CN104390947A - Fluorescence method for detecting hyaluronidase - Google Patents
Fluorescence method for detecting hyaluronidase Download PDFInfo
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- CN104390947A CN104390947A CN201410669140.8A CN201410669140A CN104390947A CN 104390947 A CN104390947 A CN 104390947A CN 201410669140 A CN201410669140 A CN 201410669140A CN 104390947 A CN104390947 A CN 104390947A
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- hyaluronidase
- conjugated polymer
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Abstract
The invention discloses a fluorescence method for detecting hyaluronidase. The method is characterized in that a cationic conjugated polymer is firstly combined with hyaluronic acid which is connected with fluorescence quencher adriamycin and has negative charge by virtue of electrostatic attraction; the fluorescence of the conjugated polymer is quenched due to the electron transfer between the conjugated polymer and the adriamycin, and the fluorescence intensity of the conjugated polymer is weakened due to the increasing of concentration of the adriamycin; when the hyaluronidase is added, hyaluronic acid chains are hydrolyzed, so that the combination between the cationic conjugated polymer and hyaluronic acid is weakened, the distance is increased, adriamycin is released, the fluorescence quenching effect is weakened, the fluorescence of the conjugated polymer is gradually restored, and the fluorescence recovery degree of the conjugated polymer is correlated to the concentration of the hyaluronidase. The method is simple to operate, high in response speed, low in cost and high in selectivity and sensitivity and has an important significance on biological detection and early diagnosis and treatment of diseases.
Description
Technical field
The invention belongs to bio-sensing and analysis field, particularly a kind of fluorescent method utilizing cationic conjugated polymer and hyaluronic acid probe in detecting hyaluronidase.
Background technology
The common trait of conjugated polymer has large π-electron-conjugated system in molecule, provide the condition that electric charge moves on a large scale, because the π of its main chain-pi-conjugated structure with the atom of p track can be provided (as N, S etc.) the pi-conjugated structure of p-that is connected defines delocalized pi-bond along macromolecular chain, and electric charge moves along macromolecular chain by this delocalized pi-bond.Therefore, conjugated polymer is one energy/electron transfer mediator very efficiently, and feature that it has " molecular wire ", the exciton produced that is namely stimulated can move rapidly along conjugated main chain.The hydrophilic radicals such as ammonium root, carboxylate radical, sulfonate group or oligomeric ethylene glycol be covalently attached to conjugated polymer side chain can make it have water-soluble.By introducing cation group, whole piece polymer chain can be made positively charged in aqueous, can interact with electronegative biomolecule.
Hyaluronic acid is a kind of electronegative linear glutinous polysaccharide, is repeated to connect to form by the dissacharide units of (1-β-4) D-glucuronic acid (1-β-3) N-acetyl group-D-aminoglucose.First Columbia Univ USA ophthalmology professor Meyer in 1934 etc. isolate this material from bovine vitreous body, have also discovered its existence subsequently in human body.In body, hyaluronic acid demonstrates multiple important physiological function: as maintained institutional framework integrality, formed around each cell high degree of hydration matrix, to promote between cell migration and transfer and mediates cell (the Rafal Fudala such as signal, Mark E.Mummert, Zygmunt Gryczynski, et al.Journal ofPhotochemistry and Photobiology B:Biology, 2012, (106): 69 – 73).It with protein, nucleic acid is the same, be all the base substance of life process, be extensively present in the soft connective tissue of biosome.The a large amount of carboxyls, hydroxyl, the amide group that contain in hyaluronan molecule, under physiological condition or suitable pH environment, carboxyl fully can be dissociated into negative ion.Therefore, hyaluronic acid has outstanding water retention property, and also have viscoelastic double characteristic, namely its aqueous solution had both had the elasticity of gel, also had the viscosity of solution.In recent years, due to physicochemical property and the physiological function of hyaluronic acid uniqueness, it is used widely in all many-sides, such as: protection eyes, protection and lubricating joint, promotion Angiogenesis, promote the treatment etc. of wound healing and tumour.
