CN106033086A - Method and kit for sperm acrosomal enzyme activity detection based on LSPR, and applications of method and kit - Google Patents
Method and kit for sperm acrosomal enzyme activity detection based on LSPR, and applications of method and kit Download PDFInfo
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- CN106033086A CN106033086A CN201510116466.2A CN201510116466A CN106033086A CN 106033086 A CN106033086 A CN 106033086A CN 201510116466 A CN201510116466 A CN 201510116466A CN 106033086 A CN106033086 A CN 106033086A
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- perforatorium
- zymolyte
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- acrosin
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Abstract
The present invention discloses a method and a kit for sperm acrosomal enzyme activity detection based on LSPR, and applications of the method and the kit. The kit comprises: a functionalized gold nanoparticle labeled sperm acrosomal enzyme substrate, wherein the functionalized gold nanoparticle labeled sperm acrosomal enzyme substrate contains gold nanoparticles and a sperm acrosomal enzyme substrate immobilized on the surface of the gold nanoparticles by adopting chemical bonding and/or physical adsorption; and a buffer liquid for making the sperm acrosomal enzyme substrate and the sperm acrosomal enzyme be subjected to specific combination and reaction. With the kit and the detection method of the present invention, the sperm acrosomal enzyme activity can be rapidly (about 3 min), reliably, sensitively and low-costly detected.
Description
Technical field
The present invention relates to a kind of activity rating method of acrosin, particularly one based on nanogold particle surface plasma altogether
Shake (LSPR) detection method of sperm acrosin activities in assessing, test kit and application thereof, belong to field of biological detection.
Background technology
Acrosin activity can reflect sperm quality, and acrosomal enzyme vigor deficiency may cause sterile, is to evaluate sperm function and fertility
Strong and weak important indicator, in conjunction with conventional indexs such as semen routine analysis, sperm morphology dyeing, can more comprehensively reflect sperm quality.
The detection of acrosomal enzyme utilizes acrosomal enzyme antibody to carry out radioimmunology or fluorescent immune method detection, by observing sperm in early days
In acrosome, the quantity of acrosomal enzyme judges sperm fertilizing ability.Kennedy et al. first reported calculating acrosomal enzyme hydrolysis substrate later
The method of efficiency judges the activity of acrosin, and first sperm is cracked by the method, then is placed in by lysate at the bottom of addition enzyme hydrolysis
In the detection pipe of thing, by the colour developing depth after observation substrate hydrolysis, calculate the hydrolysis vigor of light absorption value detection acrosomal enzyme.This side
Method show that index more can react the physiological function of acrosin, has become standard detecting method.
Have the most again scholar according to sperm acrosome reaction deeper into understanding propose, the activity of acrosin should by occur acrosome
The sperm of reaction judges the most more to meet the actual physiological process of sperm fertilization.Currently reported advanced induction acrosome reaction, so
The activity of the acrosomal enzyme that rear detection discharges after there is acrosome reaction, indicates the acrosomal enzyme of sperm colony in whole semen sample with this
Activity.But, current detection method needs by complicated instrument, and the detection cost time is long.
Summary of the invention
Present invention is primarily targeted at and provide a kind of based on nanometer gold local surface plasma resonance detection sperm acrosin activities in assessing
Method, its can realize quick to acrosin, sensitive, detect in real time, thus overcome the deficiencies in the prior art.
Another free-revving engine of the present invention is that providing a kind of lives based on nanometer gold local surface plasma resonance detection acrosin
The test kit of property.
A further object of the present invention is to provide the application of described detection method and test kit.
For realizing aforementioned invention purpose, the technical solution used in the present invention includes:
A kind of test kit based on LSPR detection sperm acrosin activities in assessing, it comprises:
Functional gold nanoparticles labelling perforatorium zymolyte, is comprised nanometer gold and is consolidated by chemical bonding and/or physical adsorption way
It is scheduled on the perforatorium zymolyte on nanometer gold surface；
And, be suitable to make described perforatorium zymolyte and the single-minded combination of acrosin and carry out the buffer reacted.
