CN103149179A - Method of quantitatively detecting proteins by Co(NH3)6)3+-DNA resonance light scattering - Google Patents
Method of quantitatively detecting proteins by Co(NH3)6)3+-DNA resonance light scattering Download PDFInfo
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- CN103149179A CN103149179A CN2013100311307A CN201310031130A CN103149179A CN 103149179 A CN103149179 A CN 103149179A CN 2013100311307 A CN2013100311307 A CN 2013100311307A CN 201310031130 A CN201310031130 A CN 201310031130A CN 103149179 A CN103149179 A CN 103149179A
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Abstract
The invention discloses a method of quantitatively detecting proteins by (Co(NH3)6)3+-DNA resonance light scattering, a resonance light scattering signal of the protein is increased intensively by electrostatic interaction between a(Co(NH3)6)3+-DNA compound and proteins so as to detect standard bovine serum albumin (BSA). The resonance light scattering intensively which is strengthened on a wavelength of 346.0nm and the BSA are in a good linear relation when the concentration of the BSA in a range of 0.05-1.2Mu g/ml. The method is applied in a differential comparison of a response valve of the resonance light scattering of various proteins of human serum albumin and detecting total protein level of the actual human serum albumin so that good effects are gained. The method has the advantages of being high in sensitivity, accurate, reliable and simple in detection system. A practical new method is built for quantitative detecting trace protein.
Description
Technical field
The present invention relates to a kind of method that resonance light-scattering approach detects analysing protein content, belong to technical field of protein detection.
Background technology
Protein is one of most important material in biosome, is the substance basis of life.The mensuration of kinds of protein and content thereof is of great significance for biochemical analysis and medical diagnosis on disease tool.At present, traditional method of protein measurement has absorption photometry (comprising Kjeldahls method, biuret method and Coomassie brilliant blue method) and fluorimetry.Wherein the absorption photometry major defect is that sensitivity is lower, can not be used for the lower biological sample of analysing protein concentration; Fluorimetry only limits to have the system of fluorescence, and its scope of application is restricted.Some inventive methods that also occur, as European patent G01N33/68A12, US Patent No. 2010047913 etc., with liquid chromatography (LC) and mass spectrometry, although improved the sensitivity and the accuracy that detect, but due to apparatus expensive, consuming time long, operation is more complicated, has limited its use.Therefore seeking new protein detection method has great importance.
Resonant light scattering (Resonance Light Scattering, RLS) analytical approach is a kind of light-scattering analysis technology that can measure on common fluorospectrophotometer, sensitive and abundant signal occurs when the biological large molecular recognition of research, assembling and gathering, can obtain the sensitivity same high with fluorescence method commonly used.1993, Pasternack etc. realized the gathering of porphyrins on nucleic acid with resonance light-scattering approach research first.Present this technology is widely used in the quantitative detection of the biomacromolecules such as nucleic acid, protein, polysaccharide, and be used between biomacromolecule, biomacromolecule and other interaction between substances research on mechanisms.
In the report of existing document, mostly adopt organic dyestuff probe or Metal ion indicator to launch Resonance Light-Scattering Spectra to biomacromolecule protein and nucleic acid, utilize in addition the quantum dot resonant light scattering quantitatively to detect protein and other etc.But for simple metal complex compound [Co (NH
3)
6]
3+Resonant light scattering research and the application in protein analysis thereof of-DNA-protein system there is not yet report.
Summary of the invention
The purpose of this invention is to provide a kind of quantitative detection method of protein, can be fast, sensitive, detect trace amount of protein content in actual sample exactly.
Theoretical according to resonant light scattering, the increase of particle volume and strong electrostatical binding and large electron delocalization conjugation in conjunction with several height that cause all can produce strong resonant light scattering phenomenon.In the present invention, six cobaltammine ions and DNA molecular generation electrostatical binding, simultaneously because DNA is cross-linked with each other together with protein molecule, the formation bioplex.Final [Co (the NH that forms
3)
6]
3+-DNA-protein system causes the protein volume sharply to increase, and corresponding resonant light scattering signal is than protein, [Co (NH
3)
6]
3+-protein, DNA-protein system all are able to strong enhancing.The present invention utilizes above-mentioned characteristic and principle, and in certain protein concentration scope, the signal of resonant light scattering strengthens and the concentration of protein has linear relationship, thereby can detect the complex sample of the protein that contains very low concentrations.
Technical scheme comprises the steps:
1. calf thymus DNA (ctDNA) solution that adds successively 0.7mL10 μ g/mL, the Britton-Robinson of 1.5mL (BR) buffer solution (pH3.78), the 1mmol/L[Co (NH of 0.1mL
3)
6] Cl
3Solution often adds a kind of reagent and all system is mixed with vortex mixer.In above-mentioned mixed solution, adding respectively 1 histone matter content is 0～2.0 μ g/mL concentration known and the different standard group protein solution of concentration, and the experimental group protein solution of unknown concentration to be measured at last.Respectively each mixed solution is shaken up at last, then be diluted to 10mL with redistilled water.
