阅读说明：本技术 一种基于留置针的仿生型拉曼基底及其制备和应用 (Bionic Raman substrate based on indwelling needle and preparation and application thereof ) 是由 牟忠德 韩晶晶 狄文忆 高瀚 刘宝喜 于 2020-04-29 设计创作，主要内容包括：本发明提供了一种基于留置针的仿生型拉曼基底,该基底包括留置针软管和附着层,附着层包括聚多巴胺膜和单层二氧化硅纳米粒子,在单层二氧化硅纳米粒子表面吸附有特异性肿瘤标记物探针,并用牛血清白蛋白分子封闭纳米粒子表面多余位点。其制备方法包括1)盐酸多巴胺在留置针软管表面形成聚多巴胺膜；2)聚多巴胺膜表面吸附单层二氧化硅纳米粒子；3)肿瘤标记物探针吸附到二氧化硅纳米粒子表面,并用牛血清白蛋白分子封闭纳米粒子表面多余位点。该基于留置针的仿生型拉曼基底可以应用于患者血液中肿瘤标记物的检测,安全性好,不会给患者带来负担,能够反映长时间内血液中特定肿瘤标记物的平均水平。(The invention provides a bionic Raman substrate based on an indwelling needle, which comprises an indwelling needle hose and an attachment layer, wherein the attachment layer comprises a polydopamine film and single-layer silicon dioxide nano particles, a specific tumor marker probe is adsorbed on the surfaces of the single-layer silicon dioxide nano particles, and bovine serum albumin molecules are used for sealing redundant sites on the surfaces of the nano particles. The preparation method comprises 1) forming a polydopamine film on the surface of the indwelling needle hose by dopamine hydrochloride; 2) adsorbing a single-layer silicon dioxide nano particle on the surface of the polydopamine film; 3) adsorbing the tumor marker probe on the surface of the silica nanoparticle, and sealing redundant sites on the surface of the nanoparticle by using bovine serum albumin molecules. The bionic Raman substrate based on the indwelling needle can be applied to detection of tumor markers in blood of a patient, is good in safety, does not bring burden to the patient, and can reflect the average level of specific tumor markers in the blood for a long time.)

1. The utility model provides a bionical type raman basement based on indwelling needle which characterized in that: the substrate comprises an indwelling needle hose (1) and an outer surface attachment layer (3) of the indwelling needle hose (1), wherein the attachment layer (3) comprises a polydopamine film (3a) and single-layer silica nanoparticles (3b), the polydopamine film (3a) is adsorbed on the outer surface of the indwelling needle hose (1), the single-layer silica nanoparticles (3b) are adsorbed on the polydopamine film (3a), specific tumor marker probes are adsorbed on the surface of the single-layer silica nanoparticles (3b), and bovine serum albumin molecules are used for sealing redundant sites on the surfaces of the nanoparticles.

2. The biomimetic raman substrate based on an indwelling needle as recited in claim 1, wherein: the indwelling needle hose (1) is an indwelling needle sleeve or a transparent catheter part which is used for infusion and medicine delivery and can indwelling needles of different specifications and different types for more than 1 hour in a patient body.

3. The biomimetic raman substrate based on an indwelling needle as recited in claim 1, wherein: the polydopamine membrane (3a) is black and has a thickness of 30-200 nm.

4. The biomimetic raman substrate based on an indwelling needle as recited in claim 1, wherein: in the single-layer silica nanoparticles (3b), the silica nanoparticles are solid silica nanoparticles or mesoporous silica nanoparticles, and the diameter of the nanoparticles is 50-200 nm.

5. The biomimetic raman substrate based on an indwelling needle as recited in claim 1, wherein: the specific tumor marker probe is an antibody or RNA probe capable of specifically recognizing one or more antigen proteins, nucleic acids, hormones or enzymes expressed by oncogenes.

6. A preparation method of the bionic Raman substrate based on the indwelling needle as set forth in claim 1-5, wherein the preparation method comprises the following steps: the method comprises the following steps:

1) adding a dopamine hydrochloride solution into a weak alkaline solution, then soaking the remaining needle hose (1) in the dopamine hydrochloride solution, and rotating the hose to form a polydopamine film (3a) on the surface of the hose;

2) dispersing the silicon dioxide nanoparticles with the amino groups or the mercapto groups grafted on the surfaces into a weak alkaline solution to prepare a silicon dioxide nanoparticle colloidal solution with the mass fraction of 5-15%, then soaking the indwelling needle hose (1) with the polydopamine membrane (3a) on the surfaces, which is obtained in the step 1), into the colloidal solution, rotating the hose to form a single-layer silicon dioxide nanoparticle (3b) on the surface of the polydopamine membrane (3 a);

3) placing the indwelling needle hose (1) with the single-layer silicon dioxide nano particles (3b) adsorbed on the surface into a specific tumor marker probe solution, adsorbing tumor marker probe molecules on the surface of the single-layer silicon dioxide nano particles (3b), and finally sealing redundant sites on the surface of the nano particles to obtain the bionic Raman substrate based on the indwelling needle.