Further, described test kit also comprises the description of the using method recording described test kit, described using method bag
Include:
Measure mainly by described buffer and the functional gold nanoparticles labelling perforatorium zymolyte being dispersed in described buffer
The mixed solution of composition absorption spectrum in 300-1000nm wave-length coverage, and record absorbing wavelength λ that absworption peak is corresponding0；
Variable concentrations C is added in described mixed solutionnPerforatorium enzymatic solution, and measure respectively add variable concentrations essence
The different mixing reaction system absorption spectrums in 300-1000nm wave-length coverage obtained after sub-acrosomal enzyme standard solution, 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 sets up acrosin solution concentration and wavelength shift value or absworption peak deviant
Standard working curve, wherein n is the natural number more than or equal to 2；
And, described mixed solution adds unknown concentration CxPerforatorium enzymatic solution, and it is anti-to measure the mixing that is consequently formed
Answer system absorbing wavelength λ in 300-1000nm wave-length coveragex, wavelength shift value Δ λx=λx-λ0Or absworption peak deviant
ΔAx=Ax-A0, and calculate C according to described standard working curvex。
As one of preferred embodiment, described using method may also include that the described mixed solution of control is at 300-1000nm wavelength
In the range of the peak value of absworption peak between 0.6-0.8.
The detection device of a kind of sperm acrosin activities in assessing, it comprises described test kit.
A kind of detection method based on LSPR detection sperm acrosin activities in assessing, it comprises:
Functional gold nanoparticles labelling perforatorium zymolyte is provided and is suitable to make described perforatorium zymolyte special with acrosin
One combines and carries out the buffer reacted, and described functional gold nanoparticles labelling perforatorium zymolyte comprises nanometer gold and by changing
Bonding and/or physical adsorption way are fixed on the perforatorium zymolyte on nanometer gold surface；
Measure mainly by described buffer and the functional gold nanoparticles labelling perforatorium zymolyte being dispersed in described buffer
The mixed solution of composition absorption spectrum in 300-1000nm wave-length coverage, and record absorbing wavelength λ that absworption peak is corresponding0；
Variable concentrations C is added in described mixed solutionnPerforatorium enzymatic solution, and measure respectively add variable concentrations essence
The different mixing reaction system absorption spectrums in 300-1000nm wave-length coverage obtained after sub-acrosomal enzyme standard solution, 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 sets up acrosin solution concentration and wavelength shift value or absworption peak deviant
Standard working curve, wherein n is the natural number more than or equal to 2；
And, described mixed solution adds unknown concentration CxPerforatorium enzymatic solution, and it is anti-to measure the mixing that is consequently formed
Answer system absorbing wavelength λ in 300-1000nm wave-length coveragex, wavelength shift value Δ λx=λx-λ0Or absworption peak deviant
ΔAx=Ax-A0, and calculate C according to described standard working curvex。
As one of preferred embodiment, described detection method may also include that the described mixed solution of control is at 300-1000nm wavelength
In the range of the peak value of absworption peak between 0.6-0.8.
Among an embodiment of the present invention, described perforatorium zymolyte by with modify selected sense on nanometer gold surface
Group occurs coupling reaction to be fixedly attached to described nanometer gold surface.
Wherein, specific for nanometer gold functionalization thus offer and the functional group of perforatorium zymolyte coupling are not limited to certain
Kind, those skilled in the art should understand easily according to this explanation, every can modify on nanometer gold surface can be with
The functional group of acrosin substrate-function is all applicable, and wherein, the group of described functional group termini can be carboxyl, amino
Or other active groups.
