CN106370646A - Method for detecting proteins in to-be-detected sample - Google Patents
Method for detecting proteins in to-be-detected sample Download PDFInfo
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- CN106370646A CN106370646A CN201610676849.XA CN201610676849A CN106370646A CN 106370646 A CN106370646 A CN 106370646A CN 201610676849 A CN201610676849 A CN 201610676849A CN 106370646 A CN106370646 A CN 106370646A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 104
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000001514 detection method Methods 0.000 claims abstract description 72
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000010931 gold Substances 0.000 claims abstract description 26
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- 238000012360 testing method Methods 0.000 claims description 39
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- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a method for detecting proteins in a to-be-detected sample. Specifically, the invention provides a novel method for detecting proteins in a to-be-detected sample based on dependence of the color of nanometer gold/silver on the size and distance between nanometer gold/silver particles to realize distinguishing and detection of a plurality of proteins in serum and urine. Experimental results show that the method can rapidly distinguish and detect proteins in tears, urine and serum.
Description
Technical field
The present invention relates to detection field, relate more specifically to method of protein in a kind of detection testing sample.
Background technology
Protein is the ultimate unit of life, with various forms of vital movements, such as immunity and metabolism, close phase
Close.Abnormalization of protein concentration or the appearance of specific proteins, are the omens of some disease bursts, for example: albumin lacks
Mistake can cause old people's amyotrophy, and myoglobin content is the sensitive indexes of early diagnosis of acute myocardial infarction.In tear
Lysozyme content is low, be lead to that eyes are dry and astringent, the main cause of dacryocystisises and lachrymal gland blockage.Therefore, accurately, quick detection
Protein content in human body, extremely important in the field such as proteomics and medical diagnosiss.
The multiformity of protein structure and complexity are so as to detection is challenging.At present, detect protein both at home and abroad
Technology and instrument, mainly include enzyme linked immunosorbent assay, fluorescent spectrometry, near infrared spectroscopy and high performance liquid chromatography etc..
The detector of these commercializations, can qualitatively or quantitatively detect a certain protein by disposable detection, but related
Experimental principle is higher to the requirement testing each link, complex operation, and time of measuring is long, needs special laboratory technician to carry out
Detection, is related to relatively costly, these unfavorable conditions limit the widely using property of above-mentioned instrument and technology.Meanwhile, by once
Property detect that above-mentioned instrument and technology cannot distinguish between the multiple protein of detection, but it is (or same to realize normal and abnormal proteins
Protein variable concentrations) differentiation detection tend to predict certain burst disease.Therefore, the new effective protein of exploitation is distinguished
Detection method, significant.
Content of the invention
It is an object of the invention to provide method of protein in a kind of detection testing sample.
In a first aspect of the present invention, there is provided the side of protein in a kind of non-diagnostic non-therapeutic ground detection testing sample
Method, including step:
I () provides an indicator, described indicator contains the slaine being selected from the group: monovalence gold salt, monovalence silver salt or
A combination thereof, gathers color a of described indicator；
(ii) described indicator is mixed with testing sample, obtain the first mixed solution, and it is molten to gather described first mixing
Color b of liquid；
(iii) compare described color a and described color b, and extract the colour difference signal of color a and color b；
(iv) analyze described colour difference signal, obtain chromatism data △ r, △ g and △ b；
(v) analysis described chromatism data △ r, △ g and △ b, thus protein in testing sample is carried out qualitative and/or
Detection by quantitative.
In another preference, in described indicator, the concentration of monovalence gold salt is 0.02-50mmol/l, preferably 0.1-
10mmol/l.
In another preference, in step (i), described indicator is obtained in the following manner:
A () provides a trivalent gold salt aqueous solution；
B () adds reproducibility protein solution, wherein trivalent gold and reproducibility egg in described trivalent gold salt aqueous solution
The ratio (mmol:mg) of white matter is (0.1-10): (1-100), obtains the second mixed solution；
C ph that () adjusts the second described mixed solution is 7.5-12.5, and acutely shakes, so that the reduction of trivalent gold salt
For monovalence gold salt, the as the 3rd mixed solution；
D ph value that () adjusts the 3rd described mixed solution is 5.5-8.5, preferably 6.0-8.0, as described instruction
Agent.
In another preference, described reproducibility protein is selected from the group:
Collagenase, transport protein, carrier protein, lipoprotein, keratin, saccharifying enzyme, human serum albumin, liver egg
In vain, papain and trypsin.