7. The preparation method of the biomimetic Raman substrate based on the indwelling needle as recited in claim 6, wherein: the weak alkaline solution in the steps 1) and 2) is an aqueous solution with the pH value of 8.0-9.0 prepared by ammonia water, sodium hydroxide or Tris (Tris (hydroxymethyl) aminomethane); the rotating hose in the step 1) and the step 2) is a hose which rotates clockwise or anticlockwise under the conditions of normal temperature and normal pressure, the rotating speed is 10-50 r/min, and the duration is 1-24 hours.

8. The preparation method of the biomimetic Raman substrate based on the indwelling needle as recited in claim 6, wherein: adding the dopamine hydrochloride solution into the weak alkaline solution in the step 1), wherein the concentration of the dopamine hydrochloride solution is 2-20 mg/ml, and the volume ratio of the weak alkaline solution to the dopamine hydrochloride solution is 0.5: 1-2: 1.

9. The preparation method of the biomimetic Raman substrate based on the indwelling needle as recited in claim 6, wherein: the step 3) of adsorbing the tumor marker probe molecules on the surface of the single-layer silica nanoparticle (3b), and finally closing the redundant sites on the surface of the nanoparticle refers to coupling the tumor marker probe molecules with the silica nanoparticle by a chemical coupling method, and then closing the redundant sites by bovine serum albumin after cleaning by phosphate buffer solution, and the specific process is as follows: soaking the indwelling needle hose adsorbed with the single-layer silicon dioxide nano particles (3b) in a glutaraldehyde aqueous solution with the concentration of 3-5 wt% for 2-5 h in a rotating state, and then washing the indwelling needle hose with a phosphate Tween 20 buffer solution for multiple times to remove redundant glutaraldehyde; then soaking the remaining needle hose (1) in a tumor marker probe phosphate buffer solution of 50-200 mug/ml for overnight standing at the temperature of 2-4 ℃; and finally, soaking the indwelling needle hose (1) in 0.8-1 wt% of bovine serum albumin solution for blocking, removing redundant bovine serum albumin molecules by using phosphate buffer solution to obtain the bionic Raman substrate based on the indwelling needle, and placing the bionic Raman substrate in the phosphate buffer solution at the temperature of 2-6 ℃ for later use.

10. An application method of the bionic Raman substrate based on the indwelling needle as set forth in claims 1-5, wherein: the Raman substrate is applied to detection of tumor markers in blood of a patient, and the specific operation is as follows: the indwelling needle hose (1) adsorbed with the tumor marker probe is indwelling in a blood vessel of a patient, the probe continuously captures a target tumor marker in the blood of the patient, the indwelling needle hose (1) is taken out after 1-7 days, the target tumor marker is washed by phosphate buffer solution, silver-dyed in silver nanoparticle sol with the particle size of 20-30 nm for 5 min-6 h, and the phosphate buffer solution is washed again for Raman detection, so that the type and the content of the tumor marker in the blood of the patient can be determined.

Technical Field

The invention relates to a bionic Raman substrate based on an indwelling needle and preparation and application thereof, belonging to the technical field of biomedical research and clinical detection and diagnosis.

Background

The malignant tumor is second to cardiovascular and cerebrovascular diseases, becomes one of the diseases with the highest fatality rate, and seriously threatens human health. A recent article, "Cancer statistics,2020," at CA A Cancer Journal for Clinicians showed a 29% decrease in Cancer mortality in the United states from 1991 to 2017, a 2.2% decrease from 2016 to 2017, and a maximum annual decrease. The study indicates that promotion of early cancer screening and novel therapies are of great significance in reducing mortality.