Further, among an embodiment of the present invention, the preparation of described functional gold nanoparticles labelling perforatorium zymolyte
Method includes:
The nanometer gold with obvious ultraviolet-visible absorption spectroscopy absworption peak is provided,
Selected functional group is modified on described nanometer gold surface by chemistry or physical method,
Described perforatorium zymolyte and described selected functional group is made to carry out coupled reaction and be fixedly connected on described nanometer gold surface.
Further, the size range of described nanometer gold is 20m～150nm.
Further, described nanometer gold includes nanometer gold bar, nano gold spherical or nanometer gold cubic block, but is not limited to this.
Preferably, the diameter of described nanometer gold bar about 20nm, a length of 30nm～50nm, the particle diameter of described nano gold spherical is about
30～120nm, the length of side about 10nm～100nm of described nanometer gold cubic block.
Further, described perforatorium zymolyte can be can be with any suitable kind of perforatorium enzyme reaction (such as hydrolysis)
Class, such as ZP3 albumen etc..
Further, described buffer can be PBS etc., and its pH value is preferably about 7.
Compared with prior art, beneficial effect of the present invention at least that:
(1) by the size of appropriate design gold nano, the ultraviolet-visible absorption spectroscopy of nanometer gold (such as gold nanorods) is in routine
Ultraviolet-uisible spectrophotometer measurement within the scope of, it is not necessary to use special ultraviolet-uisible spectrophotometer to detect；
(2) utilizing functionalization material, particularly hyper-branched polymer is to nanometer gold, and such as gold nanorods surface is modified, can
Thinking that perforatorium zymolyte provides binding site, improve at nanometer gold, perforatorium pheron is modified on such as gold nanorods surface
The density of substrate, improves detection limit and the sensitivity (0.1～50 μm ol/L) of detection acrosin；
(3) by test kit and the detection method of the present invention, can quickly (about 3 minutes), reliable, sensitive and low cost
The activity of detection acrosin.
Accompanying drawing explanation
Fig. 1 is the schematic diagram modifying perforatorium zymolyte among the present invention one typical embodiments on gold nanorods surface；
Fig. 2 is showing of the nanometer gold bar detection acrosin that among the present invention one typical embodiments, perforatorium zymolyte is modified
It is intended to；
Fig. 3 is the transmission electron microscope photo of the nanometer gold bar of preparation among the present invention one typical embodiments；
Fig. 4 is the transmission electron microscope photo of the nano gold spherical of preparation among the present invention one typical embodiments.
Detailed description of the invention
As it was previously stated, in view of many defects of prior art, inventor, through studying for a long period of time and putting into practice in a large number, is proposed this
The technical scheme of invention, i.e. a kind of side based on nanometer gold local surface plasma resonance (LSPR) detection sperm acrosin activities in assessing
Method, it mainly by modifying perforatorium zymolyte on nanometer gold surface, utilizes perforatorium enzyme hydrolysis acrosomal enzyme substrate to cause
The change of the surface plasma resonance signal of nanometer gold, it is achieved quick to acrosin, sensitive, detect in real time.
Further saying, among an embodiment of the present invention, a kind of method based on LSPR detection sperm acrosin activities in assessing can
To comprise:
(1) nanometer gold that perforatorium zymolyte is modified is provided, including:
Nanometer gold, mainly utilizes its surface plasma resonance signal to be affected by the dielectric constant of external environment, can monitor sperm
Change during acrosomal enzyme and acrosin substrate-function；
Modify functional group on nanometer gold surface, mainly provide on nanometer gold bar surface substantial amounts of can be with perforatorium pheron
In conjunction with site, the most preferably, can use amphiphilic hyper-branched molecule (such as, polystyrene-b-polyacrylic acid, or
Polystyrene-b-polyvinyl alcohol) nanometer gold surface is functionalized, have an advantage in that: a, amphiphilic hyper-branched macromolecule can
To improve nanometer gold stability in different solvents, will not reunite；B, the periphery of hyper-branched polymer can expose more
Many functional groups, it is simple to modify further.