2. with the various mixed solutions of above-mentioned steps 1 gained, shake up rear placement 1 hour in mixing, carry out synchronous scanning (λ ex=λ em) on fluorospectrophotometer, obtain the RLS spectrum between 200.0nm～700.0nm, and in the 346.0nm mensuration RLS of place intensity.Set exciting and launching slit width of scanning and be 5nm, negative high voltage is 400V.
3. resonant light scattering intensity (Δ I) the drawing standard curve of the known protein concentration (C) of combined standard histone matter solution and its enhancing obtains measuring the range of linearity, the equation of linear regression Δ I=aC+b of protein and linearly dependent coefficient.
4. according to the regression equation of typical curve and the resonant light scattering intensity of experimental group protein solution enhancing, experiment with computing histone matter solution concentration.
The invention has the beneficial effects as follows: utilize [Co (NH
3)
6]
3+-DNA-protein system produces the strong enhancing phenomenon of resonant light scattering signal, be used for quantitatively detecting the content of different sample trace amount of protein, have highly sensitive, accurately and reliably, the characteristics such as economical and practical, can be widely used in protein trace quantitative detection field, have using value in fields such as Agricultural development quality detection, food nutrition standard detection, clinical medicine, medicals diagnosis on disease.
Description of drawings
Fig. 1 is the resonant light scattering spectrogram of different proteins system
Fig. 2 is for detecting the typical curve of bovine serum albumin(BSA) (BSA)
Embodiment
Embodiment 1: the drawing standard curve
1.DNA stock solution: accurate weighing calf thymus DNA (ctDNA, Sigma D-1501), directly be dissolved in the solution that is mixed with 10 μ g/mL in redistilled water, preserve in 0～4 ℃ of refrigerator; BSA stock solution: accurately take bovine serum albumin(BSA) (BSA), directly be dissolved in the solution that is made into 100 μ g/mL in redistilled water, preserve in 0～4 ℃ of refrigerator; A series of Britton-Robinson (BR) buffer solution of preparation pH1.8～11.9; With CoCl
26H
2O is raw material, makes [Co (NH
3)
6] Cl
3Crystal directly is dissolved in redistilled water, is mixed with 10
-3The storing solution of mol/L.
2. respectively in the 10mL color comparison tube, add successively the ctDNA stock solution of 0.7mL10 μ g/mL, the BR buffer solution (pH3.78) of 1.5mL, the [Co (NH of 0.1mL1mmol/L
3)
6] Cl
3Solution, and the 100 μ g/mL BSA stock solutions that add respectively 0.005mL, 0.008mL, 0.01mL, 0.03mL, 0.05mL, 0.08mL, 0.10mL, 0.12mL, 0.15mL, 0.20mL are as standard group solution.Often add a kind of reagent and all system is mixed with vortex mixer, at last test solution is diluted to scale with redistilled water.
3. with the BSA standard group solution of above-mentioned steps 2 gained, shake up rear placement 1 hour in mixing, carry out synchronous scanning (λ ex=λ em) on fluorospectrophotometer, obtain the RLS spectrum between 200.0nm～700.0nm, and in the 346.0nm mensuration RLS of place intensity.Set exciting and launching slit width of scanning and be 5nm, negative high voltage is 400V.
4. resonant light scattering intensity (Δ I) the drawing standard curve of the known protein concentration (C) of combined standard histone matter solution and its enhancing, as shown in Figure 2.The concentration of BSA and has good linear relationship between the RLS signal that strengthens in the scope of 0.05 μ g/mL～1.20 μ g/mL.Equation of linear regression is Δ t=88.80C-1.30, and linearly dependent coefficient is 0.991, shows that the method has very high sensitivity.
Embodiment 2: the response difference of multiple proteins
1. respectively in the 10mL color comparison tube, add successively the ctDNA solution of 0.7mL10 μ g/mL, the BR buffer solution (pH3.78) of 1.5mL, the [Co (NH of 0.1mL1mmol/L
3)
6] Cl
3Solution, and the bovine serum albumin solution, human serum albumin solution, insulin solutions, gelatin solution, Egg-white solution, the gamma globulin solution that add respectively the 100 μ g/mL of 0.1mL.Often add a kind of reagent and all system is mixed with vortex mixer, at last test solution is diluted to scale with redistilled water.
2. be different types of protein solution of 1.0 μ g/mL with above-mentioned steps 1 gained concentration, after shaking up, mixing respectively placed 1 hour, and carry out respectively synchronous scanning (λ ex=λ em) on fluorospectrophotometer, obtain the RLS spectrum between 200.0nm～700.0nm, and in the 346.0nm mensuration RLS of place intensity, as shown in table 1.Set exciting and launching slit width of scanning and be 5.0nm, negative high voltage is 400V.
