAC, NHS, MES and enzyme.
(3-4) centrifuging, collection has the nm of gold-protein compound substance of enzymatic performance, the nm of gold with enzymatic performance-protein compound substance of collecting is scattered in phosphate buffer solution (10mM, pH=7) get final product in, the concentration that is scattered in the nm of gold with the enzymatic performance-EcoRI endonuclease compound substance in the phosphate buffer solution is 4.5nmmol/L.
Nm of gold and the nm of gold-EcoRI endonuclease compound substance of the nm of gold colloid that present embodiment obtains, sulfydryl undecanoic acid root (MUA) protection obtain ultraviolet-visible absorption spectroscopy after testing.From ultraviolet-visible absorption spectroscopy, modify the nm of gold colloid compound substance no tangible plasma peak shift in ultraviolet-visible absorption spectroscopy behind the EcoRI as can be known, illustrate that the character of collaurum is not affected.
The nm of gold of present embodiment-EcoRI endonuclease compound substance identifies that through λ DNA its EcoRI endonuclease activity is not affected.
Claims (10)
1. preparation method with nm of gold-protein compound substance of enzymatic performance comprises:
(1) preparation of the nm of gold of sulfydryl undecanoic acid root protection;
The nm of gold of the sulfydryl undecanoic acid root protection that (2) step (1) is obtained is connected the described nm of gold-protein compound substance with enzymatic performance of preparation with restriction endonuclease;
Wherein, step (1) specifically comprises the steps:
(1-1) the nm of gold colloid is purged degassing 10-60 second with nitrogen;
(1-2) the nm of gold colloid after will outgasing is scattered in the phosphate buffer solution that contains polysorbas20, and stable at least 20 minutes, add sulfydryl undecanoic acid aqueous solution then, left standstill under the normal temperature 2-4 hour, obtain mixed liquor; Wherein, the mol ratio of nm of gold colloid and sulfydryl undecanoic acid is 1: (100-200);
(1-3) the mixed liquor centrifuging that step (1-2) is obtained behind the removal supernatant liquor, is dissolved in precipitation in the phosphate buffer solution ultrasonic dispersion;
(1-4) repeating step (1-3) centrifugal, go clearly, resuspended process, in supernatant liquor, do not contain the sulfydryl undecanoic acid, the nm of gold of the sulfydryl undecanoic acid root protection of collecting centrifugal is scattered in the phosphate buffer solution standby;
Step (2) specifically comprises the steps:
(2-1) phosphate buffer solution of the nm of gold that the sulfydryl undecanoic acid root that obtains in the step (1) is protected and 1-ethyl-3-(3-dimethyl amine propyl group) carbodiimide hydrochloride EDAC, N-hydroxy-succinamide NHS and restriction endonuclease join in 2-(N-morpholino) the ethyl sulfonic acid MES buffer solution, normal temperature leaves standstill reaction 2-4 hour, obtains mixed liquor;
(2-2) the mixed liquor centrifuging that step (2-1) is obtained behind the removal supernatant liquor, is dissolved in precipitation in the phosphate buffer solution ultrasonic dispersion;
(2-3) repeating step (2-2) centrifugal, go clearly, resuspended process, in supernatant liquor, do not contain EDAC, NHS, MES and restriction endonuclease;
Nm of gold-protein the compound substance with enzymatic performance is collected in (2-4) centrifuging, and the nm of gold with the enzymatic performance-protein compound substance that obtains is scattered in the phosphate buffer solution and gets final product.
2. the preparation method with nm of gold-protein compound substance of enzymatic performance as claimed in claim 1 is characterized in that, in the step (1-2), the concentration that is scattered in the nm of gold colloid in the phosphate buffer solution that contains polysorbas20 is 5-10nmol/L.
3. the preparation method with nm of gold-protein compound substance of enzymatic performance as claimed in claim 1 is characterized in that, in the step (1-2), contains in the phosphate buffer solution of polysorbas20, and polysorbas20 concentration is 0.1-1mg/mL; The concentration of sulfydryl undecanoic acid is 1-10mmol/L in the sulfydryl undecanoic acid aqueous solution.
4. the preparation method with nm of gold-protein compound substance of enzymatic performance as claimed in claim 1 is characterized in that, in the step (1-4), the concentration that is scattered in the nm of gold of the sulfydryl undecanoic acid root protection in the phosphate buffer solution is 4-5nmol/L.
5. the preparation method with nm of gold-protein compound substance of enzymatic performance as claimed in claim 1 is characterized in that, the EDAC that adds in step (2-1) mixed liquor and the mol ratio of NHS are 1: (1-5); The concentration of 2-(N-morpholino) ethyl sulfonic acid MES is 5-20mmol/L in step (2-1) mixed liquor.
6. the preparation method with nm of gold-protein compound substance of enzymatic performance as claimed in claim 1; it is characterized in that; in step (2-1) mixed liquor, the concentration of the nm of gold of sulfydryl undecanoic acid root protection is 2-3nmol/L, and the concentration of restriction endonuclease is 1000-5000U/mL.
7. the preparation method with nm of gold-protein compound substance of enzymatic performance as claimed in claim 1, it is characterized in that, in the step (2-4), be scattered in the concentration 4-5nmol/L of the nm of gold with the enzymatic performance-protein compound substance in the phosphate buffer solution.
8. the preparation method with nm of gold-protein compound substance of enzymatic performance as claimed in claim 1, it is characterized in that, step (1-2), (1-3), (1-4), (2-2) and (2-4) in used buffer solution be phosphate buffer solution, its solute concentration is 5-20mmol/L.
9. as the arbitrary described preparation method with nm of gold-protein compound substance of enzymatic performance of claim 1-8, it is characterized in that, described restriction endonuclease is restriction enzyme, is selected from EcoRI endonuclease, Nt.AlwI nicking restriction endonuclease, EcoRV restriction endonuclease, SmaI restriction endonuclease, BamHI restriction endonuclease, HhaI restriction endonuclease, XcmI restriction endonuclease and BbvCI restriction endonuclease.
10. nm of gold-protein compound substance with enzymatic performance is for making according to the arbitrary described preparation method with nm of gold-protein compound substance of enzymatic performance of claim 1-9.
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