In another preference, in described indicator, do not contain reproducibility protein.
In another preference, in step (c), add sodium hydroxide solution in the second described mixed solution, from
And adjust the ph of described second mixed solution.
In another preference, in step (d), after adjusting ph value, also include the step adding monovalence silver salt.
In another preference, the monovalence gold salt in described indicator is (1- with the ratio (mmol:mmol) of monovalence silver salt
100): (100-1), preferably (1-10): (10-1), more preferably for 10:1.
In another preference, in the first described mixed solution, the concentration of testing sample (with the content agent of protein) is
0.5-50 μ g/ml, preferably 1-20 μ g/ml, more preferably for 1-10 μ g/ml.
In another preference, in step (iii), carry out color acquisition using digital camera or scanner.
In another preference, in step (iii), collection reflection light color.
In another preference, in step (iii), the depth carrying out liquid during color acquisition is 0.5-2cm, preferably
For 1cm.
In another preference, in step (v), using multivariate statistics software (mvsp) and hierarchical cluster analysis
(hca) method analysis described chromatism data △ r, △ g and △ b.
In another preference, in step (v), the Euclidean distance of analysis described chromatism data △ r, △ g and △ b,
Thus qualitative and quantitative detection is carried out to protein in testing sample.
In another preference, methods described also includes the step making standard curve.
In another preference, after step (v), also include the analytical data that typing step (v) obtains, set up data
The step in storehouse.
In another preference, described testing sample is selected from the group:
Blood, serum, urine, tear.
In another preference, the protein that methods described detection is selected from the group:
Hemoglobin, albumin, globulin, transferrinss, pepsin, Myoglobin, chymase, lysozyme
And catalase.
In another preference, protein in the methods described testing sample that is selected from the group of detection:
Hemoglobin in (i) blood or serum；
(ii) albumin in urine；With
(iii) lysozyme in tear.
In a second aspect of the present invention, there is provided the test kit of protein in a kind of detection testing sample, described reagent
Box includes:
I () indicator, described indicator contains the slaine being selected from the group: monovalence gold salt, monovalence silver salt or its group
Close；
(ii) standard substance of the albumen being selected from the group: hemoglobin, albumin, globulin, transferrinss, pepsin,
Myoglobin, chymase, lysozyme and catalase；With
(iii) description, has recorded the method described in first aspect present invention in described description.
In a third aspect of the present invention, there is provided the system of protein in a kind of detection testing sample, described system bag
Include:
Test kit described in (i) second aspect present invention；
(ii) camera function module；
(iii) aberration comparison module；
(iv) data analysis module；With
(v) data memory module；
(vi) optionally output module.
In another preference, described camera function module is used for shooting color a of (collection) indicator and first mixed
Close color b of solution.
In another preference, described aberration comparison module is used for relatively described color a and described color b, and extracts
Color a and the colour difference signal of color b.
In another preference, described data analysis module is used for analyzing described colour difference signal, obtains chromatism data
△ r, △ g and △ b；And analyze described chromatism data △ r, △ g and △ b, thus carry out qualitative to protein in testing sample
And/or detection by quantitative.
In another preference, have recorded methods described according to a first aspect of the present invention in described data memory module and survey
The color data of the fixed protein being selected from the group:
Hemoglobin, albumin, globulin, transferrinss, pepsin, Myoglobin, chymase, lysozyme
And catalase.
In another preference, described output module is used for output data analysis and/or data storage result.
In another preference, described output module includes display.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment)
Can be combined with each other between each technical characteristic of body description, thus constituting new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description
Fig. 1 is present invention construction triple channel (△ r, △ g and △ b) sensor, realizes containing in tear, serum and urine
The step that multiple proteins distinguish detection.
Fig. 2 is that embodiment 1 uses protein to reduce the gold salt of trivalent (as haucl4·3h2O), generate the gold salt of monovalence (such as
[aucl2]-) solution (indicator) ultravioletvisible absorption (uv-vis) spectrum.
Fig. 3 is that embodiment 1 uses protein to reduce the gold salt (such as [aucl of monovalence2]-) solution, generate different size of ball
Ultravioletvisible absorption (uv-vis) spectrum of shape nanometer gold.
Fig. 4 be embodiment 1 use digital camera or scanner collection indicator (before mixing) and its with serum in 9 kinds common
The sample image of (after mixing) after protein mixing, and the aberration response signal obtaining the two using photoshop software.
