CN106033086B - Method, kit and its application based on LSPR detection sperm acrosin activities in assessing - Google Patents
Method, kit and its application based on LSPR detection sperm acrosin activities in assessing Download PDFInfo
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- CN106033086B CN106033086B CN201510116466.2A CN201510116466A CN106033086B CN 106033086 B CN106033086 B CN 106033086B CN 201510116466 A CN201510116466 A CN 201510116466A CN 106033086 B CN106033086 B CN 106033086B
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Abstract
The invention discloses a kind of method, kit and its application based on LSPR detection sperm acrosin activities in assessing.Wherein, the kit includes：Functional gold nanoparticles mark perforatorium zymolyte, and the perforatorium zymolyte of nanometer gold surface is fixed on comprising nanogold and by chemical bonding and/or physical adsorption way；And suitable for making the perforatorium zymolyte and the single-minded combination of acrosin and the buffer solution reacted., can quick (about 3 minutes or so), reliable, the sensitive and inexpensive activity for detecting acrosin by the kit and detection method of the present invention.
Description
Technical field
It is particularly a kind of based on nanogold particle surface etc. the present invention relates to a kind of activity rating method of acrosin
Ion resonance (LSPR) detects method, kit and its application of sperm acrosin activities in assessing, belongs to field of biological detection.
Background technology
Acrosin activity can reflect sperm quality, and acrosome enzyme activity deficiency may cause infertility, be evaluation sperm function and
The strong and weak important indicator of fertility, with reference to conventional indexs such as semen routine analysis, sperm morphology dyeing, it can more comprehensively reflect sperm
Quality.
The detection early stage of acrosin carries out radioimmunology using acrosome enzyme antibody more or fluorescent immune method detects, and passes through sight
The quantity for examining acrosin in perforatorium judges sperm fertilizing ability.Later Kennedy et al. first reported calculating acrosin
The method of hydrolysis substrate efficiency judges the activity of acrosin, and the method first cracks sperm, then lysate is placed in and added
In the detection pipe for entering enzyme hydrolysis substrate, by observing the colour developing depth after substrate hydrolysis, the water of light absorption value detection acrosin is calculated
Solution vigor.The method show that index can more react the physiological function of acrosin, as standard detecting method.
In recent years again have scholar according to sperm acrosome reaction deeper into understanding propose, the active Ying Youfa of acrosin
The sperm of raw acrosome reaction judges the actual physiology course for just more meeting sperm fertilization.Currently reported advanced induction acrosome
Reaction, then detection occur the activity of the acrosin discharged after acrosome reaction, sperm group in whole semen sample are indicated with this
The acrosin activity of body.However, current detection method needs the instrument by complexity, detection spends time length.
The content of the invention
It is a kind of based on nanogold local surface plasma resonance detection perforatorium it is a primary object of the present invention to provide
The method of enzymatic activity, it can realize quick to acrosin, sensitive, real-time detection, so as to overcome the deficiencies in the prior art.
Another free-revving engine of the present invention is that providing a kind of nanogold local surface plasma resonance that is based on detects sperm
The kit of acrosin activity.
A further object of the present invention is the application for providing the detection method and kit.
To realize aforementioned invention purpose, the technical solution adopted by the present invention includes：
A kind of kit based on LSPR detection sperm acrosin activities in assessing, it is included：
Functional gold nanoparticles mark perforatorium zymolyte, are inhaled comprising nanogold and by chemical bonding and/or physics
Subsidiary formula formula is fixed on the perforatorium zymolyte of nanometer gold surface；
And suitable for making the perforatorium zymolyte and the single-minded combination of acrosin and the buffer solution reacted.
