CN110672567B - Low molecular weight heparin gold nano material and application thereof in heparanase detection - Google Patents
Low molecular weight heparin gold nano material and application thereof in heparanase detection Download PDFInfo
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Abstract
The invention discloses a low molecular weight heparin gold nano material and application thereof in heparanase detection, wherein gold nanoparticles and gold nanorods are adopted to carry out covalent double labeling on low molecular weight heparin with the diameter less than 10nm, and a fluorescence energy resonance transfer (FRET) mode is utilized to detect the change of FRET value of a substrate after heparanase degradation so as to reflect the activity value of the heparanase, so that the influence of secondary cracking on the activity determination of the heparanase is effectively avoided.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to gold nano-material double-labeled low-molecular-weight heparin and application thereof in heparanase detection.
Background
Heparan sulfate is a long-chain glycosaminoglycan composed of uronic acid and glucosamine alternately, widely exists in tumor Extracellular Matrix (ECM) and cell surface, is a main component of basement membrane, and can provide attachment points for growth factors, vascular endothelial growth factors, protein kinases and the like in vivo. Heparanase is the only endogenous hydrolase found in human body which can hydrolyze heparan sulfate so far, and the activity of the heparanase is closely related to tumor. Multiple studies show that the expression level of heparanase is obviously higher than the normal level in the later stage of tumor development, and the over-expressed heparanase can promote the growth of tumor cells, the generation of blood vessels, infiltration and migration, so that the survival rate of tumor patients is greatly reduced. Therefore, the activity detection of heparanase is an important link for judging the occurrence and development of tumors.
The activity detection is a key detection index in the research process of heparanase, and the time of enzyme catalytic reaction, the reaction dosage of enzyme and substrate and the like can be calculated by measuring the activity of the enzyme. Two methods are commonly used for the determination of heparanase activity: one is to detect the change of the content of the substrate or product before and after the catalytic reaction by means of HPLC and the like; the other is to detect structural changes of the substrate and product before and after catalysis by absorbance. Because of the complex structure of heparin substrate and the lack of characteristic absorption groups, neither of the above two methods can be used to establish a direct method for measuring heparanase activity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art for heparanase activity detection, and provides gold nano-material double-labeled low-molecular-weight heparin and application thereof in heparanase detection. The gold nanoparticles and the gold nanorods are adopted to carry out covalent double labeling on the low molecular weight heparin with the diameter less than 10nm, and the FRET value change of the substrate after the degradation of the heparinase is detected by utilizing a fluorescence energy resonance transfer (FRET) mode to reflect the activity value of the heparanase, so that the influence of secondary cracking on the activity determination of the heparanase is effectively avoided.
In order to achieve the purpose, the invention adopts the following technical scheme:
a gold nano material of low molecular weight heparin is characterized in that gold nano particles are connected with the non-reduction end of the low molecular weight heparin, and gold nano rods are connected with the reduction end of the low molecular weight heparin.
Further, the low molecular weight heparin has a diameter of less than 10 nm.
Further, the gold nanoparticles are connected to the non-reducing end of the low molecular weight heparin in a Michael addition manner through sulfydryl, and the gold nanorods are combined to the reducing end of the low molecular weight heparin through an amino group in a reductive amination manner.
The application of the low molecular weight heparin gold nano material in heparanase activity detection.
The application of the low molecular weight heparin gold nano material in heparanase detection.
The invention adopts the characteristic of combining gold nano-material and polysaccharide, obtains the FRET probe substrate for the heparinase activity determination by carrying out covalent double labeling on the low molecular weight heparin with the diameter less than 10nm based on the fluorescence quenching effect characteristics of the gold nano-particles and the gold nano-rods with high quantum efficiency, and effectively utilizes the advantages of wide excitation spectrum, narrow emission spectrum, adjustable emission wavelength along with the size, long fluorescence life, high photochemical stability and the like of the gold nano-material. Compared with fluorescent micromolecules, the double-labeled low-molecular heparin is used for detecting the activity of heparanase, has higher sensitivity and low toxicity, and reduces the risk of clinical use. Meanwhile, the fluorescence has long service life and is not easy to bleach, and the sensitivity of enzyme activity detection is improved.
In addition, the technical means of carrying out covalent double labeling by adopting gold nanoparticles and gold nanorod energy transfer molecules can carry out specific covalent modification on other in-vivo proteins so as to realize positioning tracing on polysaccharide and quantitative analysis on protein.