3. as shown in Table 1, the size of each protein response signal meets the order of their molecular size ranges substantially, that is to say that the response signal of this analytical approach depends primarily on the size of protein volume, and this is consistent with the resonance light scattering theory.
The resonant light scattering response of table 1 different proteins
Embodiment 3: the mensuration of total protein content in the human serum sample
1. 2 parts of fresh human serum samples (effluent north agriculture university hospital provide) 0.2mL is provided respectively is placed in the 100mL volumetric flask, be diluted to scale with redistilled water, shake up standby.
2. in the 10mL color comparison tube, add successively the ctDNA solution of 0.7mL10 μ g/mL, the BR buffer solution (pH3.78) of 1.5mL, the [Co (NH of 0.1mL1mmol/L
3)
6] Cl
3Solution adds respectively the human serum sample 0.1mL that stocks in above-mentioned steps 1 at last, often adds a kind of reagent and all system is mixed with vortex mixer, at last test solution is diluted to scale with redistilled water.
3. with the human serum sample solution of above-mentioned steps 2 gained, after shaking up, mixing respectively placed 1 hour, and carry out respectively synchronous scanning (λ ex=λ em) on fluorospectrophotometer, obtain the RLS spectrum between 200.0nm～700.0nm, and in the 346.0nm mensuration RLS of place intensity.Set exciting and launching slit width of scanning and be 5.0nm, negative high voltage is 400V.
4. according to the enhancing value (Δ I) of measuring RLS intensity in sample, find the content (C) of human serum gross protein on the typical curve that obtains in embodiment 1.
5. carry out the mensuration of total protein content in above-mentioned 2 parts of human serum samples with traditional Coomassie brilliant blue method (CBB G-250), and result and the inventive method measurement result are compared.Adopt RLS method of the present invention respectively two sample triplicates to be measured, the average recovery rate of measurement result and relative standard deviation (RSD) are all listed in table 2.
6. the RLS method from table 2 can be found out the average recovery rate of two sample triplicate measurement results and the numerical value of relative standard deviation (RSD) respectively, analytical approach of the present invention accurately and reliably, can be used for the mensuration of actual sample, be with a wide range of applications.
The analysis of table 2 human serum total protein content
Claims (5)
1. utilization [Co (NH
3)
6]
3+-DNA resonant light scattering probe quantitative detects method of protein, it is characterized in that, comprises the steps:
(1) add successively calf thymus ctDNA solution, Britton-Robinson (BR) buffer solution, [Co (NH
3)
6] Cl
3Solution adds respectively the standard group protein solution of 1 group of concentration known at last, and the experimental group protein solution of unknown concentration to be measured, and each mixed solution shakes up dilution;
(2) after each mixed solution of step (1) gained is placed respectively 1 hour, carry out synchronous fluorescence scanning under 200.0nm～700.0nm, obtain its resonant light scattering spectrum, and in 346.0nm place mensuration resonant light scattering intensity;
(3) resonant light scattering intensity (Δ I) the drawing standard curve of the concentration (C) of combined standard histone matter solution and its enhancing, obtain the equation of linear regression between Δ I and C, and the method is measured the range of linearity of protein;
(4) according to the regression equation of measurement protein typical curve and the resonant light scattering intensity of experimental group protein solution enhancing, the concentration of experiment with computing histone matter solution.
2. the described utilization of claim 1 [Co (NH
3)
6]
3+-DNA resonant light scattering probe in detecting method of protein is characterized in that, in described step (1), the amount ratio of mix reagent is DNA: the BR damping fluid: [Co (NH
3)
6] Cl
3=(7 μ g: 1.5mL: 10
-4Mmol)/10mL.
3. the described utilization of claim 1 [Co (NH
3)
6]
3+-DNA resonant light scattering probe in detecting method of protein is characterized in that, in described step (1), adopting pH is the acidity that 3.78 Britton-Robinson buffer solution is controlled solution to be measured.
4. the described utilization of claim 1 [Co (NH
3)
6]
3+-DNA resonant light scattering probe in detecting method of protein is characterized in that, in described step (1), add the order of several reagent, and often add a kind of reagent all the system vortex to be mixed.
5. the described utilization of claim 1 [Co (NH
3)
6]
3+-DNA resonant light scattering probe in detecting method of protein, it is characterized in that, verified that this inventive method can be applied to bovine serum albumin(BSA), human serum albumins, insulin, gelatin, Egg-white, gamma globulin, and the detection of total protein content in the human serum sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113720812A (en) * | 2020-05-26 | 2021-11-30 | 重庆福莱鲨生物技术有限公司 | Method for measuring protein solubility |
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Application publication date: 20130612 |