Perforatorium zymolyte (such as ZP3 albumen), by physics mode or with the functional group reactions on nanometer gold surface, the most excellent
Choosing is latter approach and is fixed on the surface of nanometer gold.
(2) detection of the plasmon resonance signal of nanometer gold: perforatorium enzyme hydrolysis acrosin substrate (such as ZP3 albumen)
After, cause the dielectric constant of nanometer gold (such as gold nanorods) surrounding to change, can by ultraviolet-visible absorption spectroscopy
Clearly to detect this change.
Among one more specifically embodiment, a kind of method based on LSPR detection sperm acrosin activities in assessing specifically includes:
A. nanometer gold is prepared；
B. nanometer gold is surface-functionalized.Outside nanometer gold, modify Shangguan can roll into a ball by chemistry or physical method；
C. functional gold nanoparticles labelling perforatorium zymolyte.Perforatorium zymolyte is by the sense modified middle with step (2)
Group's reaction or physisorption are fixed on nanometer gold surface.
D. nanometer gold/perforatorium zymolyte hybrid systems detection acrosomal enzyme.Measure the nanometer gold modified by perforatorium zymolyte
Ultraviolet-visible absorption spectroscopy, wavelength corresponding to its ultraviolet and visible absorption peak is designated as λa, after adding acrosin, perforatorium
Zymolyte is inhaled by acrosin partial hydrolysis, the UV, visible light of the nanometer gold measuring the perforatorium zymolyte modification of partial hydrolysis
Receiving spectrum, the wavelength that its longitudinal absworption peak is corresponding is λb, the difference of absworption peak wavelength is designated as Δ λ=λa-λb, acrosin
Concentration c be directly proportional to Δ λ.
E. the preparation of detection by quantitative equipment and evaluation.
Wherein, the pattern of nanometer gold includes nanometer gold bar, nano gold spherical, nanometer gold cubic block etc..
Wherein, the functionalization of nanometer gold (such as gold nanorods) is not limited to certain specifically reaction and method, every can be in nanometer
It can be all feasible with the usability methods of acrosin substrate-function functional group that gold surface is modified.
Further, described perforatorium zymolyte be mainly characterized by can combination single-minded with acrosin.Any one sperm
Acrosomal enzyme substrate can serve as by the substrate of nano gold mark.
Among aforesaid embodiment of the present invention, mainly by the change of surface plasma resonance and the acrosin of nanometer gold
Concentration be directly proportional and carry out detecting.But, those skilled in the art should know, utilizes other ginsengs that plasma resonance changes
Number as nanometer gold absworption peaks intensity may also be used for detection perforatorium enzyme concentration.
Additionally, the acrosin being suitable for utilizing the test kit of the present invention and detection method to carry out detecting should be not limited to the essence of the mankind
Son, and can also is that the acrosin of the animals such as general mammal such as pig, Canis familiaris L., horse.
Also, in an exemplary embodiments of the present invention, technical scheme can be accomplished in that
(1). prepare nanometer gold bar: the preparation of nanometer gold bar is broadly divided into two steps: seed solution preparation and gold plant growth.
I) prepared by seed solution: by 5mL 0.5mM HAucl4Solution and the mixing of 5mL 0.2M CTAB solution, be stirred vigorously
Under the conditions of (1200r/min) by 0.01M sodium borohydride (NaBH newly configured for 0.6mL4) solution is diluted to 1mL and is then added to
In above-mentioned mixed solution, stopping after stirring 2min, gained solution is as the seed of 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, adds 70 μ L 0.0788M ascorbic acid after being uniformly mixed；Finally take 12 μ L seeds
Solution joins in growth-promoting media, stands 6h at 30 DEG C.The gold nanorods of preparation is centrifugal 10min under the conditions of 8000r/min,
To remove the CTAB on gold nanorods surface.