Further, the kit also includes the specification for the application method for recording the kit, the use
Method includes：
Measure mainly marks sperm top by the buffer solution and the functional gold nanoparticles being dispersed in the buffer solution
Absorption spectrum of the mixed solution of body zymolyte composition in 300-1000nm wave-length coverages, and record and absorbed corresponding to absworption peak
Wavelength X0；
Various concentrations C is added in the mixed solutionnPerforatorium enzyme solutions, and determine respectively add it is different dense
Absorption of the different mixing reaction systems obtained after the acrosin standard liquid of degree in 300-1000nm wave-length coverages
Spectrum, record absworption peak corresponding wavelength λnWith absorption peak An, wavelength shift value is designated as Δ λn=λn-λ0, absworption peak deviant note
For Δ An=An-A0, find out CnWith Δ λnOr Δ AnIt is directly proportional, and thus establish acrosin solution concentration and wavelength shift value
Or the standard working curve of absworption peak deviant, wherein n are the natural number more than or equal to 2；
And unknown concentration C is added in the mixed solutionxPerforatorium enzyme solutions, and determine and be consequently formed
Absorbing wavelength λ of the hybrid reaction system in 300-1000nm wave-length coveragesx, wavelength shift value Δ λx=λx-λ0Or absworption peak is inclined
Shifting value Δ Ax=Ax-A0, and calculate C according to the standard working curvex。
As one of preferred embodiment, the application method may also include：The mixed solution is controlled in 300-
The peak value of absworption peak in 1000nm wave-length coverages is between 0.6-0.8.
A kind of detection means of sperm acrosin activities in assessing, it includes described kit.
A kind of detection method based on LSPR detection sperm acrosin activities in assessing, it is included：
There is provided functional gold nanoparticles mark perforatorium zymolyte and suitable for making the perforatorium zymolyte and sperm
The single-minded combination of acrosin and the buffer solution reacted, the functional gold nanoparticles mark perforatorium zymolyte include nanogold
And the perforatorium zymolyte of nanometer gold surface is fixed on by chemical bonding and/or physical adsorption way；
Measure mainly marks sperm top by the buffer solution and the functional gold nanoparticles being dispersed in the buffer solution
Absorption spectrum of the mixed solution of body zymolyte composition in 300-1000nm wave-length coverages, and record and absorbed corresponding to absworption peak
Wavelength X0；
Various concentrations C is added in the mixed solutionnPerforatorium enzyme solutions, and determine respectively add it is different dense
Absorption of the different mixing reaction systems obtained after the acrosin standard liquid of degree in 300-1000nm wave-length coverages
Spectrum, record absworption peak corresponding wavelength λnWith absorption peak An, wavelength shift value is designated as Δ λn=λn-λ0, absworption peak deviant note
For Δ An=An-A0, find out CnWith Δ λnOr Δ AnIt is directly proportional, and thus establish acrosin solution concentration and wavelength shift value
Or the standard working curve of absworption peak deviant, wherein n are the natural number more than or equal to 2；
And unknown concentration C is added in the mixed solutionxPerforatorium enzyme solutions, and determine and be consequently formed
Absorbing wavelength λ of the hybrid reaction system in 300-1000nm wave-length coveragesx, wavelength shift value Δ λx=λx-λ0Or absworption peak is inclined
Shifting value Δ Ax=Ax-A0, and calculate C according to the standard working curvex。
As one of preferred embodiment, the detection method may also include：The mixed solution is controlled in 300-
The peak value of absworption peak in 1000nm wave-length coverages is between 0.6-0.8.
Among the embodiment of the present invention, the perforatorium zymolyte passes through with modifying the choosing in nanometer gold surface
Determine functional group coupling reaction occurs and is fixedly attached to the nanometer gold surface.
Wherein, for nanogold functionalization to be not limited into certain so as to provide the functional group being coupled with perforatorium zymolyte
Specific species, those skilled in the art be able to should easily understand according to this explanation, every to modify in nanometer golden watch
Simultaneously the functional group of energy and acrosin substrate-function is all applicable in face, wherein, the group of the functional group termini can be
Carboxyl, amino or other active groups.