Fig. 5 is through 5 parallel laboratory tests in embodiment 1, extracts the color of Fig. 4 collection using photoshop software, obtains
△ r, △ g, △ b statistical data.
Fig. 6 is that aberration (△ rgb) response signal data gathering is imported multivariate statistics software (mvsp) by embodiment 1,
Realize the differentiation detection of 9 kinds of protein in serum.
Fig. 7 is that aberration (△ rgb) response signal data of collection in embodiment 1 imports hierarchical cluster analysis (hca) software,
Realize the differentiation detection of 9 kinds of protein in serum.
Fig. 8 a is that in embodiment 2, synthesis indicator (gold salt solution of monovalence) detects that the protein of variable concentrations (such as turns ferrum
Albumen and pepsin), corresponding color change and color difference analysis figure.
Fig. 8 b is the root-mean-square valve Euclidean distance (eds) of respective response signal value △ rgb in embodiment 2, with tested
Survey the increase of protein transferrin concentration, assume linear increment trend.
Fig. 8 c is corresponding Euclidean distance (eds) in embodiment 2, with the increasing of detected protein pepsin concn
Plus, assume linear increment trend.
Fig. 8 d is that Fig. 8 a chromatism data (△ rgb) gathering is imported multivariate statistics software (mvsp) by embodiment 2, realizes
The differentiation detection of variable concentrations and dissimilar protein (as transferrinss and pepsin) in serum.
Fig. 9 be in embodiment 3 using digital camera or scanner collection synthesis indicator (before mixing) and its with urine in 9
Plant the sample image of (after mixing) after common protein mixes, and the aberration response letter obtaining the two using photoshop software
Number.
Figure 10 is through 5 parallel laboratory tests in embodiment 3, extracts the color of Fig. 9 collection using photoshop software, obtains
Obtain △ r, △ g, △ b statistical data.
Figure 11 is that aberration (△ rgb) response signal data of collection in embodiment 3 imports multivariate statistics software (mvsp),
Realize the differentiation detection of common 9 kinds of protein in urine.
Figure 12 is that aberration (△ rgb) response signal data importing hierarchical cluster analysis (hca) of collection in embodiment 3 is soft
Part, realizes the differentiation detection of common 9 kinds of protein in urine.
Figure 13 is different big by after the mixing of the indicator of development and variable concentrations lysozyme, prepare in embodiment 4
Ultravioletvisible absorption (uv-vis) spectrum of coccoid solution of gold nanoparticles.
Figure 14 is using lysozyme content in this inventive method Quantitative detection tear in embodiment 4.The instruction of development
Agent and tear, variable concentrations lysozyme mixed color change situation.
Figure 15 is to utilize uv-visible absorption spectra instrument (existing universal tester) test to be diluted 10 times in embodiment 4
Lysozyme content in tear.
Figure 15 a is the result of the absorption intensity detecting variable concentrations lysozyme in embodiment 4.
Figure 15 b is the light absorption value result detecting variable concentrations lysozyme in embodiment 4.
Specific embodiment
The present inventor, through extensively and in depth studying, is surprised to find that a kind of differentiation detects multiple proteins threeway first
Road sensor.Specifically, the dependency to its particle size and spacing for the color based on nanometer gold/silver for the present invention, devises one
Kind of novel sensor, realizes the differentiation detection of multiple proteins containing in serum and urine.Experiment shows, the sensing of the present invention
The protein containing in detection tear, urine and serum can quickly be distinguished by device.Complete the present invention on this basis.
Inventive sensor
The invention provides method of protein in a kind of detection testing sample.
Present invention also offers a kind of threeway road sensor distinguishing detection multiple proteins, this sensor can quick area
Go-on-go surveys the protein containing in tear, urine and serum.
As used herein, test sample is treated in the detection of described " the threeway road sensors of detection multiple proteins " as present invention
Method of protein in product.
Sensor constructed by the present invention, the dependency to its particle size and spacing for the color based on nanometer gold/silver, with
And the difference of different proteins reproducibility, the nanoparticle of different size, different shape, variable concentrations is generated for reducing metal salt
Son.
Sensor constructed by the present invention, its sensing passage is red (△ r), green (△ g), blue (△ b) color response signal.
Sensor constructed by the present invention, the as triple channel visualized array sensor based on single indicator, have
The features such as simple structure, use easy to spread.