(2) technology path (Fig. 1) at the hyper-branched polymer that nanometer gold bar surface modified end is hydroxyl is as follows:
A) first nanometer gold bar [1-(2-bromo-2-methyl-propionyloxy)] ethane (DTBE) double with 2,2'-dithio is by sulfur gold
Key (S-Au) reaction absorption is around gold nanorods, and it is poly-that macromole evocating agent causes styrene (PS) monomer polymerization reactions to generate
Styrene.10mg DTBE is dissolved in 100mL DMF, adds 1mL above-mentioned removal CTAB under intense agitation
Gold nanorods solution, add 30mg styrene at 40 DEG C, stir 12h.
B) 2-((bromine butyryl) epoxide) ethyl propylene acid esters (BBEA) is caused to carry out Self condensation vinyl polymerization further anti-
Answer (SCVP), high molecular polymer hyperbranched on gold surface is modified.Add 15mg acrylic monomers (AA) more single
Body is polymerized, substantial amounts of carboxyl on gold nanorods periphery is modified.
(3) perforatorium zymolyte is modified: 96mg EDC and 29mg NHS is dissolved in 10mL deionized water, and 3mL is peripheral
Nanometer gold bar containing a large amount of carboxyls centrifugal 10min under the conditions of 5000r/min adds after being concentrated to 500 μ L under rapid mixing conditions
Enter in above-mentioned solution, utilize PBS to adjust pH to 7.1, be subsequently adding 400 μ L 2mg/mL perforatorium zymolyte ZP3 eggs
In vain, at 25 DEG C, 1h is cultivated.Reactant liquor is centrifugal 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 acrosomal enzyme.Initially set up a working curve, take appropriate
The gold nanorods solution of perforatorium enzyme modification, in quartz colorimetric utensil, controls the concentration of above-mentioned solution with guarantee fund's nanometer rods longitudinally
The peak value of absworption peak is advisable between 0.6-0.8, measures the gold nanorods of perforatorium enzyme modification in 300-1000nm wave-length coverage
Absorption spectrum, the wavelength that its longitudinal absworption peak is corresponding is designated as λ0, add containing variable concentrations C in above-mentioned solutionnSperm top
The solution of body enzyme, measures the ultraviolet-visible absorption spectroscopy λ of the acrosin adding variable concentrations respectivelyn, wavelength shift value is designated as
Δλn=λn-λ0, set up CnWith Δ λnBetween relation.Acrosin for unknown concentration c, it is possible to use the Δ λ of mensuration counts
Calculate.
Below in conjunction with some preferred embodiments the technical solution of the present invention is further explained explanation, but experiment condition therein
It is not construed as the limitation to basic technical scheme of the present invention with setup parameter.And protection scope of the present invention is not limited to following reality
Execute example.
Embodiment 1: use the gold nanorods detection acrosin that perforatorium zymolyte is modified
1. prepare nanometer gold bar: the preparation of nanometer gold bar is broadly divided into two steps: seed solution preparation and gold plant growth.1) seed
Prepared by solution: by 5mL 0.5mM HAucl4Solution and the mixing of 5mL 0.2M CTAB solution, under intense agitation
(1200r/min) by 0.01M sodium borohydride (NaBH newly configured for 0.6mL4) solution is diluted to 1mL and is then added to above-mentioned mixed
Closing in solution, stop after stirring 2min, gained solution is as the seed of growth gold nanorods；2) seed growth: 25 DEG C of conditions
Lower by 5mL 0.2M CTAB solution and 0.2mL 4mM AgNO3Mixing, adds 5.0mL 1mM HAuCl4Solution, stirs
70 μ L 0.0788M ascorbic acid are added after mixing mix homogeneously；Finally take 12 μ L seed solutions and join in growth-promoting media, at 30 DEG C
Lower standing 6h.The gold nanorods of preparation is centrifugal 10min under the conditions of 8000r/min, to remove the CTAB on gold nanorods surface.