Further, among the embodiment of the present invention, the functional gold nanoparticles mark perforatorium zymolyte
Preparation method include：
Nanogold with obvious ultraviolet-visible absorption spectroscopy absworption peak is provided,
Functional group is selected in the nanogold surface modification by chemistry or physical method,
The perforatorium zymolyte is carried out coupled reaction with the selected functional group and be fixedly connected on the nanometer
Gold surface.
Further, the size range of the nanogold is 20m~150nm.
Further, the nanogold includes nanometer gold bar, nano gold spherical or nanogold cubic block, but not limited to this.
Preferably, the diameter of the nanometer gold bar about 20nm, length are 30nm~50nm, and the particle diameter of the nano gold spherical is about
30~120nm, the length of side about 10nm~100nm of the nanogold cubic block.
Further, the perforatorium zymolyte can be can be with any conjunction of perforatorium enzyme reaction (such as hydrolysis)
Suitable species, such as ZP3 albumen etc..
Further, the buffer solution can be PBS etc., and its pH value is preferably 7 or so.
Compared with prior art, beneficial effect of the present invention at least that：
(1) by rationally designing the size of gold nano, the ultraviolet-visible absorption spectroscopy of nanogold (such as gold nanorods) exists
Within the measurement range of conventional ultraviolet-uisible spectrophotometer, it is not necessary to carried out using special ultraviolet-uisible spectrophotometer
Detection；
(2) nanogold, such as gold nanorods surface are repaiied using functionalization material, particularly hyper-branched polymer
Decorations, can provide binding site for acrosin substrate, improve in nanogold, such as gold nanorods surface modification perforatorium
The density of zymoprotein substrate, improve test limit and the sensitivity (0.1~50 μm of ol/L) of detection acrosin；
(3), can quick (3 minutes or so), reliable, sensitive and inexpensive by the kit and detection method of the present invention
Detect the activity of acrosin.
Brief description of the drawings
Fig. 1 is in the signal of gold nanorods surface modification perforatorium zymolyte among an of the invention typical embodiments
Figure；
Fig. 2 is the nanometer gold bar detection perforatorium of perforatorium zymolyte modification among a typical embodiments of the invention
The schematic diagram of enzyme；
Fig. 3 is the transmission electron microscope photo of the nanometer gold bar prepared among an of the invention typical embodiments；
Fig. 4 is the transmission electron microscope photo of the nano gold spherical prepared among an of the invention typical embodiments.
Embodiment
As it was previously stated, in view of many defects of prior art, inventor are carried through studying for a long period of time and largely putting into practice
Go out technical scheme, i.e. one kind is based on nanogold local surface plasma resonance (LSPR) detection perforatorium enzyme activity
Property method, it is mainly by nanogold surface modification perforatorium zymolyte, utilizing perforatorium enzyme hydrolysis acrosin
Substrate causes the change of the surface plasma resonance signal of nanogold, realizes quick to acrosin, sensitive, real-time detection.
Further say, it is a kind of that sperm acrosin activities in assessing is detected based on LSPR among the embodiment of the present invention
Method can include：
(1) nanogold of perforatorium zymolyte modification is provided, including：
Nanogold, mainly influenceed using its surface plasma resonance signal by the dielectric constant of external environment, Ke Yijian
Survey acrosin and the change during acrosin substrate-function；
The functional group in nanometer gold surface is modified, mainly being provided on nanometer gold bar surface largely can be with perforatorium
The site that zymoprotein combines, more preferably, amphiphilic hyper-branched molecule can be used (for example, polystyrene-b- polypropylene
Acid, or polystyrene-b- polyvinyl alcohol) nanometer gold surface is functionalized, it the advantage is that：A, amphiphilic hyper-branched height
Molecule can improve stability of the nanogold in different solvents, will not reunite；B, the periphery of hyper-branched polymer can be with
More functional groups are exposed, are easy to further modify.