Hyaluronidase (Hyaluronidase, HAase) be the class glycosidase being distributed widely in occurring in nature, it can disconnect the glycosidic bond between NAG in hyaluronic acid sugar chain and D-glucose carboxylic acid, by its depolymerization be hydrolyzed into low molecular weight hyaluronic acid or oligosaccharides.Hyaluronidase Late Cambrian is in nineteen twenty-nine, Duran-Reynals finds a kind of " invasin " that promote the diffusions such as vaccine, dyestuff, toxin in mammal testis and hetero-organization extract thereof, be accredited as hyaluronidase (F.Duran-Reynals.Tissue permeability and the spreading factor in infection:Acontribution to the host:parasite problem [J] .Bacteriol Rev subsequently, 1942,6 (4): 197-252).Hyaluronidase has overexpression in many different cancer cell, such as carcinoma of urinary bladder, prostate cancer, malignant mela noma etc.Several carcinogenic cells behavior, comprises the humidification etc. of tissue invasion and revascularization, also increases relevant with the activity of hyaluronidase.In addition, hyaluronidase also can be used for anti-cancer chemotherapeutic agents, hyaluronidase add the resistance to the action of a drug that can reduce tumour cell.Therefore, carry out detecting efficiently to hyaluronidase, in the early diagnosis and therapy etc. of tumour, there is important Theory and applications researching value.
The detection method of the hyaluronidase reported at present has (the Dan Cheng such as nephelometry, viscosimetry, zymography, immunoassay and some other chemical method, Weiye Han, Kuncheng Yang, et al.Talanta, 2014 (130): 408 – 414).Wherein apply more method nephelometry, nephelometry based on the hyaluronate of macromolecule, sedimentation phenomenon occurs in acidified serum to set up, but this method needs a large amount of enzymes, and measurement result is mutually inaccurate.Zymography is comparatively simple but be not suitable for responsive quantitative test.Immunoassays rule needs specialty and expensive reagent.Therefore find one can specific detection hyaluronidase, easy and simple to handle, quick, detection method that cost is low, becomes problem demanding prompt solution again.
Summary of the invention
Technical matters: the technical problem to be solved in the present invention is the deficiency for existing detection hyaluronic acid enzyme method, there is provided a kind of utilize hyaluronic acid probe to be combined with cation type water-soluble conjugated polymer utilize fluorescence signal to detect the method for hyaluronidase, sensitivity and specificity good, and easy and simple to handle, quick, cost is low.
Technical scheme: the present invention is a kind of fluorescent method based on cation type water-soluble conjugated polymer and hyaluronic acid probe in detecting hyaluronidase, uses the method can realize detecting the quantitative and qualitative analysis of hyaluronidase.The core of this technology utilizes before and after hyaluronidase and combined with fluorescent quencher hyaluronic acid probe have an effect, and the fluorescent quenching of cation type water-soluble conjugated polymer and the degree of recovery carry out the detection of hyaluronidase.When hyaluronidase concentration is zero, conjugated polymer is combined by electrostatic interaction with hyaluronic acid probe, and the fluorescence of conjugated polymer is because the electro transfer between the adriamycin on hyaluronic acid probe is by quencher; When hyaluronidase concentration increases, hyaluronidase and hyaluronic acid are had an effect, and hyaluronic acid chain is hydrolyzed into fragment, largely reducing the electrostatical binding effect of conjugated polymer and hyaluronic acid probe, both distances are increased, and therefore the fluorescence of conjugated polymer is recovered; The degree that conjugated polymer fluorescence recovers is relevant to the concentration of hyaluronidase, therefore quantitatively can detect hyaluronidase according to this method.
The fluorescent method of detection hyaluronidase of the present invention, based on cation type water-soluble conjugated polymer and hyaluronic acid HA probe, comprises the following steps:
1) the hyaluronic acid probe of combined with fluorescent quencher is prepared;
2) cation type water-soluble conjugated polymer is combined with described hyaluronic acid probe, carries out fluoroscopic examination;
3) in hyaluronic acid probe, add hyaluronidase, after itself and hyaluronidase effect, add cation water-soluble conjugated polymer wherein, again carry out fluoroscopic examination.
Fluorescence quencher comprises the small molecule fluorescent quencher of adriamycin DOX.
Described cation type water-soluble conjugated polymer, its fluorescent emission due to the electro transfer between doxorubicin fluorescence quencher can by quencher.
Described cation type water-soluble conjugated polymer and hyaluronic acid probe are combined by electrostatic attraction effect.
The effect of described hyaluronic acid probe and hyaluronidase is that hyaluronic acid chain is hydrolyzed to the process of hyaluronic acid short chain by hyaluronidase.