Described indicator is: the gold salt of monovalence is (as haucl2) and monovalence silver salt (as agcl) one or two mixing
Thing.
Described protein is: Collagenase, transport protein, carrier protein, lipoprotein, keratin, saccharifying enzyme, human blood
Pure albumen, orgotein, papain and trypsin.
The described building method of triple channel (△ r, △ g and △ b) sensor based on single indicator and its differentiation inspection
The method surveying multiple proteins, is shown in Fig. 1, comprises the steps:
(1) synthesis of single indicator: reduce the gold salt of trivalent (as haucl using protein4·3h2O), generate monovalence
Gold salt (as [aucl2]-) solution, its own or the mixture with monovalence silver salt (as agcl), as build multichannel sensor
Single indicator.
(2) preparation of nanometer gold/silver: the single finger prepared using the difference of different proteins reproducibility, reduction step (1)
Show agent (monovalence gold/silver salt), generate different colours, different size, different shape, the nanometer gold/silver particles of variable concentrations.
(3) color acquisition: add and without protein using in digital camera or scanner acquisition step (1) indicator
When color.
(4) color difference analysis: using photoshop software shear effective picture, the color that then extraction step (3) gathers,
Obtain △ r, △ g, △ b data.
(5) the differentiation detection of protein
△ r, △ g, △ b data importing multivariate statistics software (mvsp) and the hierarchical cluster analysis that step (4) is obtained
(hca) in software, realize the differentiation detection of the different types of protein of variable concentrations.
(6) foundation of data base: by distinguish detection the different types of protein of variable concentrations test data (△ r, △ g,
△ b value) input database, to call at any time.
Following table shows protein content normal concentration range in adult human blood and urine, and pre- beyond normal range
Show relevant disease
Nanometer gold/silver
Gold/silver nano particle has excellent optics with good biocompatibility so as to become in drug delivery, medical science
The fields such as picture, medical diagnosis on disease, detection and analysis are with a wide range of applications.At present, increasing research worker is using gold/silver
The nanoparticle detection and analysis ion related to environment water security, food safety, life and health etc., biomolecule and albumen etc..
Nanometer gold/silver directly affects its solution colour.This feature is so that nanometer gold/silver has potentially in a lot of fields
Application, such as optical imagery, zwitterion or biomolecule detection analysis etc..Due to heavy metal ion, some toxicity aniones,
Biological micromolecule (as aminoacid, Glutathione and dopamine) and protein etc., direct relation environment water security, food safety
Or the health problem of the mankind etc..So, a lot of research groups are applying the excellent optical characteristics of nanometer gold/silver, realize ion,
Biological micromolecule and the detection and analysis of protein.
Euclidean distance
Euclidean distance, i.e. euclidean metric (euclidean metric), it is a distance definition generally adopting, refer to
The actual distance between two points in m-dimensional space, or the natural length (i.e. this point is to the distance of initial point) of vector.In two dimension
With the Euclidean distance in three dimensions is exactly the actual range between 2 points.
The formula of two-dimensional space is: 0 ρ=sqrt ((x1-x2) ^2+ (y1-y2) ^2) | x |=√ (x2+y2)
Three-dimensional formula is: 0 ρ=√ ((x1-x2) ^2+ (y1-y2) ^2+ (z1-z2) ^2) | x |=√ (x2+y2+
z2)
Aberration corresponding signal
As used herein, described " aberration corresponding signal " refer to the indicator of the present invention mix with protein before and after face
Aberration.
Chromatism data
As used herein, described " chromatism data " refers to red (△ r), green (△ g), blue (△ b) value of chromatism, wherein, △
Red, green, the blue color change that r, △ g and △ b refers to before and after the indicator of the present invention and protein mixing respectively quantifies
Value.
One group of chromatism data in the application specific embodiment is as shown in the table:
Remarks: blank (blank), albumin (ba), catalase (cat), hemoglobin (bhb), globulin (gb),
Lysozyme (lys), chymase (cht), pepsin (pep), Myoglobin (mb) and transferrinss (trf).
Correlated performance test of the present invention is as follows:
(1) protein is used to reduce the gold salt of trivalent (as haucl4·3h2O), generate the gold salt (such as [aucl of monovalence2]-)
Solution, its own or the mixture with monovalence silver salt (as agcl), as build the single indicator of multichannel sensor.This refers to
Show ultravioletvisible absorption (uv-vis) spectrum of agent, see Fig. 2.Using the dissimilar albumen with variable concentrations of this indicator detection
Matter.Meanwhile, (after after gathering indicator (before image) and its mix with protein using digital camera or scanner
Image sample image), shears effective picture using photoshop software, then according to the color change of above-mentioned sample image
Data (as △ rgb), builds threeway road sensor, sees Fig. 4 and Fig. 9.This sensor can be distinguished in detection serum and urine and contain
Multiple proteins.