Perforatorium zymolyte (such as ZP3 albumen), the functional group reactionses by physics mode or with nanometer gold surface, especially
It is preferably latter approach and is fixed on the surface of nanogold.
(2) detection of the plasmon resonance signal of nanogold：Perforatorium enzyme hydrolysis acrosin substrate (such as ZP3
Albumen) after, cause the dielectric constant of nanogold (such as gold nanorods) surrounding environment to change, pass through ultravioletvisible absorption light
Spectrum can clearly detect this change.
Among one more specifically embodiment, a kind of method based on LSPR detection sperm acrosin activities in assessing is specifically wrapped
Include：
A. nanogold is prepared；
B. nanogold is surface-functionalized.Shangguan is modified outside nanogold and can be rolled into a ball by chemistry or physical method；
C. functional gold nanoparticles mark perforatorium zymolyte.Perforatorium zymolyte by with step (2) in modification
Functional group reactionses or physisorption are fixed on a nanometer gold surface.
D. nanogold/perforatorium zymolyte hybrid systems detection acrosin.What measure was modified by perforatorium zymolyte
The ultraviolet-visible absorption spectroscopy of nanogold, wavelength corresponding to its ultraviolet and visible absorption peak are designated as λa, after adding acrosin, essence
Sub- acrosome zymolyte determines the purple of the nanogold of the perforatorium zymolyte modification of partial hydrolysis by acrosin partial hydrolysis
Outer visible absorption spectra, wavelength corresponding to its longitudinal absworption peak are λb, the difference of absworption peak wavelength is designated as Δ λ=λa-λb, sperm
The concentration c of acrosin is directly proportional to Δ λ.
E. the preparation and evaluation of detection device are quantified.
Wherein, the pattern of nanogold includes nanometer gold bar, nano gold spherical, nanogold cubic block etc..
Wherein, the functionalization of nanogold (such as gold nanorods) is not limited to certain specific reaction and method, it is every can be
Energy and the usability methods of acrosin substrate-function functional group are all feasible in nanogold surface modification.
Further, the perforatorium zymolyte be mainly characterized by can be with the single-minded combination of acrosin.It is any one
Kind perforatorium zymolyte can serve as by the substrate of nano gold mark.
Among foregoing embodiment of the present invention, mainly using nanogold surface plasma resonance change and sperm
The concentration of acrosin is directly proportional to be detected.But those skilled in the art should know, plasma resonance change is utilized
The other specification such as intensity of nanogold absworption peak may also be used for detection perforatorium enzyme concentration.
In addition, the acrosin for being adapted to kit and detection method using the present invention to be detected should be not limited to people
The sperm of class, and can also be the acrosin of the general mammal such as animal such as pig, dog, horse.
Also, in the exemplary embodiments of the present invention, technical scheme can be realized in the following way：
(1) prepares nanometer gold bar：The preparation of nanometer gold bar is broadly divided into two steps:Seed solution prepares and Jin Zhongsheng
It is long.
I) prepared by seed solution：By 5mL 0.5mM HAucl4Solution and the mixing of 5mL 0.2M CTAB solution, are acutely being stirred
0.01M sodium borohydrides (the NaBH that (1200r/min) newly configures 0.6mL under the conditions of mixing4) solution is diluted to 1mL and is then added to
In above-mentioned mixed solution, stop after stirring 2min, seed of the resulting solution as growth gold nanorods；
Ii) seed growth：By 5mL 0.2M CTAB solution and 0.2mL 4mM AgNO under the conditions of 25 DEG C3Mixing, then add
Enter 5.0mL 1mM HAuCl4Solution, 70 μ L 0.0788M ascorbic acid are added after being uniformly mixed；Finally take 12 μ L seeds
Solution is added in growth-promoting media, and 6h is stood at 30 DEG C.The gold nanorods of preparation centrifuge under the conditions of 8000r/min
10min, to remove the CTAB on gold nanorods surface.