In described fluoroscopic examination, the buffer solution used is 0.15M NaCl, 0.02M PBS damping fluid, pH=5.6.
In described fluoroscopic examination, temperature is 25 DEG C.
Described fluoroscopic examination adds after hyaluronidase solution hatches 30 minutes at 37 DEG C to measure fluorescence emission spectrum.
Described fluoroscopic examination detects with fluorospectrophotometer, and excitation wavelength is at cation type water-soluble conjugated polymer maximal ultraviolet-visible absorbance peak place, and emission wavelength sweep limit is 415-600nm.
Beneficial effect: fluorescence detection method of the present invention is based on soluble conjugated polymer signal multiplication and the real-time advantage detected, and adriamycin the fluorescence of quencher soluble conjugated polymer and hyaluronidase can be hydrolyzed hyaluronic feature, utilize before and after hyaluronidase and hyaluronic acid probe have an effect, the degree that the fluorescence of cation type water-soluble conjugated polymer recovers carries out the detection of hyaluronidase.The method is easy and simple to handle, fast response time, and cost is lower, and has higher selectivity and sensitivity, is the detection method that a kind of analysis efficiency is higher.
Accompanying drawing explanation
Fig. 1 is the molecular structural formula of cation type water-soluble conjugated polymer (PFEP), hyaluronic acid (HA), adriamycin (DOX).
Fig. 2 is the fundamental diagram utilizing cation type water-soluble conjugated polymer and hyaluronic acid probe in detecting hyaluronidase.
Fig. 3 is the fluorescence spectrum figure detected based on the hyaluronidase of fluorescence detection method to variable concentrations of cation type water-soluble conjugated polymer and hyaluronic acid probe.Hyaluronidase concentration scope is 0 ~ 1.85U/mL.
Fig. 4 is that variable concentrations hyaluronidase adds before and after system to be measured, the corresponding relation curve map of system fluorescence intensity change.Explain: little figure represents the linear relationship chart of the hyaluronidase in system fluorescence intensity and 0 ~ 1.30U/mL concentration range.
Fig. 5 is specificity analyses figure.Be respectively to detect in end liquid and add hyaluronidase, fibrin ferment (Thrombin), the comparing result of lysozyme (Lysozyme) and PBS buffer solution.
Embodiment
In order to understand content of the present invention better, below for the experiment of cation type water-soluble conjugated polymer and fluorescein-labeled hyaluronic acid probe in detecting hyaluronidase, the embodiment of the technology of the present invention is described.
This technology is used to comprise following three steps:
1) preparation is in conjunction with the hyaluronic acid probe of adriamycin;
2) cation type water-soluble conjugated polymer is combined with described hyaluronic acid probe, carries out fluoroscopic examination;
3) in hyaluronic acid probe, add hyaluronidase, after itself and hyaluronidase effect, in system, add cation water-soluble conjugated polymer, again carry out fluoroscopic examination.
Wherein:
Step 1 reaction mechanism is: the carboxyl on hyaluronic acid and the amino on adriamycin under the catalytic action of 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) and N-hydroxy-succinamide (NHS), condensation reaction occur and links together.The molecular structural formula of hyaluronic acid and adriamycin as shown in Figure 1.
Cation type water-soluble conjugated polymer in step 2 can refer to document (Huang Y Q, Fan Q L, Lu X M, Fang C, Liu S J, Li H, Wen Y, Wang L H, Huang W.Journal ofPolymer Science:PartA:Polymer Chemistry2006,44 (19), 5778-5794) prepare, its concentration represents with the volumetric molar concentration of repetitive.The concentration of hyaluronic acid probe also represents with the volumetric molar concentration of its repetitive.This conjugated polymer is combined with hyaluronic acid probe by electrostatic attraction effect, makes the fluorescence of this conjugated polymer by adriamycin quencher, thus fluorescence signal is significantly weakened.The fluorescence recovery extent of the conjugated polymer ratio of the fluorescence intensity added before and after hyaluronidase, i.e. I
c/ I
0represent.When hyaluronidase concentration is zero, the fluorescence intensity I of conjugated polymer
0represent.
