CN102230896B - Method for quantitative detection of ultramicro-protein based on principle of biomineralization - Google Patents
Method for quantitative detection of ultramicro-protein based on principle of biomineralization Download PDFInfo
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- CN102230896B CN102230896B CN 201110089936 CN201110089936A CN102230896B CN 102230896 B CN102230896 B CN 102230896B CN 201110089936 CN201110089936 CN 201110089936 CN 201110089936 A CN201110089936 A CN 201110089936A CN 102230896 B CN102230896 B CN 102230896B
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Abstract
The invention discloses a method for quantitative detection of ultramicro-protein based on the principle of nano gold particle biomineralization, which comprises the following steps: firstly, by taking the first generation of PPIHA dendrimer and chloroauric acid (HAuCl4) as raw materials, preparing nano gold colloid with surface modified by amine by means of a microwave reflux heating method, and combining the nano gold colloid with to-be-detected protein to form conjugate at a normal temperature; secondly, carrying out biomineralization on the nano gold-protein conjugate in a sodium citrate-chloroauric acid system; and then carrying out resonance scattering spectral analysis on the biomineralization product by using a fluorescence spectrum spectrograph. Because of the linear relation between the peak strength of the resonance scattering spectrum of the product at the characteristic wavelength and the protein concentration, the ultramicro-protein can be quantitatively detected. The method is easy and quick and has high sensitivity, high specificity and high accuracy. In addition, the method has a good portability and can be used for early tumor diagnosis, quick food safety detection, water quality monitoring and the like.
Description
Technical field
The present invention relates to the ultramicron quantification of protein detection method based on the biomineralization principle.
Background technology
In recent years, the noble metal nano particulate is widely used in the biological detection analytical technology, for important impetus has been played in the development of life science.Be core with the noble metal nano particulate, develop and a series of new bio analysis of molecules detection techniques, in each relevant research field of biomedicine, particularly aspects such as ultramicron molecular recognition, early diagnosis of tumor, food security fast detecting, water quality monitoring play an important role, and the development of ultramicron biomolecule analysis technology has been produced far-reaching influence.
Wherein, nm of gold (AuNPs) is widely used in the design of Photobiology probe with the dependent physicochemical characteristics of its yardstick.The design of nm of gold optical probe is core with using common nm of gold with finishing bio-identification sensing assembly, carries out bio-identification and realizes covalent coupling with detected object.The specificity of molecular recognition component and target molecule is combined in the nm of gold microparticle surfaces and produces corresponding physics (chemistry) variation, and then conversion is enlarged into detectable optics or electrical signal.Thereby, the strong approach that is designed to trace biomolecular identification detection of nano gold biological optical probe.
Yet, rely on electrostatic interaction or surface tension effects between the nm of gold of existing surperficial unmodified and testing protein, with non-covalent mode coupling, be easy to dissociate the company of sticking, the agglomerations of existing more, and to the surrounding environment sensitivity, reduce the sensitivity and the specificity that detect.And the recognition capability to specific objective albumen of existing quantification of protein technology is limited, and detection sensitivity is lower, less stable, and sense cycle is long, the cost height.Address the above problem, the detection reagent that preparation cost is cheap is set up corresponding simple analyzing method simultaneously just becomes pressing for of market.
Summary of the invention
The object of the present invention is to provide a kind of ultramicron quantification of protein detection method based on nm of gold particulate biomineralization principle.
Technical scheme of the present invention is achieved in that
(1), the nm of gold seed of surface amination preparation
With tree-shaped body (PPIHA) and gold chloride (HAuCl
4) be raw material, use microwave backflow heating to prepare the nm of gold seed colloidal sol of surface amination.With 1-2mL, 0.67% chlorauric acid solution is dissolved in the 100-150mL ultrapure water, adds 85-95 μ L, the toluene solution of 5%PPIHA; After fully mixing, with microwave backflow heating 3-6min, make the nm of gold seed colloidal sol of surface amination, 4 ℃ of sealings are preserved.In this step on the one hand, PPIHA makes Au in the gold chloride as reductive agent
3+Be reduced to the Au nanoparticle; On the other hand, PPIHA is at reduction Au
3+The time nm of gold surface carried out the amination modification, be beneficial to it with the covalent coupling of protein.Thus, make the nm of gold seed colloidal sol of surface amination.