(2) it is as follows for the technology path (Fig. 1) of the hyper-branched polymer of hydroxyl in nanometer gold bar surface modified end：
A) nanometer gold bar passes through with the thiobis of 2,2'- bis- [1- (the bromo- 2- methyl-propionyloxies of 2-)] ethane (DTBE) first
Around gold nanorods, macromole evocating agent triggers the generation of styrene (PS) monomer polymerization reactions for sulphur gold key (S-Au) reaction absorption
Polystyrene.10mg DTBE are dissolved in 100mL DMF, add the above-mentioned removal CTAB of 1mL gold under intense agitation
Nanometer rods solution, add 30mg styrene and stir 12h at 40 DEG C.
B) further trigger 2- ((bromine butyryl) epoxide) ethyl propylene acid esters (BBEA) progress Self condensation vinyl polymerization anti-
Answer (SCVP), the hyperbranched high molecular polymer in gold surface modification.The progress of 15mg acrylic monomers (AA) monomer is added again
Polymerization, the substantial amounts of carboxyl in the modification of gold nanorods periphery.
(3) perforatorium zymolyte is modified：96mg EDC and 29mg NHS are dissolved in 10mL deionized waters, 3mL peripheries
Nanometer gold bar containing a large amount of carboxyls centrifuges 10min under the conditions of 5000r/min and is concentrated to after 500 μ L under rapid mixing conditions
It is added in above-mentioned solution, adjusts pH to 7.1 using PBS, then add 400 μ L 2mg/mL perforatorium zymolytes ZP3
Albumen, 1h is cultivated at 25 DEG C.Reaction solution centrifuges under the conditions of 10000r/min, and removing is not coupled to gold nanorods surface
Antibody.
(4) nanometer gold bar-perforatorium zymolyte hybrid systems detection acrosin.A working curve is initially set up, is taken
The gold nanorods solution of appropriate perforatorium enzyme modification controls the concentration of above-mentioned solution to ensure gold nano in quartz colorimetric utensil
The peak value of rod longitudinal direction absworption peak is advisable between 0.6-0.8, and perforatorium enzyme modification is determined in 300-1000nm wave-length coverages
Gold nanorods absorption spectrum, wavelength corresponding to its longitudinal absworption peak is designated as λ0, added in above-mentioned solution containing different dense
Spend CnAcrosin solution, respectively measure add various concentrations acrosin ultraviolet-visible absorption spectroscopy λn, ripple
Long deviant is designated as Δ λn=λn-λ0, establish CnWith Δ λnBetween relation.For unknown concentration c acrosin, Ke Yili
Calculated with the Δ λ of measure.
Below in conjunction with some preferred embodiments the technical solution of the present invention is further explained explanation, but reality therein
Test condition and setup parameter is not construed as limitation to basic technical scheme of the present invention.And protection scope of the present invention is not limited to
Following embodiments.
Embodiment 1：The gold nanorods modified using perforatorium zymolyte detect acrosin
1. prepare nanometer gold bar：The preparation of nanometer gold bar is broadly divided into two steps:Prepared by seed solution and gold kind grows.
1) prepared by seed solution：By 5mL 0.5mM HAucl4Solution and the mixing of 5mL 0.2M CTAB solution, under intense agitation
0.01M sodium borohydrides (the NaBH that (1200r/min) newly configures 0.6mL4) solution is diluted to 1mL and is then added to above-mentioned mixing
In solution, stop after stirring 2min, seed of the resulting solution as growth gold nanorods；2) seed growth：Under the conditions of 25 DEG C
By 5mL 0.2M CTAB solution and 0.2mL 4mM AgNO3Mixing, adds 5.0mL 1mM HAuCl4Solution, it is stirred
70 μ L 0.0788M ascorbic acid are added after even；Finally take 12 μ L seed solutions to be added in growth-promoting media, 6h is stood at 30 DEG C
.The gold nanorods of preparation centrifuge 10min under the conditions of 8000r/min, to remove the CTAB on gold nanorods surface.Prepare
The TEM of nanometer gold bar is as shown in Figure 3.