Fluoroscopic examination instrument used is Shimadzu RF-5301PC fluorospectrophotometer.Fluorescence spectral measuring condition: xenon lamp excites, excitation wavelength is 404nm, and emission scan scope is 415-600nm.Detect 0.15M NaCl, 0.02M PBS damping fluid that damping fluid is pH=5.6, measure with 4mL quartz colorimetric utensil, sample volume 2.7mL; Cation type water-soluble conjugated polymers substrate concentration is 1.5 × 10
-7mol/L, the concentration of hyaluronic acid probe is 0 ~ 3.1 × 10
-6mol/L, detected temperatures 25 DEG C.
Step 3 adds hyaluronidase in hyaluronic acid probe, after hatching 30 minutes, carries out fluoroscopic examination after adding cation water-soluble conjugated polymer in system at 37 DEG C.Because hyaluronic acid chain is hydrolyzed by hyaluronidase, weaken the electrostatical binding effect of conjugated polymer and hyaluronic acid probe, both distances are increased, and the fluorescence quenching of adriamycin to conjugated polymer weakens, and the fluorescence of conjugated polymer is recovered.Use I
crepresent the fluorescence intensity adding conjugated polymer after hyaluronidase, calculate the relative value Δ I of fluorescence intensity change:
ΔI＝I
C/I
0
Δ I is relevant to the concentration of hyaluronidase, and the relation curve according to both quantitatively can detect hyaluronidase.
Fluoroscopic examination instrument used, fluorescence spectral measuring condition, detect damping fluid with step 2, measure with 700uL quartz colorimetric utensil, sample volume 540uL; Cation type water-soluble conjugated polymers substrate concentration is 1.5 × 10
-7mol/L, the concentration of hyaluronic acid probe is 1.48 × 10
-6mol/L, the concentration of hyaluronidase is 0 ~ 1.85U/mL, detected temperatures 25 DEG C.
Embodiment 1 detects the hyaluronidase of variable concentrations
It is 25 DEG C in temperature, in the PBS damping fluid of pH=5.6, to containing 8 × 10
-5add the hyaluronidase of a series of difference amount in the end liquid of mol/L hyaluronic acid probe, at 37 DEG C, hatch 30 minutes, being diluted to hyaluronic acid concentration and probe concentration is afterwards 1.48 × 10
-6mol/L, adds 1.5 × 10
-7mol/L cation type water-soluble conjugated polymer, scans its fluorescence emission spectrum, the final concentration of experimental result hyaluronidase is as shown in Figure 5 respectively 0,0.19,0.38,0.56,0.74,0.93,1.11,1.30,1.49,1.85U/mL.As seen from the figure along with the concentration of hyaluronidase increases, the fluorescence recovery extent of cation type water-soluble conjugated polymer is higher.Calculate Δ I, the concentration of hyaluronidase is mapped to obtain Fig. 4.It is have the good range of linearity within the scope of 0 ~ 1.30U/mL that this figure shows at hyaluronidase concentration, is greater than this scope, and the curve trend of Δ I is mild, represents that the concentration adding hyaluronidase is tending towards saturated.
The detectability formula of the method: LOD=3S
0/ S, wherein S
0represent the standard deviation of blank sample, S represents sensitivity (Eggins B R, the Chemical Sensors and Biosensors in the method range of linearity, John Wiley & Sons Ltd, West Sussex, England, 2002).This standard deviation of testing blank sample is 0.04, and the Calculation of Sensitivity of joint line linearity curve obtains detection and is limited to 0.075U/mL.
Embodiment 2 specificity analyses
Detect 1: be that the hyaluronidase of 0.38U/mL adds and detects in end liquid by concentration, other experiment condition is with embodiment 1.
Meanwhile, following test is done as a comparison.
Detect 2: be that the lysozyme (Lysozyme) of hyaluronidase 5 times and fibrin ferment (Thrombin) are tested by equal-volume concentration, as negative control, other experiment condition is with embodiment 1.
Detect 3: replace hyaluronidase to test with isopyknic blank buffer solution, as blank, other experiment condition is with embodiment 1.
Result as shown in Figure 5.The fluorescence recovery extent adding the conjugated polymer of the detection system of hyaluronidase is significantly higher than other 3 kinds of control systems, shows that the detection of the method to hyaluronidase has higher specificity.
Claims (9)
1. detect a fluorescent method for hyaluronidase, it is characterized in that the method is based on cation type water-soluble conjugated polymer and hyaluronic acid HA probe, comprises the following steps:
1) the hyaluronic acid probe of combined with fluorescent quencher is prepared;
2) cation type water-soluble conjugated polymer is combined with described hyaluronic acid probe, carries out fluoroscopic examination;
3) in hyaluronic acid probe, add hyaluronidase, after itself and hyaluronidase effect, add cation water-soluble conjugated polymer wherein, again carry out fluoroscopic examination.