(2) use the sensor that (1) builds, (present invention is only repeatedly to distinguish the protein containing in detection serum and urine
Select 9 kinds of protein as object of study), extract △ rgb response signal using photoshop software, this statistical data analysis is shown in
Fig. 5 and Figure 10, and the scan data that this is gathered imports multivariate statistics software (mvsp) and hierarchical cluster analysis (hca) software
In, the differentiation detection of the common protein realizing containing in serum and urine, see Fig. 6, Fig. 7, Figure 11 and Figure 12.
(3) use the sensor that (1) builds, quantification area go-on-go surveys the protein of variable concentrations (as transferrinss stomach function regulating egg
White enzyme), the transferrinss of the indicator researched and developed and variable concentrations and pepsin mix before and after color change, and aberration
Response signal figure, is shown in Fig. 8 a.With the increase of detected transferrinss and pepsin concn, indicator color change is deepened.
The root-mean-square valve Euclidean distance of respective response signal value △ rgb, with detected transferrinss and pepsin concn
Increase, assume linear increment trend.This linear relationship, the protein that can be used for detection by quantitative differentiation variable concentrations (such as turns ferrum egg
White and pepsin).
Coherent detection Analysis on Mechanism of the present invention is as follows:
Using ultraviolet-uisible spectrophotometer, the prepared single indicator of test and the mixed UV, visible light of protein
Absorb (uv-vis) spectrum, see Fig. 3.According to ultraviolet-visible absorption spectroscopy (uv-vis) peak position, corresponding absorbing wavelength, reflect
Reduce indicator using different proteins, obtain different size of spherical nanoparticle.As reduced monovalence using hemoglobin
Gold salt (such as [aucl2]-) solution, the absworption peak of the spherical nanometer gold of acquisition is 530nm, prepares nanometer metallographic using other albumen
The corresponding relation answered is: albumin 535nm, globulin 537nm, transferrinss 555nm, pepsin
551nm, Myoglobin 555nm, chymase 553nm, lysozyme 560nm, catalase
622nm.
Main advantages of the present invention include:
(a) need not be complicated modification, separate and purification process.
B () is without the instrument and equipment of Large expensive.
C () utilizes this development method, it is possible to achieve the major protein containing in quick, Sensitive Detection tear, urine, blood
Matter, is expected to be used for clinical medicine detection.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.
Embodiment 1
The differentiation detection of triple channel sensor design and Proteins in Serum
In the present embodiment, the step of triple channel sensor design and its differentiation detection Proteins in Serum is as follows:
The synthesis of the single indicator of a
In room temperature environment, take the three hydration gold chloride (haucl that 6ml molar concentration is 1mm (mmol/l)4·3h2O) molten
Liquid, adds the pectase that 20 μ l concentration are 1mg/ml, acutely shakes mix homogeneously, prepared monovalence gold salt (such as [aucl2]-) solution,
As indicator, ultravioletvisible absorption (uv-vis) spectrum of this solution is shown in Fig. 2.
The preparation of b nanometer gold
In room temperature environment, take the pbs buffer of the 10mm ph=7.6 of 10 part of 200 μ l, be separately added into 20 μ l pure water, dense
Spend hemoglobin for 1mg/ml, albumin, globulin, transferrinss, pepsin, Myoglobin, chymase, molten
Bacterium enzyme and catalatic (pure) aqueous solution, are subsequently added indicator prepared by 0.5ml step (a), add after mix homogeneously
The deionized water of 0.4ml, is incubated half an hour in 37 DEG C of environment, obtains different size of spherical solution of gold nanoparticles, and it is purple
Outer visible absorbance (uv-vis) spectrum is shown in Fig. 3.
C color acquisition
Using digital camera or scanner collection indicator (before image) and its with 9 kinds of common proteins in aqueous solution
The sample image of (after image) after matter mixing, and the aberration response signal of the two is obtained using photoshop software, see
Fig. 4.
D color difference analysis
The aberration response signal being gathered using photoshop software extraction step (c), repeats to test five times, obtains aberration
Data, i.e. △ r, △ g, △ b.Its statistical data analysis, is shown in Fig. 5.