It is 2. as follows for the technology path (referring to Fig. 1) of the hyper-branched polymer of hydroxyl in nanometer gold bar surface modified end：
A) nanometer gold bar passes through with the thiobis of 2,2'- bis- [1- (the bromo- 2- methyl-propionyloxies of 2-)] ethane (DTBE) first
Around gold nanorods, macromole evocating agent triggers the generation of styrene (PS) monomer polymerization reactions for sulphur gold key (S-Au) reaction absorption
Polystyrene.10mg DTBE are dissolved in 100mL DMF, add the above-mentioned removal CTAB of 1mL gold under intense agitation
Nanometer rods solution, add 30mg styrene and stir 12h at 40 DEG C.B) 2-((bromine butyryl) epoxide) ethyls third are further triggered
Olefin(e) acid ester (BBEA) carries out Self condensation vinyl polymerization reaction (SCVP), the hyperbranched high molecular polymerization in gold surface modification
Thing.Addition 15mg acrylic monomers (AA) monomer is polymerize, the substantial amounts of carboxyl in the modification of gold nanorods periphery.
3. the modification of perforatorium zymolyte：96mg EDC and 29mg NHS are dissolved in 10mL deionized waters, 3mL peripheries contain
The nanometer gold bar for having a large amount of carboxyls centrifuges 10min under the conditions of 5000r/min and is concentrated to after 500 μ L to be added under rapid mixing conditions
Enter into above-mentioned solution, adjust pH to 7.1 using PBS, then add 400 μ L 2mg/mL perforatorium zymolyte ZP3 eggs
In vain, 1h is cultivated at 25 DEG C.Reaction solution centrifuges under the conditions of 10000r/min, removes and is not coupled to the anti-of gold nanorods surface
Body.
4. nanometer gold bar-perforatorium zymolyte hybrid systems detection acrosin.A working curve is initially set up, is taken suitable
The gold nanorods solution of perforatorium enzyme modification is measured in quartz colorimetric utensil, controls the concentration of above-mentioned solution to ensure gold nanorods
The peak value of longitudinal absworption peak is advisable between 0.6-0.8, and perforatorium enzyme modification is determined in 300-1000nm wave-length coverages
The absorption spectrum of gold nanorods, wavelength corresponding to its longitudinal absworption peak are designated as λ0, added in above-mentioned solution and contain various concentrations Cn
Acrosin solution, respectively measure add various concentrations acrosin ultraviolet-visible absorption spectroscopy λn, wavelength
Deviant is designated as Δ λn=λn-λ0, establish CnWith Δ λnBetween relation.For unknown concentration c acrosin, can utilize
The Δ λ of measure is calculated
5. referring to done canonical plotting, the concentration of acrosin is 5 μM in determination sample, detection range 0.01
μM~50 μM.
Embodiment 2：The nano gold spherical modified using perforatorium zymolyte detects acrosin
1. prepare nano gold spherical：Take 100mL 2.5 × 10-4mol/L HAuCl4Solution is heated to 100-150 DEG C, adds
The citric acid three sodium solutions of 10mL 1%, when the color of solution is changed into transparent claret completely, stop after continuing backflow 10min
Heating, room temperature is cooled to, 4 DEG C of preservations, obtained nano gold spherical ultraviolet spectra absorbing wavelength is 526nm.The transmission of nano gold spherical
Electron microscope is shown in Fig. 4.
2. the mark of nanogold particle：10mg DTBE are dissolved in 100mL DMF, added under intense agitation
The nearly ball solution of the above-mentioned nanometers of 20mL, 12h is stirred at 40 DEG C adding 30mg styrene.Then 40mg BBEA monomers are added, are drawn
Hair polymerization, forms hyper-branched polymer, after reacting a period of time, 15mg acrylic monomers (AA) is added, from outside gold nanorods
Enclose the upper substantial amounts of carboxyl of modification.