Has the advantages that:
1. the activity of heparanase is detected by using low molecular weight heparin to replace expensive heparan sulfate, so that the cost of an experiment is reduced;
2. compared with HPLC detection of heparanase activity, the method is simple, convenient and quick, and saves a large amount of time; compared with the method using absorbance for detection, the method improves the detection sensitivity, can efficiently and quickly monitor the activity of heparanase, and can be used for screening other enzyme proteins at high flux.
Drawings
FIG. 1 is a schematic diagram of fluorescence resonance energy transfer of double-labeled low molecular weight heparin in the invention.
FIG. 2 is the excitation/emission spectrum of gold nanoparticles (AuNCs @ GSH-cys) and gold nanorods in example 1（AuNRs/side-SiO2/end-NH2) The absorption spectrum of (2).
FIG. 3 shows the activity of heparanase on the surface of different cells in example 2.
Detailed Description
The technical scheme of the invention is further explained by combining specific examples, and the experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
As shown in figure 1, based on the fluorescence quenching effect characteristics of the gold nanoparticles (A) and the gold nanorods (B) with high quantum efficiency, the method disclosed by the invention can be used for reflecting the activity value of heparanase by carrying out covalent double labeling on low-molecular-weight heparin with the diameter less than 10nm and detecting the change of FRET value of a substrate after the degradation of the heparanase in a fluorescence energy resonance transfer (FRET) mode, so that the influence of secondary cracking on the activity determination of the heparanase is effectively avoided.
The gold nanoparticles and the gold nanorods can be specifically and respectively covalently bonded to the non-reduction end and the reduction end of the low molecular weight heparin to prepare the low molecular weight heparin modified by the gold nanomaterial, thereby providing good support for researching the expression and activity detection of heparanase on the surface of tumor cells. Meanwhile, the gold nanoparticles or gold nanorods can also carry out specific covalent modification on other in-vivo proteins, and a novel monitoring strategy is provided for the research of the interaction between the proteins and the conformational change of the proteins.
Fluorescence Resonance Energy Transfer (FRET) refers to the phenomenon of energy transfer that occurs when two fluorescent molecules are close together (less than 10 nm), and is commonly used to detect the positional relationship of two different small molecules. The length of the low molecular weight heparin (LWMH) selected in the invention is less than 10nm, so that the gold nanoparticles marked at both ends of the LWMH and the gold nanorods can generate energy resonance transfer.
Example 1
Preparation of double-labeled Low molecular weight heparin
1. Preparation of gold nanoparticles (AuNCs @ GSH-cys)
78.7 mg of HAuCl was taken4·4H2O was dissolved in 20 mL of double distilled water, and the solution was sufficiently dissolved with stirring to prepare a 10 mM aqueous tetrachloroaurate solution. And (3.0) mL of the solution is taken, 4.15 mL of double distilled water and 2.85 mL of 10 mM reduced Glutathione (GSH) solution are added, the mixture is fully stirred, the temperature is set to be 90 ℃, the reaction is carried out for 50 min, and then AuNCs @ GSH solution is obtained, and a fluorescence spectrophotometer is used for measuring the optimal excitation wavelength and the optimal emission wavelength. In order to obtain the optimal fluorescence intensity, the molar ratio of glutathione to tetrachloroauric acid is groped to improve the fluorescence property of the gold nanoclusters. The molar ratio of the two is set in the experiment, [ GSH]/[HAuCl4]The molar ratio is 0.8:1-1.1: 1. After the reaction is finished, centrifuging at 12000 r/min to remove large particles, dialyzing overnight by using a dialysis membrane with the molecular weight of 500 Da, then re-suspending the gold nanoparticles by double distilled water, and storing at 4 ℃ in a dark place for later use.
In order to obtain gold nanoparticles with sulfydryl on the surface, the AuNCs @ GSH is subjected to cysteine amide condensation reaction. 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, carboxyl activating reagent) and N-hydroxysuccinimide (NHS, coupling agent), wherein the molar ratio of [ EDC ]/[ NHS ] is set to be 1: 2.5 in an experiment, the EDC is ensured to be added with the concentration which is about 10 times of the concentration of the gold nanoparticles, the equimolar cysteine is added, the gold nanoparticles are slowly mixed for 2 hours at room temperature, and after overnight dialysis is carried out by using a dialysis membrane with the molecular weight of 500 Da, double distilled water is used for re-suspending the gold nanoparticles, and the gold nanoparticles AuNCs @ GSH-cys are kept away from light at 4 ℃ for later use, so that the gold nanoparticles AuNCs @ GSH-cys are obtained.