The TEM of the nanometer gold bar of preparation is as shown in Figure 3.
2. the technology path (refering to Fig. 1) at the hyper-branched polymer that nanometer gold bar surface modified end is hydroxyl is as follows:
A) first nanometer gold bar [1-(2-bromo-2-methyl-propionyloxy)] ethane (DTBE) double with 2,2'-dithio is by sulfur gold key
(S-Au) reaction absorption is around gold nanorods, and macromole evocating agent causes styrene (PS) monomer polymerization reactions to generate polyphenyl
Ethylene.10mg DTBE is dissolved in 100mL DMF, adds 1mL above-mentioned removal CTAB under intense agitation
Gold nanorods solution, add 30mg styrene at 40 DEG C, stir 12h.B) 2 ((bromine butyryl) epoxides) are caused further
Ethyl propylene acid esters (BBEA) carries out Self condensation vinyl polymerization reaction (SCVP), height hyperbranched on gold surface is modified
Molecularly Imprinted Polymer.Add 15mg acrylic monomers (AA) monomer to be polymerized, substantial amounts of carboxylic on gold nanorods periphery is modified
Base.
3. perforatorium zymolyte is modified: 96mg EDC and 29mg NHS is dissolved in 10mL deionized water, and 3mL contains periphery
The nanometer gold bar of a large amount of carboxyls centrifugal 10min under the conditions of 5000r/min joins after being concentrated to 500 μ L under rapid mixing conditions
In above-mentioned solution, utilize PBS to adjust pH to 7.1, be subsequently adding 400 μ L 2mg/mL perforatorium zymolyte ZP3 albumen,
1h is cultivated at 25 DEG C.Reactant liquor is centrifugal under the conditions of 10000r/min, removes the antibody not being coupled to gold nanorods surface.
4. nanometer gold bar-perforatorium zymolyte hybrid systems detection acrosomal enzyme.Initially set up a working curve, take appropriate sperm
The gold nanorods solution that acrosomal enzyme is modified is in quartz colorimetric utensil, and the concentration controlling above-mentioned solution longitudinally absorbs with guarantee fund's nanometer rods
The peak value at peak is advisable between 0.6-0.8, measures the suction of the gold nanorods of perforatorium enzyme modification in 300-1000nm wave-length coverage
Receiving spectrum, the wavelength that its longitudinal absworption peak is corresponding is designated as λ0, add containing variable concentrations C in above-mentioned solutionnAcrosin
Solution, measure the ultraviolet-visible absorption spectroscopy λ of acrosin adding variable concentrations respectivelyn, wavelength shift value is designated as
Δλn=λn-λ0, set up CnWith Δ λnBetween relation.Acrosin for unknown concentration c, it is possible to use the Δ λ of mensuration counts
Calculate
5., with reference to the canonical plotting done, measuring the concentration of acrosin in sample is 5 μMs, and detection range is
0.01 μM～50 μMs.
Embodiment 2: use the nano gold spherical detection acrosin that perforatorium zymolyte is modified
1. prepare nano gold spherical: take 100mL 2.5 × 10-4mol/L HAuCl4Solution is heated to 100-150 DEG C, adds 10mL 1%
Citric acid three sodium solution, when the color of solution becomes transparent claret completely, stops heating, cooling after continuing backflow 10min
To room temperature, 4 DEG C of preservations, the nano gold spherical ultraviolet spectra absorbing wavelength obtained is 526nm.The transmission electron microscope picture of nano gold spherical is shown in figure
4。
2. the labelling of nanogold particle: 10mg DTBE is dissolved in 100mL DMF, adds 20mL under intense agitation
Above-mentioned nanometer nearly ball solution, stirs 12h adding 30mg styrene at 40 DEG C.It is subsequently adding 40mg BBEA monomer, causes
Polymerization, forms hyper-branched polymer, after reaction a period of time, adds 15mg acrylic monomers (AA), from gold nanorods
Enclose the upper substantial amounts of carboxyl of modification.