3. the gold nano of perforatorium zymolyte modification：96mg EDC and 29mg NHS are dissolved in 10mL deionized waters,
Nano gold spherical of the 10mL peripheries containing a large amount of carboxyls centrifuges 10min under the conditions of 5000r/min, is concentrated to after 500 μ L quick
It is added under stirring condition in above-mentioned solution, adjusts pH to 7.1 using PBS, then add 400 μ L 2mg/mL sperms tops
Body zymolyte ZP3 albumen, 1h is cultivated at 25 DEG C.Reaction solution centrifuges 10min under the conditions of 10000r/min, and removing is not coupled
To the antibody on nano gold spherical surface.
4. nano gold spherical-perforatorium zymolyte hybrid systems detection acrosin.A working curve is initially set up, is taken
The nano gold spherical solution of 10mL perforatorium enzyme modifications is with quartz colorimetric utensil, controlling the concentration of above-mentioned solution to ensure nanogold
The peak value of absworption peak is advisable between 0.6-0.8, and the nanometer of perforatorium enzyme modification is determined in 300-1000nm wave-length coverages
The absorption spectrum of gold goal, wavelength corresponding to its absworption peak are designated as λ0, added in above-mentioned solution and contain various concentrations CnSperm top
The ultraviolet-visible absorption spectroscopy λ of the solution of body enzyme, the respectively acrosin of measure addition various concentrationsn, wavelength shift value note
For Δ λn=λn-λ0, establish CnWith Δ λnBetween relation.For the acrosin c of unknown concentration, the Δ determined can be utilized
λ is calculated.
5. referring to done canonical plotting, the concentration of acrosin is 7 μM in determination sample, detection range 0.01
μM~30 μM.
Finally, it is to be noted that, term " comprising ", "comprising" or its any other variant be intended to it is non-exclusive
Property includes, so that process, method, article or equipment including a series of elements not only include those key elements, and
Also include the other element that is not expressly set out, or also include for this process, method, article or equipment inherently
Key element.
It will be appreciated by those skilled in the art that the embodiment of present invention described above, is not formed to the present invention
The restriction of protection domain.Any technique according to the invention design made various other corresponding changes and deformation, all should
Comprising within the scope of the invention as claimed.
Claims (5)
1. a kind of kit based on LSPR detection sperm acrosin activities in assessing, it is characterised in that include：
Functional gold nanoparticles mark perforatorium zymolyte, comprising nanogold and pass through chemical bonding and/or physical absorption side
Formula is fixed on the perforatorium zymolyte of nanometer gold surface, and the nanogold is selected from nanometer gold bar, nano gold spherical or nanometer Gionee
A diameter of 20nm of square, wherein nanometer gold bar, length are 30nm~50nm, and the particle diameter of nano gold spherical is 30~120nm, nanometer
The length of side of Gionee square is 10nm~100nm；
Suitable for making the perforatorium zymolyte and the single-minded combination of acrosin and the buffer solution reacted；And
The specification of the application method of the kit is recorded, described application method includes：
Control mainly marks acrosin by the buffer solution and the functional gold nanoparticles being dispersed in the buffer solution
The concentration of the mixed solution of substrate composition, makes the peak value of absworption peak of the mixed solution in 300-1000nm wave-length coverages exist
Between 0.6-0.8；Absorption spectrum of the mixed solution in 300-1000nm wave-length coverages is determined, and it is corresponding to record absworption peak
Absorbing wavelength λ0And absorption peak strength A0；
Various concentrations C is added in the mixed solutionnPerforatorium enzyme solutions, and respectively determine and to add various concentrations
Absorption spectrum of the different mixing reaction systems obtained after acrosin standard liquid in 300-1000nm wave-length coverages,
Record absworption peak corresponding wavelength λnWith absorption peak An, wavelength shift value is designated as Δ λn=λn-λ0, absworption peak deviant is designated as Δ An
=An-A0, find out CnWith Δ λnOr Δ AnIt is directly proportional, and thus establish acrosin solution concentration and wavelength shift value or absorption
The standard working curve of peak deviant, wherein n are the natural number more than or equal to 2；
And unknown concentration C is added in the mixed solutionxPerforatorium enzyme solutions, and it is anti-to determine the mixing that is consequently formed
Answer absorbing wavelength λ of the system in 300-1000nm wave-length coveragesx, wavelength shift value Δ λx=λx-λ0Or absworption peak offset value delta
Ax=Ax-A0, and calculate C according to the standard working curvex。
2. kit according to claim 1, it is characterised in that：The perforatorium zymolyte passes through with modifying in nanometer
The selected functional group of gold surface occurs coupling reaction and is fixedly attached to the nanometer gold surface, the selected functional group source in
Amphiphilic hyper-branched molecule, the amphiphilic hyper-branched molecule include polystyrene-b- polyacrylic acid, or polystyrene-b- gathers
Vinyl alcohol.