(2), nm of gold-protein conjugate (AuNPs-BSA) preparation
With 180-200 μ L, 1% bovine serum albumin(BSA) (Bovine serum albumin, BSA) solution joins in the nm of gold seed colloidal sol that 3-5mL step (1) makes, and adds 110-130 μ L 1% hydrochloric acid, shake up the back and be diluted to 6ml with ultrapure water, make the AuNPs-BSA conjugate.Under these conditions, the amino acid residues such as surperficial carboxyl of nm of gold surface amino groups and BSA mutually combine by covalent coupling, make the AuNPs-BSA conjugate then.
(3), the biomineralization of AuNPs-BSA conjugate
For realizing the mineralising of AuNPs-BSA conjugate, getting 1-2mL AuNPs-BSA conjugate is that 7.0 phosphate buffers mix with 0.6-0.8mL pH, shakes up, and is diluted to 10mL with ultrapure water, standby.
Get the above-mentioned AuNPs-BSA conjugate of a certain amount of (in the 0-180 μ L scope), join in the 3.00mL ultrapure water.Add 1-2mL 0.02% chlorauric acid solution then successively, sodium citrate-phosphate buffer of 1-3mL pH 2.26 and 2-3mL 8.0 * 10
-3Oxammonium hydrochloride solution (the NH of mol/L
2OHHCL), be diluted to 8-10mL with ultrapure water again.Hatch 18-23min with being placed on 38 ℃, the biomineralization product that obtains the AuNPs-BSA conjugate for detection of.In this process, the BSA of nm of gold surface coupling forms the Au in the specific electric field attracts solution
3+Ion is at nm of gold conjugate surface enrichment, makes the reaction velocity of nm of gold surface gold chloride-oxammonium hydrochloride long response time system accelerate, and impels gold chloride to be reduced to golden simple substance under the effect of oxammonium hydrochloride and is deposited on nm of gold conjugate surface.AuNPs-BSA conjugate generation biomineralization reaction, Au
3+Become Au at nm of gold conjugate surface reduction, selectivity has enlarged the particle diameter of nm of gold conjugate, the corresponding characteristic peaks intensity that strengthens the compound resonance scattering spectroscopy.In 0-180 μ L scope, maintenance favorable linearity corresponding relation between the characteristic peaks intensity BSA concentration of nm of gold conjugate biomineralization product, thereby the resonance scattering spectroscopy characteristic peaks intensity that can utilize product is used for the quantitative detection of BSA as detection signal.
(4), resonance scattering spectroscopy working curve preparation
In 0-180 μ L scope, 10 μ L at interval make the biomineralization product of the AuNPs-BSA conjugate of a series of variable concentrations according to the described method of step (3); In the spectral range of 200nm to 800nm, measure the resonance scattering spectroscopy of product, get the intensity of characteristic peak BSA concentration is done curve, obtain to detect the working curve of 0-180 μ L BSA.This curve can be used for the quantitative detection of ultramicron protein.
(5), measure the actual sample method
According to step (2), 180 μ L BSA solution to be measured is made the AuNPs-BSA conjugate.Obtain its biomineralization product according to step (4), detect its resonance scattering spectroscopy, read respective concentration at working curve.
It is the nano gold sol that raw material one step preparation surface amino groups is modified that the key problem in technology that the present invention solves is to select tree-shaped body and gold chloride, and the nm of gold uniform particle diameter that makes is evenly distributed.Simultaneously, use the method for biomineralization to realize that the selectivity of nm of gold-protein conjugate resonance scattering signal significantly strengthens, improved the selectivity and the sensitivity that detect, guaranteed specificity and the stability of this method quantitative test.The present invention is not limited only to the quantitative detection of ultramicron BSA, also can promote the quantitative test for other trace protein.At different protein, need the corresponding pH value condition of nm of gold in the regulating step (2)-protein conjugate preparation and the minimum amount that conjugate forms desired protein, prepare corresponding stabilized nanoscale gold conjugate then and carry out subsequent step.
Effect of the present invention is embodied in:
(1) preparation method of the surface amino groups decorated nanometer aurosol of the present invention's proposition is starting material with tree-shaped body and gold chloride, utilizes one step of microwave backflow heating to make, and equipment requires low, and preparation technology is simple and direct, and cost is low, is easy to realize industrialization production.
(2) adopt the prepared nano gold sol system of the technology of the present invention, nm of gold diameter of particle homogeneous (13-16nm), dispersion degree height and optical property are stable.
(3) the present invention has solved nm of gold-protein conjugate poor stability, easy shortcoming of reuniting in the mode of covalent coupling.Utilize the biomineralization method to realize that the selectivity of nm of gold-protein conjugate resonance light scattering spectrum signal strengthens, the characteristic peaks signal strengthens 322 times.