The differentiation detection of e protein
△ r, △ g, △ b data importing multivariate statistics software (mvsp) and the hierarchical cluster analysis that step (d) is obtained
(hca) in software, realize the differentiation detection of the dissimilar protein that aqueous solution contains, see Fig. 6 and Fig. 7.
Protein in f detection actual sample
In room temperature environment, take the pbs buffer of the 10mm ph=7.6 of 10 part of 200 μ l, be separately added into 20 μ l pure bloods clear,
Containing the hemoglobin for 1mg/ml for the concentration, albumin, globulin, transferrinss, pepsin, Myoglobin, pancreas in serum
Chrymotrypsin, lysozyme and catalase, are subsequently added indicator prepared by 0.5ml step (a), add after mix homogeneously
The deionized water of 0.4ml, is incubated half an hour in 37 DEG C of environment, obtains different size of spherical solution of gold nanoparticles, and it is purple
Outer visible absorbance (uv-vis) spectrum, result is basically identical with Fig. 3.The color difference analysis value extracted is imported mvsp and hca statistical
Analysis software, realizes the differentiation detection of above-mentioned protein in serum, and result is basically identical with Fig. 6 and Fig. 7.
Embodiment 2
Triple channel sensor design and the qualitative and detection by quantitative of Proteins in Serum
In the present embodiment, triple channel sensor design and its qualitative and detection by quantitative Proteins in Serum step are as follows:
The synthesis of the single indicator of a
In room temperature environment, take the four hydration gold chloride (haucl that 10ml molar concentration is 0.5mm (mmol/l)4·4h2o)
Solution, is separately added into the naoh for 10m (mol/l) for the Collagenase and 50 μ l molar concentrations that 20 μ l concentration are 1mg/ml molten
Liquid, acutely shakes mix homogeneously, prepared monovalence gold salt (such as [aucl2]-) solution, as indicator, the UV, visible light of this solution is inhaled
Receive (uv-vis) spectrum and see Fig. 2.
The preparation of b nanometer gold
In room temperature environment, take the hepes buffer of the 2mm ph=8.0 of 6 part of 100 μ l, be separately added into 30 μ l pure water, no
Protein (transferrinss or the stomach egg of same concentration (1 μ g/ml, 5 μ g/ml, 10 μ g/ml, 15 μ g/ml, 20 μ g/ml, 25 μ g/ml)
White enzyme) (pure) aqueous solution, be subsequently added indicator prepared by 1ml step (a), after mix homogeneously, add the deionization of 1.5ml
Water, is incubated one hour in 37 DEG C of environment, obtains different size, the spherical solution of gold nanoparticles of different colours, sees Fig. 8 a.
C color acquisition
Using digital camera or scanner collection indicator (before image) and its different dense from contain in pure water
The sample image of (after image) after degree protein (as transferrinss and pepsin) mixing, and soft using photoshop
Part obtains corresponding aberration response signal, sees Fig. 8 a.
D color difference analysis
Extract, using photoshop software, the aberration response signal that above-mentioned steps (c) gather, repeat to test three times, obtain
Chromatism data, i.e. △ r, △ g, △ b.
The qualitative and detection by quantitative of e protein
The root-mean-square valve Euclidean distance (eds) of the response signal value △ rgb that above-mentioned steps (d) obtain, with detected
The increase of protein (as transferrinss and pepsin) concentration, assumes linear increment trend；According to this linear relationship, can achieve
The detection by quantitative of protein (as transferrinss and pepsin) is contained in serum.
△ r that above-mentioned steps (d) are obtained, △ g, △ b data import in multivariate statistics software (mvsp), achievable water
The variable concentrations containing in solution, the differentiation of dissimilar protein (as transferrinss and pepsin) detect, see Fig. 8 d.
Protein in f detection actual sample
In room temperature environment, take the hepes buffer of the 2mm ph=8.0 of 6 part of 100 μ l, be separately added into 30 μ l pure bloods clear,
In serum, the protein containing variable concentrations (as transferrinss and pepsin), is subsequently added instruction prepared by 1ml step (a)
Agent, adds the deionized water of 1.5ml after mix homogeneously, be incubated one hour in 37 DEG C of environment, obtains different size, different colours
Spherical solution of gold nanoparticles, result is basically identical with Fig. 8 a.The color difference analysis value extracted is imported mvsp and hca statistical
Analysis software, realizes the differentiation detection of above two protein in serum, and result is basically identical with Fig. 8 d.