2. a kind of fluorescent method detecting hyaluronidase according to claim 1, is characterized in that fluorescence quencher comprises the small molecule fluorescent quencher of adriamycin DOX.
3. a kind of fluorescent method detecting hyaluronidase according to claim 1, is characterized in that described cation type water-soluble conjugated polymer, its fluorescent emission due to the electro transfer between doxorubicin fluorescence quencher can by quencher.
4. a kind of fluorescent method detecting hyaluronidase according to claim 1, is characterized in that described cation type water-soluble conjugated polymer and hyaluronic acid probe are combined by electrostatic attraction effect.
5. a kind of fluorescent method detecting hyaluronidase according to claim 1, is characterized in that the effect of described hyaluronic acid probe and hyaluronidase, is that hyaluronic acid chain is hydrolyzed to the process of hyaluronic acid short chain by hyaluronidase.
6. a kind of fluorescent method detecting hyaluronidase according to claim 1, is characterized in that in described fluoroscopic examination, and the buffer solution used is 0.15M NaCl, 0.02M PBS damping fluid, pH=5.6.
7. a kind of fluorescent method detecting hyaluronidase according to claim 1, is characterized in that in described fluoroscopic examination, and temperature is 25 DEG C.
8. a kind of fluorescent method detecting hyaluronidase according to claim 1, is characterized in that described fluoroscopic examination, is to add after hyaluronidase solution hatches 30 minutes at 37 DEG C to measure fluorescence emission spectrum.
9. a kind of fluorescent method detecting hyaluronidase according to claim 1, it is characterized in that described fluoroscopic examination detects with fluorospectrophotometer, excitation wavelength is at cation type water-soluble conjugated polymer maximal ultraviolet-visible absorbance peak place, and emission wavelength sweep limit is 415-600nm.
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| CN105136786A (en) * | 2015-08-04 | 2015-12-09 | 南京邮电大学 | Method utilizing gold nano particles and thioflavine T to detect G-enriched nucleic acid sequence |
| CN105675573A (en) * | 2016-03-16 | 2016-06-15 | 安徽师范大学 | Hyaluronidase detection method |
| CN107973901A (en) * | 2017-12-07 | 2018-05-01 | 南京邮电大学 | The preparation and application method of double Photobiology window targeted nano bioprobes |
| CN111777616A (en) * | 2020-08-11 | 2020-10-16 | 湖南科技大学 | Porphyrin derivative capable of detecting hyaluronidase based on self-assembly, preparation method and application thereof |
| CN112179875A (en) * | 2019-07-04 | 2021-01-05 | 长沙理工大学 | Preparation and application of type I hyaluronidase fluorescent nano sensor |
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2014
- 2014-11-20 CN CN201410669140.8A patent/CN104390947A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105136786A (en) * | 2015-08-04 | 2015-12-09 | 南京邮电大学 | Method utilizing gold nano particles and thioflavine T to detect G-enriched nucleic acid sequence |
| CN105675573A (en) * | 2016-03-16 | 2016-06-15 | 安徽师范大学 | Hyaluronidase detection method |
| CN105675573B (en) * | 2016-03-16 | 2019-05-14 | 安徽师范大学 | The detection method of hyaluronidase |
| CN107973901A (en) * | 2017-12-07 | 2018-05-01 | 南京邮电大学 | The preparation and application method of double Photobiology window targeted nano bioprobes |
| CN112179875A (en) * | 2019-07-04 | 2021-01-05 | 长沙理工大学 | Preparation and application of type I hyaluronidase fluorescent nano sensor |
| CN112179875B (en) * | 2019-07-04 | 2022-06-21 | 长沙理工大学 | Preparation and application of type I hyaluronidase fluorescent nano sensor |
| CN111777616A (en) * | 2020-08-11 | 2020-10-16 | 湖南科技大学 | Porphyrin derivative capable of detecting hyaluronidase based on self-assembly, preparation method and application thereof |
| CN111777616B (en) * | 2020-08-11 | 2023-04-07 | 湖南科技大学 | Porphyrin derivative capable of detecting hyaluronidase based on self-assembly, preparation method and application thereof |
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