(4) the ultramicron quantification of protein sensitivity of analytical method height of the present invention's foundation, detection limit is low to moderate 3.00 * 10
-9Mol/L is far below existing commercialization quantification of protein analytical approach.Simultaneously, the sensing range of this method is 3.00 * 10
-9Mol/L to 2.40 * 10
-8Mol/L is better than similar detection method in the market, and detection time is short, detects stability and accuracy height.
Embodiment
Embodiment one:
With tree-shaped body (PPIHA) and gold chloride (HAuCl
4) be raw material, microwave backflow heating prepares the nm of gold seed colloidal sol of surface amination.With 1.5mL, 0.67% chlorauric acid solution is dissolved in the 100mL ultrapure water, adds 90 μ L, the toluene solution of 5%PPIHA; After fully mixing, with microwave backflow heating 5min.
Add 180 μ L in the nm of gold seed colloidal sol that makes to 4mL, (Bovineserum albumin, BSA) solution add 120 μ L, 1% hydrochloric acid to 1% bovine serum albumin(BSA) again, shake up the back and are diluted to 6ml with ultrapure water, and reaction generates the AuNPs-BSA conjugate.
Getting 1.8mL AuNPs-BSA conjugate is that 7.0 phosphate buffers mix with 0.6mL pH, shakes up, and is diluted to 10mL with ultrapure water, standby.10 μ L get the above-mentioned AuNPs-BSA conjugate of a certain amount of (in the 0-180 μ L scope) at interval, join in the 3.00mL ultrapure water.Add 2mL 0.02% chlorauric acid solution then successively, the oxammonium hydrochloride solution of sodium citrate-phosphate buffer of 2mL pH 2.26 and 2.4mL 8.0 * 10-3mol/L, be diluted to 10mL with ultrapure water again, hatch the biomineralization product that 20min makes the AuNPs-BSA conjugate of a series of variable concentrations with being placed on 38 ℃; In the spectral range of 200nm to 800nm, measure the resonance scattering spectroscopy of product, get the intensity of characteristic peak BSA concentration is done curve, obtain to detect the working curve of 0-180 μ L BSA.In this example in 40nmol/L to the 240nmol/L scope 548nm place resonance peak intensity keep the good linear relation with conjugate concentration.Its linear fit curvilinear equation is:
I
548nm＝2.02E6*C
BSA+1.23E6，R
2＝0.9915
Repeat preparation process, can read respective concentration at working curve by detecting resonance scattering spectroscopy.
Embodiment two:
With tree-shaped body (PPIHA) and gold chloride (HAuCl
4) be raw material, microwave backflow heating prepares the nm of gold seed colloidal sol of surface amination.The chlorauric acid solution of 1.2mL 0.67% is dissolved in the 100mL ultrapure water, adds 85 μ L, the toluene solution of 5%PPIHA; After fully mixing, with microwave backflow heating 5min.
With 180 μ L, 1% bovine serum albumin(BSA) (Bovine serum albumin, BSA) solution joins in the nm of gold seed colloidal sol that 4.5mL step (1) makes, and adds 115 μ L, 1% hydrochloric acid, shake up the back and be diluted to 6ml with ultrapure water, make the AuNPs-BSA conjugate.
Getting 1.5mL AuNPs-BSA conjugate is that 7.0 phosphate buffers mix with 0.6mL pH, shakes up, and is diluted to 10mL with ultrapure water, standby.10 μ L get the above-mentioned AuNPs-BSA conjugate of a certain amount of (in the 0-180 μ L scope) at interval, join in the 3.00mL ultrapure water.Add 1.6mL 0.02% chlorauric acid solution then successively, the oxammonium hydrochloride solution (NH2OHHCL) of sodium citrate-phosphate buffer of 2mL pH 2.26 and 2.5mL 8.0 * 10-3mol/L is diluted to 10mL with ultrapure water again.Hatch 20min with being placed on 38 ℃, obtain the biomineralization product of AuNPs-BSA conjugate; In the spectral range of 200nm to 800nm, measure the resonance scattering spectroscopy of product, get the intensity of characteristic peak BSA concentration is done curve, obtain to detect the working curve of 0-180 μ L BSA.In this example in 40nmol/L to the 240nmol/L scope 548nm place resonance peak intensity keep the good linear relation with conjugate concentration.Its linear fit curvilinear equation is:
I
548nm＝1.93E6*C
BSA+1.01E6，R
2＝0.9930
Repeat preparation process, can read respective concentration at working curve by detecting resonance scattering spectroscopy.