2. Gold nanorods (AuNRs/side-SiO)2/end-NH2) Preparation of
The gold nanorods were prepared according to a seed growth method. The method comprises the following specific steps: to 0.25 mL 10 mM HAuCl4·4H29.5 mL of 10 mM cetyltrimethylammonium bromide (CTAB) solution was slowly dropped into the O aqueous solution, and after sufficiently stirring, 0.6 mL of 10 mM NaBH precooled was slowly dropped4And (3) mixing the solution uniformly, rapidly and violently stirring for 2 min, and standing for 2 h at room temperature to obtain the gold seed solution. Another 9.5 mL 10 mM CTAB solution was added with 0.5 mL 10 mM HAuCl4Solution, 140. mu.L 10 mM AgNO3Solution, 0.055 mL 0.1M ascorbic acid solution, stirring well. And slowly dripping 12 mu L of gold seed solution into the solution, stirring for 10 s, and standing for 24h to obtain a gold nanorod (AuNRs) solution. Can be adjusted according to AgNO3The added amount adjusts the maximum absorbance of the gold nanorods. Provided with AgNO3The amounts added were 80. mu.L, 100. mu.L, 120. mu.L, 140. mu.L, 180. mu.L, 210. mu.L, and 240. mu.L. After the reaction is finished, centrifuging at 10000 rpm for 15 min, removing unreacted and excessive reagents in the supernatant, repeating twice, and suspending into double distilled water for later use.
To obtain gold nanorods with amino groups on the surface, taking the AuNRs solution, adding 100 mu L of 10 mM cysteamine solution, stirring for 3 h at room temperature, centrifuging for 15 min at 10000 rpm after the reaction is finished, removing unreacted and excessive reagents in supernatant, repeating twice, and resuspending into double distilled water for later use to obtain the AuNRs-NH with aminated surface2And (3) solution.
Because the surface of low molecular weight heparin (LWMH) has negative charges, AuNRs-NH2The solution has positive charge, and in order to avoid charge interaction, the invention adopts silicification AuNRs-NH2The solution is made to have a negative charge on its surface. The method comprises the following specific steps: to 3 mL of AuNRs-NH2Adding TEOs solution (1.75 wt% dissolved in ethanol) 80 μ L and 0.1M NaOH solution 30 μ L sequentially, and stirring at room temperature for 10 hr to obtain AuNRs/side-SiO2/end-NH2 And (3) solution. The surface area of the gold nanorods subjected to silicification is adjusted by changing the addition amount of the TEOs solution, and finally, the gold nanorods are wrapped with silicon while two ends of the gold nanorods are exposed. The amounts of TEOs solution added were 60. mu.L, 80. mu.L, 100. mu.L, 120. mu.L, 140. mu.L for the experimental settings. After the reaction is finished, centrifuging at 10000 rpm for 15 min, removing unreacted and excessive reagents in the supernatant, repeating twice, and suspending into double distilled water for later use.
3. Preparation of double-labeled Low molecular weight heparin (LWMH)
The reactivity of terminal double bonds is increased by esterifying carboxyl groups on LWMH, then gold nanoparticles (A) having mercapto groups on the surface are site-specifically bonded to non-reducing terminals of LWMH by Michael addition reaction catalyzed by boric acid catalyst, and finally the protective ester is removed under alkaline conditions, thereby forming non-reducing terminal-labeled A-LWMH. On the basis, gold nanorods (B) with amino groups on the surface are marked on the reduction end of LWMH-M by using catalyst sodium borohydride to obtain FRET probe substrate A-LWMH-B for detecting heparinase activity.
(1) Preparation of LWMH benzethonium chloramide ester: dissolving 500 mg LWMH in 5 mL water (solution A), dissolving 1.25 g benzylchloramine in 15 mL water (solution B), slowly dropping solution B into solution A to produce a white solid, stirring at 1000 rpm for 1 h, standing for 1 h, removing supernatant, adding equal volume of water, stirring for 15 min, standing for 30 min, repeating once, filtering, and drying under reduced pressure at 30 ℃ for 20 h. After 0.59 g of LWMH benzethonium chloride salt was completely dissolved in 23.15 mL of dichloromethane, 4.915 mL of benzyl chloride was slowly added dropwise and stirred at 30 ℃ and 400 rpm for 24 hours to obtain 52 mg of LWMH benzethonium chloride ester.
(2) Preparation of A-LWMH: adding 45 mg of LWMH benzethonium chloride into 10 mL of formamide solution, heating at 50 ℃ until the mixture is completely dissolved, adding gold nanoparticle solid (58.8 mg, 0.3 mmol) with sulfydryl on the surface and boric acid (18.6 mg, 0.3 mmol) into the formamide solution, and reacting at 50 ℃ in a dark place for 24 hours. And after the reaction is finished, dialyzing for 48 hours in 50% ethanol by using a dialysis bag with the molecular weight cutoff of 500, removing unreacted gold nanoparticle molecules, and freeze-drying to obtain the gold nanoparticle-marked A-LWMH.
