阅读说明：本技术 低分子量肝素金纳米材料及其在乙酰肝素酶检测中的应用 (Low molecular weight heparin gold nano material and application thereof in heparanase detection ) 是由 顾亚云 曾旭辉 丁伟华 孙斐 彭利忠 陈晨 于 2019-09-26 设计创作，主要内容包括：本发明公开了一种低分子量肝素金纳米材料及其在乙酰肝素酶检测中的应用,通过采用金纳米颗粒和金纳米棒对直径小于10 nm的低分子量肝素进行共价双标记,利用荧光能量共振转移(FRET)的方式检测肝素酶降解后底物的FRET值变化来反映乙酰肝素酶活性值,从而有效避免二次裂解对乙酰肝素酶活性测定带来的影响。(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.)

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.

2. The low molecular weight heparin gold nanomaterial of claim 1, wherein: the low molecular weight heparin has a diameter of less than 10 nm.

3. 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.

4. The use of the low molecular weight heparinized gold nanomaterial of claim 1 in heparanase detection.

5. The use of the low molecular weight heparinized gold nanomaterial of claim 1 in the detection of heparanase activity.

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 (AuNRs/side-SiO) in example 1₂/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.