Embodiment three:
With tree-shaped body (PPIHA) and gold chloride (HAuCl
4) be raw material, microwave backflow heating prepares the nm of gold seed colloidal sol of surface amination.With 2mL, 0.67% chlorauric acid solution is dissolved in the 135mL ultrapure water, adds 95 μ L, the toluene solution of 5%PPIHA; After fully mixing, with microwave backflow heating 5min.
Add 200 μ L in the nm of gold seed colloidal sol that makes to 5mL, (Bovineserum albumin, BSA) solution add 130 μ L, 1% hydrochloric acid to 1% bovine serum albumin(BSA) again, shake up the back and are diluted to 6ml with ultrapure water, and reaction generates the AuNPs-BSA conjugate.
Getting 2mL AuNPs-BSA conjugate is that 7.0 phosphate buffers mix with 0.8mL pH, shakes up, and is diluted to 12mL with ultrapure water, standby.15 μ L get the above-mentioned AuNPs-BSA conjugate of a certain amount of (in the 0-180 μ L scope) at interval, join in the 3.00mL ultrapure water.Add 2mL 0.02% chlorauric acid solution then successively, sodium citrate-phosphate buffer of 2.8mL pH 2.26 and 3mL 8.0 * 10
-3The oxammonium hydrochloride solution of mol/L is diluted to 10mL with ultrapure water again, hatches the biomineralization product that 20min makes the AuNPs-BSA conjugate of a series of variable concentrations with being placed on 38 ℃; In the spectral range of 200nm to 800nm, measure the resonance scattering spectroscopy of product, get the intensity of characteristic peak BSA concentration is done curve, obtain to detect the working curve of 0-180 μ L BSA.In this example in 40nmol/L to the 240nmol/L scope 548nm place resonance peak intensity keep the good linear relation with conjugate concentration.Its linear fit curvilinear equation is:
I
548nm＝2.21E6*C
BSA+1.20E6，R
2＝0.9902
Repeat preparation process, can read respective concentration at working curve by detecting resonance scattering spectroscopy.
Claims (1)
1. the ultramicron quantification of protein detection method based on nm of gold particulate biomineralization principle is characterized in that, utilizes the variation of the resonance scattering spectroscopy of nm of gold to detect, and comprises following steps:
(1) the nm of gold seed of surface amination preparation
With tree-shaped body PPIHA and gold chloride HAuCl
4Be raw material, use microwave backflow heating to prepare the nm of gold seed colloidal sol of surface amination, with 1-2mL, percentage by weight is that 0.67% chlorauric acid solution is dissolved in the 100-150mL ultrapure water, add 85-95 μ L, percentage by weight is the toluene solution of 5%PPIHA; After fully mixing, with microwave backflow heating 3-6min, make the nm of gold seed colloidal sol of surface amination, 4 ℃ of sealings are preserved;
(2) nm of gold-protein conjugate AuNPs-BSA preparation
With 180-200 μ L, percentage by weight is 1% bovine serum albumin(BSA) Bovine serum albumin, BSA solution joins in the nm of gold seed colloidal sol that 3-5mL step (1) makes, adding 110-130 μ L percentage by weight is 1% hydrochloric acid, shake up the back and be diluted to 6ml with ultrapure water, make the AuNPs-BSA conjugate;
(3) biomineralization of AuNPs-BSA conjugate
Getting 1-2mL AuNPs-BSA conjugate is that 7.0 phosphate buffers mix with 0.6-0.8mL pH, shakes up, and is diluted to 10mL with ultrapure water, standby;
Get the above-mentioned AuNPs-BSA conjugate of 0-180 μ L, join in the 3.00mL ultrapure water, add 1-2mL 0.02% chlorauric acid solution then successively, sodium citrate-phosphate buffer of 1-3mL pH 2.26 and 2-3mL 8.0 * 10
-3The oxammonium hydrochloride solution NH of mol/L
2OHHCL is diluted to 8-10mL with ultrapure water again, hatches 18-23min with being placed on 38 ℃, the biomineralization product that obtains the AuNPs-BSA conjugate for detection of;
(4) resonance scattering spectroscopy working curve preparation
In 0-180 μ L scope, 10 μ L at interval make the biomineralization product of the AuNPs-BSA conjugate of a series of variable concentrations according to the method for step (3); Measure the resonance scattering spectroscopy of product in the spectral range of 200nm to 800nm, get the intensity of characteristic peak BSA concentration is done curve, obtain detecting the working curve of 0-180 μ L BSA, this curve is used for the quantitative detection of ultramicron protein;
(5) measure the actual sample method
According to step (2), 180 μ L BSA solution to be measured is made the AuNPs-BSA conjugate, obtain its biomineralization product according to step (4), detect its resonance scattering spectroscopy, read respective concentration at working curve.
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