In recent years, tumor markers have the advantages of being early in appearance time, easy to obtain repeatedly, convenient to detect and the like in the cancer evolution process, so that the tumor markers become important references for early detection and prevention of cancers, and survival rate can be greatly improved and death rate can be greatly reduced by aiming at early detection of the tumor markers. Tumor development and proliferation are a gradually evolving process, and tumor markers (tumor markers) refer to a class of active substances directly synthesized, secreted or produced by tumor cells in the process of tumor development and related to tumor intimacy, and antigen proteins, nucleic acids, hormones, enzymes and other active substances mainly generated by oncogene expression. Various detection means have been developed for tumor markers, such as radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), fluoroimmunoassay, chemiluminescence immunoassay (CLIA), mass spectrometry, tag-amplified deep sequencing (TAM-Seq), bead emulsion amplification (BEAMing), and the like. However, the clinically used techniques of serological examination at the molecular level do not reflect the long-term average level of tumor markers in the blood of patients. In addition, radioimmunoassay has potential radiation hazard, analytical methods such as ELISA and CLIA require secondary labeling, the steps are complicated, and the equipment required by mass spectrometry is expensive. Therefore, it is still a challenge to achieve rapid and highly sensitive detection of low-concentration tumor markers in early stage of tumor development and to improve the clinical diagnosis level of malignant tumors in early stage.

Disclosure of Invention

The technical problem is as follows: the invention aims to provide a bionic Raman substrate based on an indwelling needle and preparation and application thereof, and the technology can ensure that a detection array is firmly adsorbed on an indwelling needle hose and is not influenced in the using process. The indwelling needle can accurately capture and enrich tumor markers in blood in the using process, and finally, rapid and efficient detection is realized.

The technical scheme is as follows: in order to achieve the aim, the invention provides a bionic Raman substrate based on an indwelling needle, which comprises an indwelling needle hose and an attaching layer on the outer surface of the indwelling needle hose, wherein the attaching layer comprises a poly-dopamine film and single-layer silica nanoparticles, the poly-dopamine film is adsorbed on the outer surface of the indwelling needle hose, the single-layer silica nanoparticles are adsorbed on the poly-dopamine film, specific tumor marker probes are adsorbed on the surfaces of the single-layer silica nanoparticles, and bovine serum albumin molecules are used for sealing redundant sites on the surfaces of the nanoparticles.

Wherein:

the indwelling needle hose is an indwelling needle sleeve or a transparent catheter part which is used for infusion and medicine delivery and can indwelling needles of different specifications and different types for more than 1 hour in a patient body.

The polydopamine membrane is black, is attached to a catheter of an indwelling needle, is 30-200 nm thick, and contains abundant primary amine, secondary amine and catechol chemical groups.

In the single-layer silicon dioxide nano particles, the silicon dioxide nano particles are solid silicon dioxide nano particles or mesoporous silicon dioxide nano particles, and the diameter of the nano particles is 50-200 nm.

The specific tumor marker probe is an antibody or RNA probe capable of specifically recognizing one or more antigen proteins, nucleic acids, hormones or enzymes expressed by oncogenes.

The invention also provides a preparation method of the bionic Raman substrate based on the indwelling needle, which comprises the following steps:

1) adding a dopamine hydrochloride solution into a weak alkaline solution, then soaking the remaining needle hose in the dopamine hydrochloride solution, rotating the hose, and carrying out oxidative autopolymerization on dopamine hydrochloride to form a poly-dopamine film on the surface of the hose;

2) dispersing the silicon dioxide nanoparticles with the amino groups or the mercapto groups grafted on the surfaces into a weak alkaline solution to prepare a silicon dioxide nanoparticle colloidal solution with the mass fraction of 5-15%, then soaking the remaining needle hose with the polydopamine membrane on the surface obtained in the step 1) into the colloidal solution, and rotating the hose to form firm single-layer silicon dioxide nanoparticles on the surface of the polydopamine membrane;

3) placing an indwelling needle hose with a monolayer of silica nanoparticles adsorbed on the surface into a specific tumor marker probe solution, adsorbing tumor marker probe molecules on the surface of the monolayer of silica nanoparticles, and finally sealing redundant sites on the surface of the nanoparticles to obtain the bionic Raman substrate based on the indwelling needle.

Wherein:

the weak alkaline solution in the steps 1) and 2) is an aqueous solution with the pH value of 8.0-9.0 prepared by ammonia water, sodium hydroxide or Tris (Tris (hydroxymethyl) aminomethane); the rotating hose in the step 1) and the step 2) is a hose which rotates clockwise or anticlockwise under the conditions of normal temperature and normal pressure, the rotating speed is 10-50 r/min, and the duration is 1-24 hours.

Adding the dopamine hydrochloride solution into the weak alkaline solution in the step 1), wherein the concentration of the dopamine hydrochloride solution is 2-20 mg/ml, and the volume ratio of the weak alkaline solution to the dopamine hydrochloride solution is 0.5: 1-2: 1.