3. a kind of detection means of sperm acrosin activities in assessing, it is characterised in that include the examination any one of claim 1-2
Agent box.
4. a kind of kit for being applied to the detection method based on LSPR detection sperm acrosin activities in assessing, the kit include：
Functional gold nanoparticles mark perforatorium zymolyte, and it includes nanogold and by chemical bonding and/or physical absorption
Mode is fixed on the perforatorium zymolyte of nanometer gold surface, and the nanogold is selected from nanometer gold bar, nano gold spherical or nanogold
A diameter of 20nm of cubic block, wherein nanometer gold bar, length are 30nm~50nm, and the particle diameter of nano gold spherical is 30~120nm, is received
The length of side of meter Jin Li squares is 10nm~100nm, and
Suitable for making the perforatorium zymolyte and the single-minded combination of acrosin and the buffer solution reacted；
Described detection method includes：
Control mainly marks acrosin by the buffer solution and the functional gold nanoparticles being dispersed in the buffer solution
The peak value of absworption peak of the mixed solution of substrate composition in 300-1000nm wave-length coverages is between 0.6-0.8；
Absorption spectrum of the mixed solution in 300-1000nm wave-length coverages is determined, and records and ripple is absorbed corresponding to absworption peak
Long λ0；
Various concentrations C is added in the mixed solutionnPerforatorium enzyme solutions, and respectively determine and to add various concentrations
Absorption spectrum of the different mixing reaction systems obtained after acrosin standard liquid in 300-1000nm wave-length coverages,
Record absworption peak corresponding wavelength λnWith absorption peak An, wavelength shift value is designated as Δ λn=λn-λ0, absworption peak deviant is designated as Δ An
=An-A0, find out CnWith Δ λnOr Δ AnIt is directly proportional, and thus establish acrosin solution concentration and wavelength shift value or absorption
The standard working curve of peak deviant, wherein n are the natural number more than or equal to 2；
And unknown concentration C is added in the mixed solutionxPerforatorium enzyme solutions, and it is anti-to determine the mixing that is consequently formed
Answer absorbing wavelength λ of the system in 300-1000nm wave-length coveragesx, wavelength shift value Δ λx=λx-λ0Or absworption peak offset value delta
Ax=Ax-A0, and calculate C according to the standard working curvex。
5. kit according to claim 4, it is characterised in that the functional gold nanoparticles mark perforatorium zymolyte
Preparation method include：
Nanogold with obvious ultraviolet-visible absorption spectroscopy absworption peak is provided,
Functional group is selected in the nanogold surface modification by chemistry or physical method, the selected functional group source is in two
Parent's property super branched molecule, the amphiphilic hyper-branched molecule include polystyrene-b- polyacrylic acid, or the poly- second of polystyrene-b-
Enol,
The perforatorium zymolyte is carried out coupled reaction with the selected functional group and be fixedly connected on the nanometer golden watch
Face.
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