3. the gold nano that perforatorium zymolyte is modified: 96mg EDC and 29mg NHS is dissolved in 10mL deionized water, 10mL
The peripheral nano gold spherical containing a large amount of carboxyls is centrifugal 10min under the conditions of 5000r/min, is quickly stirring bar after being concentrated to 500 μ L
Join in above-mentioned solution under part, utilize PBS to adjust pH to 7.1, be subsequently adding 400 μ L 2mg/mL perforatorium zymolytes
ZP3 albumen, cultivates 1h at 25 DEG C.Reactant liquor is centrifugal 10min under the conditions of 10000r/min, removes and is not coupled to nanometer gold
The antibody on ball surface.
4. nano gold spherical-perforatorium zymolyte hybrid systems detection acrosomal enzyme.Initially set up a working curve, take 10mL essence
The nano gold spherical solution that sub-acrosomal enzyme is modified, with quartz colorimetric utensil, controls the concentration of above-mentioned solution to ensure nanometer gold absworption peak
Peak value is advisable between 0.6-0.8, measures the absorbing light of the nano gold spherical of perforatorium enzyme modification in 300-1000nm wave-length coverage
Spectrum, the wavelength that its absworption peak is corresponding is designated as λ0, add containing variable concentrations C in above-mentioned solutionnThe solution of acrosin,
Measure the ultraviolet-visible absorption spectroscopy λ of the acrosin adding variable concentrations respectivelyn, wavelength shift value is designated as Δ λn=λn-λ0, set up
CnWith Δ λnBetween relation.Acrosin c for unknown concentration, it is possible to use the Δ λ of mensuration calculates.
5., with reference to the canonical plotting done, measuring the concentration of acrosin in sample is 7 μMs, and detection range is
0.01 μM～30 μMs.
Finally, in addition it is also necessary to explanation, term " includes ", " comprising " or its any other variant are intended to nonexcludability
Comprise, so that include that the process of a series of key element, method, article or equipment not only include those key elements, but also wrap
Include other key elements being not expressly set out, or also include the key element intrinsic for this process, method, article or equipment.
It will be appreciated by those skilled in the art that the detailed description of the invention of present invention described above, be not intended that and the present invention is protected model
The restriction enclosed.Any according to other changes accordingly various done by the technology design of the present invention and deformation, should be included in originally
In invention scope of the claims.
Claims (10)
1. a test kit based on LSPR detection sperm acrosin activities in assessing, it is characterised in that comprise:
Functional gold nanoparticles labelling perforatorium zymolyte, is comprised nanometer gold and is consolidated by chemical bonding and/or physical adsorption way
It is scheduled on the perforatorium zymolyte on nanometer gold surface；
And, be suitable to make described perforatorium zymolyte and the single-minded combination of acrosin and carry out the buffer reacted.
Test kit the most according to claim 1, it is characterised in that also comprise the saying of using method recording described test kit
Bright book, described using method includes:
Measure mainly by described buffer and the functional gold nanoparticles labelling perforatorium zymolyte being dispersed in described buffer
The mixed solution of composition absorption spectrum in 300-1000nm wave-length coverage, and record absorbing wavelength λ that absworption peak is corresponding0And
Absorption peak strength A0；
Variable concentrations C is added in described mixed solutionnPerforatorium enzymatic solution, and measure respectively add variable concentrations essence
The different mixing reaction system absorption spectrums in 300-1000nm wave-length coverage obtained after sub-acrosomal enzyme standard solution, 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 sets up acrosin solution concentration and wavelength shift value or absworption peak deviant
Standard working curve, wherein n is the natural number more than or equal to 2；
And, described mixed solution adds unknown concentration CxPerforatorium enzymatic solution, and it is anti-to measure the mixing that is consequently formed
Answer system absorbing wavelength λ in 300-1000nm wave-length coveragex, wavelength shift value Δ λx=λx-λ0Or absworption peak deviant
ΔAx=Ax-A0, and calculate C according to described standard working curvex。
Test kit the most according to claim 1, it is characterised in that described using method also includes: control described mixed solution
The peak value of the absworption peak in 300-1000nm wave-length coverage is between 0.6-0.8.