Embodiment 3
The differentiation detection of protein in triple channel sensor design and urine
In the present embodiment, in triple channel sensor design and its differentiation detection urine, the step of protein is as follows:
The synthesis of the single indicator of a
In room temperature environment, take the three hydration gold chloride (haucl that 10ml molar concentration is 5mm (mmol/l)4·3h2O) molten
Liquid, is separately added into the naoh for 0.1m (mol/l) for the Collagenase and 150 μ l molar concentrations that 50 μ l concentration are 6mg/ml molten
Liquid, acutely shakes mix homogeneously, prepared monovalence gold salt (such as [aucl2]-) solution, then with monovalence silver salt (agcl) with 10:1's
Concentration mixes, and this mixed liquor as indicator, compose, basically identical with Fig. 2 by the ultravioletvisible absorption (uv-vis) of this solution.
The preparation of b nanometer gold silver
In room temperature environment, take the pbs buffer of the 20mm ph=7.5 of 10 parts of 0.5ml, be separately added into 50 μ l pure water, dense
Spend hemoglobin for 6mg/ml, albumin, globulin, transferrinss, pepsin, Myoglobin, chymase, molten
Bacterium enzyme and catalatic (pure) aqueous solution, are subsequently added indicator prepared by 1.5ml step (a), add after mix homogeneously
The deionized water of 1.5ml, is incubated half an hour in 37 DEG C of environment, obtains different size of spherical gold silver nano-particle solution, its
Ultravioletvisible absorption (uv-vis) is composed, basically identical with Fig. 3.
C color acquisition
Using digital camera or scanner collection indicator (before image) and its with 9 kinds of common protein in pure water
The sample image of (after image) after mixing, and the aberration response signal of the two is obtained using photoshop software, see figure
9.
D color difference analysis
The aberration response signal being gathered using photoshop software extraction step (c), repeats to test five times, obtains aberration
Data, i.e. △ r, △ g, △ b.Its statistical data analysis, is shown in Figure 10.
The differentiation detection of e protein
△ r, △ g, △ b data importing multivariate statistics software (mvsp) and the hierarchical cluster analysis that step (d) is obtained
(hca), in software, realize variable concentrations, the differentiation detection of dissimilar protein containing in aqueous solution, see Figure 11 and Figure 12.
Protein in f detection actual sample
By standardizition so that containing the multiple proteins with concentration in above-mentioned pure water in urine.Using above-mentioned development
Method detects the above-mentioned protein containing in urine, and its testing result is basically identical with Fig. 9, Figure 10, Figure 11 and Figure 12.
Embodiment 4
The quick detection of lysozyme in triple channel sensor design and tear
In the present embodiment, in triple channel sensor design and its quick detection tear, the step of lysozyme is as follows:
The synthesis of the single indicator of a
In room temperature environment, take the three hydration gold chloride (haucl that 6ml molar concentration is 1mm (mmol/l)4·3h2O) molten
Liquid, being separately added into the Collagenase that 20 μ l concentration are 4mg/ml and 50 μ l molar concentrations is the naoh solution of 4m (mol/l), acute
Violent shock swings mix homogeneously, until mixed liquor becomes colorless from light yellow, using this colourless liquid as indicator, the ultraviolet of this solution
Visible absorbance (uv-vis) is composed, basically identical with Fig. 2.
The preparation of b nanometer gold
In room temperature environment, take the pbs buffer of the 6mm ph=7.4 of 11 parts of 0.5ml, be separately added into 50 μ l tear, pure
Water, (pure) aqueous solution of variable concentrations lysozyme, are subsequently added indicator prepared by 0.5ml step (a), add after mix homogeneously
The deionized water of 1ml.In mixed liquor, the ultimate density of lysozyme is 1 μ g/ml, 9 μ g/ml, 14 μ g/ml, 22 μ g/ml and 35 μ
g/ml.Above-mentioned mixed liquor, is incubated 20 minutes in 37 DEG C of environment, obtains different size of spherical solution of gold nanoparticles, and it is purple
Outer visible absorbance (uv-vis) spectrum, is shown in Figure 13.
C color acquisition
Using digital camera or scanner collection indicator (before image) and its with variable concentrations bacteriolyze in pure water
The sample image of (after image) after enzyme, tear mixing, and the aberration response letter of the two is obtained using photoshop software
Number, see Figure 14.