In the silica nanoparticles with the surfaces grafted with amino groups or mercapto groups in the step 2), the amino groups or mercapto groups are grafted on the surfaces of the silica nanoparticles by a chemical coupling method.

In the step 2), the principle of forming the firm single-layer silica nanoparticles on the surface of the polydopamine film is that the catechol group of the polydopamine and amino or sulfhydryl carry out Michael addition reaction and/or Schiff base reaction under the weak alkaline condition, and the single-layer silica nanoparticles are adsorbed on the surface of the polydopamine film through chemical coupling.

The step 3) of adsorbing the tumor marker probe molecules on the surface of the single-layer silica nanoparticles and finally closing the redundant sites on the surface of the nanoparticles refers to coupling the tumor marker probe molecules with the silica nanoparticles by a chemical coupling method, then cleaning the coupled sites by phosphate buffer solution and closing the redundant sites by bovine serum albumin, and the specific process is as follows: soaking the remaining needle hose adsorbed with the single-layer silicon dioxide nano particles in a 3-5 wt% glutaraldehyde aqueous solution for 2-5 h in a rotating state, and then washing the remaining needle hose with a phosphate Tween 20 buffer solution for multiple times to remove redundant glutaraldehyde; then soaking the remaining needle hose in 50-200 mug/ml tumor marker probe phosphate buffer solution for overnight standing at 2-4 ℃; and finally, soaking the indwelling needle hose in 0.8-1 wt% of bovine serum albumin solution for blocking, removing excessive bovine serum albumin molecules by using phosphate buffer solution to obtain the bionic Raman substrate based on the indwelling needle, and placing the bionic Raman substrate in the phosphate buffer solution at the temperature of 2-6 ℃ for later use.

In the coupling of the tumor marker probe molecules and the silica nanoparticles by a chemical coupling method, the chemical coupling method is a condensation reaction of amino and aldehyde groups.

The invention also provides an application method of the bionic Raman substrate based on the indwelling needle, the Raman substrate is applied to detection of a tumor marker in blood of a patient, and the specific operation is as follows: the indwelling needle hose adsorbed with the tumor marker probe is indwelling in the blood vessel of the patient through the conventional puncture operation, and the probe can continuously capture the target tumor marker in the blood of the patient; after the indwelling needle is kept for 1-7 days, the indwelling needle hose is taken out, washed by phosphate buffer solution, silver-stained in silver nanoparticle sol with the particle size of 20-30 nm for 5 min-6 h, washed again by the phosphate buffer solution, and subjected to Raman detection to capture Raman signals of the tumor markers, so that the types and the content of the tumor markers in the blood of the patient can be judged.

Has the advantages that: the bionic Raman substrate based on the indwelling needle is prepared from polydopamine and silicon dioxide nanoparticles and is used for detecting tumor markers in blood, and compared with the prior art, the bionic Raman substrate based on the indwelling needle has the following advantages:

1. due to the use of the bionic polydopamine material, the biocompatibility is good, and the prepared Raman substrate can be tightly adsorbed on the surface of the indwelling needle hose and cannot fall off due to puncture or blood washing, so that the safety of a patient in use is ensured;

2. the surface of the hose adsorbs a single-layer silica nano particle, so that the biocompatibility is further improved, and the interference of polydopamine on a Raman signal of a tumor marker can be isolated; the silicon dioxide nano particles are periodically arranged in the indwelling needle hose, so that the effect of enhancing the Raman signal of the tumor marker is achieved, the signal repeatability is good, and the signal repeatability is improved;

3. the Raman substrate prepared on the surface of the indwelling needle hose does not influence the normal use of the indwelling needle, does not bring extra burden to the patient, and can accurately reflect the average content of the tumor marker in the blood of the patient for a long time compared with blood drawing detection.

Drawings

FIG. 1 is a general schematic view of a biomimetic Raman substrate based on an indwelling needle provided in the present invention;

FIG. 2 is a cross-sectional view of the portion of the needle of FIG. 1 taken along line AA;

FIG. 3 is an electron microscope image of an indwelling needle based biomimetic Raman substrate, wherein A is an indwelling needle hose and B is a single layer of silica nanoparticles;

in the figure: 1 is an indwelling needle hose, 2 is a steel needle inside the indwelling needle hose, 3 is an adhesion layer, 3a is a polydopamine film, and 3b is a single-layer silica nanoparticle.

Detailed Description

The invention provides a bionic Raman substrate based on an indwelling needle, and preparation and application thereof. The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.