Test kit the most according to claim 1, it is characterised in that described perforatorium zymolyte by with modify at nanometer gold
The selected functional group on surface occurs coupling reaction to be fixedly attached to described nanometer gold surface.
Test kit the most according to claim 1, it is characterised in that described nanometer gold comprises nanometer gold bar, nano gold spherical or receives
Meter Jin Li square.
6. the detection device of a sperm acrosin activities in assessing, it is characterised in that comprise the reagent according to any one of claim 1-5
Box.
7. a detection method based on LSPR detection sperm acrosin activities in assessing, it is characterised in that comprise:
Functional gold nanoparticles labelling perforatorium zymolyte is provided and is suitable to make described perforatorium zymolyte special with acrosin
One combines and carries out the buffer reacted, and described functional gold nanoparticles labelling perforatorium zymolyte comprises nanometer gold and by changing
Bonding and/or physical adsorption way are fixed on the perforatorium zymolyte on nanometer gold surface；
Measure mainly by described buffer and the functional gold nanoparticles labelling perforatorium zymolyte being dispersed in described buffer
The mixed solution of composition absorption spectrum in 300-1000nm wave-length coverage, and record absorbing wavelength λ that absworption peak is corresponding0；
Variable concentrations C is added in described mixed solutionnPerforatorium enzymatic solution, and measure respectively add variable concentrations essence
The different mixing reaction system absorption spectrums in 300-1000nm wave-length coverage obtained after sub-acrosomal enzyme standard solution, 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 sets up acrosin solution concentration and wavelength shift value or absworption peak deviant
Standard working curve, wherein n is the natural number more than or equal to 2；
And, described mixed solution adds unknown concentration CxPerforatorium enzymatic solution, and it is anti-to measure the mixing that is consequently formed
Answer system absorbing wavelength λ in 300-1000nm wave-length coveragex, wavelength shift value Δ λx=λx-λ0Or absworption peak deviant
ΔAx=Ax-A0, and calculate C according to described standard working curvex。
Detection method based on LSPR detection sperm acrosin activities in assessing the most according to claim 7, it is characterised in that also wrap
Include: control the peak value of described mixed solution absworption peak in 300-1000nm wave-length coverage between 0.6-0.8.
Detection method based on LSPR detection sperm acrosin activities in assessing the most according to claim 7, it is characterised in that described merit
The preparation method that can change nano gold mark perforatorium zymolyte includes:
The nanometer gold with obvious ultraviolet-visible absorption spectroscopy absworption peak is provided,
Selected functional group is modified on described nanometer gold surface by chemistry or physical method,
Described perforatorium zymolyte and described selected functional group is made to carry out coupled reaction and be fixedly connected on described nanometer gold surface.
10. according to the detection method based on LSPR detection sperm acrosin activities in assessing described in claim 7 or 9, it is characterised in that
Described nanometer gold comprises nanometer gold bar, nano gold spherical or nanometer gold cubic block.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510116466.2A CN106033086B (en) | 2015-03-17 | 2015-03-17 | Method, kit and its application based on LSPR detection sperm acrosin activities in assessing |
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| CN110231473A (en) * | 2019-06-17 | 2019-09-13 | 华东理工大学 | A kind of biological marker object detecting method and its application based on gold nano microballoon plasma resonance |
| CN113358868A (en) * | 2021-05-24 | 2021-09-07 | 迪瑞医疗科技股份有限公司 | Sperm acrosome enzyme chemiluminescence immunoassay kit and preparation method thereof |
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