D color difference analysis
The aberration response signal being gathered using photoshop software extraction step (c), repeats to test three times, obtains aberration
Data, i.e. △ r, △ g, △ b, thus calculate Euclidean distance (eds).Eds, with the increase of detected lysozyme concentration, assumes line
Property increasing trend.
The detection by quantitative of lysozyme in e tear
Obtain indicator and tear mixed value of chromatism △ rgb, and calculate corresponding Euclidean distance by c, Step d
(eds).Then, the multiple (80.5 times) that is diluted of tear in the linear relationship being obtained using step e and detection process, is calculated
In tear, lysozyme content is 0.89 ± 0.11mg/ml.For checking the reliability of the inventive method, after tear are diluted 10 times,
It is diluted lysozyme content in tear using uv-visible absorption spectra instrument (existing universal tester) test, see Figure 15.
(existing general with using uv-visible absorption spectra instrument using lysozyme content value in the inventive method detection tear
Tester) test value 0.83 ± 0.13mg/ml is very close to illustrating the inventive method, reliable results, have certain practical application
It is worth.
The all documents referring in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can
To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (10)
1. a kind of non-diagnostic non-therapeutic ground detects method of protein in testing sample it is characterised in that including step:
I () provides an indicator, described indicator contains the slaine being selected from the group: monovalence gold salt, monovalence silver salt or its group
Close, gather color a of described indicator；
(ii) described indicator is mixed with testing sample, obtain the first mixed solution, and gather described first mixed solution
Color b；
(iii) compare described color a and described color b, and extract the colour difference signal of color a and color b；
(iv) analyze described colour difference signal, obtain chromatism data △ r, △ g and △ b；
V () analysis described chromatism data △ r, △ g and △ b, thus carry out qualitative and/or quantitative to protein in testing sample
Detection.
2. the method for claim 1 it is characterised in that in described indicator the concentration of monovalence gold salt be 0.02-
50mmol/l, preferably 0.1-10mmol/l.
3. the method for claim 1 is it is characterised in that in step (i), described indicator is made in the following manner
:
A () provides a trivalent gold salt aqueous solution；
B () adds reproducibility protein solution, wherein trivalent gold and reproducibility protein in described trivalent gold salt aqueous solution
Ratio (mmol:mg) be (0.1-10): (1-100), obtain the second mixed solution；
C ph that () adjusts the second described mixed solution is 7.5-12.5, and acutely shakes, so that trivalent gold salt is reduced to one
Valency gold salt, the as the 3rd mixed solution；
D ph value that () adjusts the 3rd described mixed solution is 5.5-8.5, preferably 6.0-8.0, as described indicator.
4. the method for claim 1 is it is characterised in that in step (v), using multivariate statistics software (mvsp) and
The analysis of hierarchical cluster analysis (hca) method described chromatism data △ r, △ g and △ b.
5. the method for claim 1 is it is characterised in that in step (v), analyze described chromatism data △ r, △ g
With the Euclidean distance of △ b, thus qualitative and quantitative detection is carried out to protein in testing sample.
6. the method for claim 1 is it is characterised in that described testing sample is selected from the group:
Blood, serum, urine, tear.
7. the method for claim 1 is it is characterised in that the methods described protein that is selected from the group of detection:
Hemoglobin, albumin, globulin, transferrinss, pepsin, Myoglobin, chymase, lysozyme and mistake
Hydrogen oxide enzyme.
8. the method for claim 1 is it is characterised in that methods described detects the albumen in the testing sample being selected from the group
Matter:
Hemoglobin in (i) blood or serum；
(ii) albumin in urine；With
(iii) lysozyme in tear.
9. a kind of test kit detecting protein in testing sample is it is characterised in that described test kit includes:
I () indicator, described indicator contains the slaine being selected from the group: monovalence gold salt, monovalence silver salt or a combination thereof；
(ii) standard substance of the albumen being selected from the group: hemoglobin, albumin, globulin, transferrinss, pepsin, flesh are red
Albumen, chymase, lysozyme and catalase；With
(iii) description, has recorded the method described in claim 1 in described description.
10. a kind of system detecting protein in testing sample is it is characterised in that described system includes:
Test kit described in (i) claim 2；
(ii) camera function module；
(iii) aberration comparison module；
(iv) data analysis module；With
(v) data memory module；
(vi) optionally output module.
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