(3) Preparation of A-LWMH-B: 15 mg of A-LWMH marked by gold nanoparticles is dissolved in 560 mu L of distilled water, 4 mg of gold nanorods with amino groups on the surface are dissolved in 158 mu L of acetic acid/DMSO (3: 17) solution, after the two are mixed uniformly, 25 mg of sodium borohydride solution is added, and the reaction is carried out for 16 h at 37 ℃. After the reaction, 500. mu.L of DMSO was added to dissolve the precipitated precipitate, and the resulting solution was dialyzed for 24 hours using a dialysis bag having a molecular weight cut-off of 500 to remove unreacted substances, followed by lyophilization to obtain FRET substrate A-LWMH-B.
As shown in FIG. 2, the excitation wavelength and emission wavelength of gold nanoparticles (AuNCs @ GSH-cys) were scanned by a fluorescence spectrophotometer, and the optimal excitation wavelength (. lamda.ex) was 560 nm and the optimal emission wavelength was foundThe emission wavelength (lambda em) is 824 nm, and the gold nanorods (AuNRs/side-SiO) are measured by an ultraviolet spectrophotometer2/end-NH2) The optimal absorption peak is 825 nm, and the result shows that the emission wavelength of the gold nanoparticle (AuNCs @ GSH-cys) and the gold nanorod (AuNRs/side-SiO)2/end-NH2) The longitudinal surface plasma resonance absorption peak (LSPR peak) overlapping rate can reach more than 95 percent, so gold nano-rod (AuNRs/side-SiO) is selected2/end-NH2) As the acceptor group of gold nanoparticles (AuNCs @ GSH-cys), an energy resonance transfer molecule pair is formed.
Example 2
Application of A-LWMH-B in detection of heparanase on tumor cell surface
Preparing 1 mg/mL A-LWMH-B double-labeled fluorescent substrate by using DMEM, taking 100 uL of the substrate solution, adding 100 uL cultured for 24h respectively, wherein the cell density is 1 x 105Different tumor cells (cervical cancer cell Hela/breast cancer cell MCF-7/breast cancer cell MAB-MD-231/lung cancer cell A549) and normal cell 293t cell, 5% CO at 37 deg.C2The cells were incubated for 12 hours and the change in fluorescence under 560 nm excitation was recorded.
Because heparanase has high expression in various malignant tumors and is closely related to invasion and metastasis of malignant tumor cells, the expression and activity conditions of the heparanase on the surface of the tumor cells can be monitored by using the gold nanoparticle and gold nanorod double-modified low-molecular-weight heparin prepared by the invention, namely the metastasis condition result of the tumor cells is judged by monitoring the change condition of the fluorescence value at the excitation wavelength of the gold nanoparticle of 560 nm. After the double-modified low-molecular-weight heparin is mixed with tumor cells, heparanase on the surface of the double-modified low-molecular-weight heparin specifically degrades the double-modified low-molecular-weight heparin, as shown in fig. 3, compared with a normal cell group human renal epithelial cell line (293 t), FRET fluorescence values of cancer cell groups are increased to different degrees, for example, fluorescence value (RFU) in a breast cancer cell (MAB-MD-231) system is increased to 7824 compared with normal cells, further, the expression level of the heparanase on the surface of the breast cancer cell (MAB-MD-231) is the highest, the transfer capacity of the heparanase is stronger, and the cancer cell (Hela) is the next cervical cancer cell.
Claims (4)
1. A low molecular weight heparin gold nano-material is characterized in that: the non-reduction end of the low molecular weight heparin is connected with gold nanoparticles, and the reduction end of the low molecular weight heparin is connected with gold nanorods;
the diameter of the low molecular weight heparin is less than 10 nm;
the gold nanorods are used as acceptor groups of the gold nanoparticles to form an energy resonance transfer molecular pair, and the change of the FRET value of a substrate after heparinase degradation is detected by utilizing a fluorescence energy resonance transfer FRET mode to reflect the activity value of heparanase.
2. The low molecular weight heparin gold nanomaterial of claim 1, wherein: the gold nanoparticles are connected to the non-reduction end of the low molecular weight heparin in a Michael addition mode through sulfydryl, and the gold nanorods are combined to the reduction end of the low molecular weight heparin through an amino group in a reductive amination mode.
3. The use of the low molecular weight heparinized gold nanomaterial of claim 1 in heparanase detection.
4. The use of the low molecular weight heparinized gold nanomaterial of claim 1 in the detection of heparanase activity.
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