CN110261359B

CN110261359B - Cancer marker imaging method based on laser confocal microscope

Info

Publication number: CN110261359B
Application number: CN201910565325.7A
Authority: CN
Inventors: 朱丽娜; 刘芸; 乔亮; 刘宝红
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2019-06-27
Filing date: 2019-06-27
Publication date: 2022-01-28
Anticipated expiration: 2039-06-27
Also published as: CN110261359A

Abstract

The invention discloses a cancer marker imaging method based on a laser confocal microscope. The method specifically recognizes cancer markers by the aptamer, and can be used for imaging research after the quantum dots are combined. By using the method, epithelial cell adhesion molecules on the surface of the circulating tumor cells are taken as targets, and imaging under a laser confocal microscope is realized. The method is rapid, sensitive, excellent in specificity, clear and accurate in imaging, and has potential application prospects.

Description

Cancer marker imaging method based on laser confocal microscope

Technical Field

The invention belongs to the technical field of microscope research, and particularly relates to a cancer marker imaging method based on a laser confocal microscope.

Background

Cancer is the largest killer that endangers human health (Brindle K. New appaaces for imaging Cancer to treatment [ J ]. Nature Reviews Cancer, 2008, 8(2): 94-107). Related data show that 13% of people die of cancer in 2007, and this data rises to 12 hundred million in 2030 (Scheinberg, D. A.; Villa, C. H.; Escoria, F. E.; McDexit, M. R., Conscripts of the infinite armada: systematic cancer therapy using nanomaterials Nat Rev Clin Oncol 2010,7 (5), 266-. Therefore, it is important to establish an effective early cancer detection and diagnosis method. According to the reports in the literature, more and more scientists pay attention to the ultra-sensitive detection of low-concentration cells, and can detect proteins on the cell surface as important biomarkers, and the change of the quantity and the state of the proteins can indicate the occurrence and the development of the diseases.

The laser confocal microscope is a high-precision laser source microscope, utilizes laser as a light source, adopts the principle of conjugate focusing on the basis of the traditional optical microscope, and utilizes a computer to carry out digital image processing on an observed and analyzed object. The microscope can greatly surpass the traditional microscope, and has prominent advantages in the research of multiple fields in recent years.

Disclosure of Invention

The invention aims to provide a cancer marker imaging method based on a laser confocal microscope.

The invention takes epithelial cell adhesion molecules (EpCAM) as a target research object, adopts aptamer (aptamer) to specifically identify the epithelial cell adhesion molecules, and further marks quantum dot probes, thereby realizing imaging observation under a laser confocal microscope.

The invention provides a cancer marker imaging method based on a laser confocal microscope, which comprises the following specific steps:

(1) modifying biotin at the tail end of the nucleic acid aptamer sequence to obtain nucleic acid aptamer-biotin (aptamer-biotin);

(2) modifying Streptavidin (SA) on the quantum dot CdSe to obtain QDs-SA;

(3) modifying 13nm gold nanoparticles on the cleaned ITO glass, dripping aptamer, incubating at room temperature for 14 hours, adding mercaptoethanol, sealing for 30 minutes, and finally adding a cancer marker. After each binding step, the cells were washed with buffer (50 mM Tris-HCl) and blown dry with nitrogen.

(4) Adding the aptamer-biotin obtained in the step (1) into the substrate in the step (3), incubating for 1 hour at room temperature, washing with a buffer solution (50 mM Tris-HCl), and drying with nitrogen.

(5) Adding the QDs-SA obtained in the step (2) into the step (4), incubating at room temperature for 1 hour, washing with a buffer solution (50 mM Tris-HCl), and drying with nitrogen.

(6) Under a laser confocal microscope, setting the excitation with the wavelength of 458nm, and acquiring image data within the range of 500-600nm

In the present invention, the cancer marker in step (3) is epithelial cell adhesion molecule (EpCAM).

According to the invention, the concentration of the epithelial cell adhesion molecule in the step (3) is 10aM-500nM, and the volume is 5 muL-10 muL.

In the invention, the volume of aptamer-biotin in the step (3) is 1 muL-5 muL, and the concentration is 5 muM-10 muM.

In the invention, the volume of QDs-SA in the step (3) is 1 muL-5 muL, and the concentration is 5 muM-10 muM.

In the present invention, the setting of the microscope parameters in step (6) is 405-488nm for the excitation light.

The invention provides an application of the cancer marker obtained by the preparation method in laser confocal imaging.

The invention adopts the aptamer to identify the cancer marker for the first time and images under a laser confocal microscope. The aptamer has excellent specificity and can sensitively capture cancer markers. The fluorescence efficiency of the quantum dots is higher than that of a common fluorescent probe, and the quantum dots are beneficial to imaging of a target object under a microscope.

Aptamers (aptamers) are specifically screened for a target molecule and are therefore characterized by high specificity and high affinity. The Aptamer can bind a variety of molecules, from small molecules to proteins, and even cells, so the range of applications of Aptamer-based biosensors is very wide. Once the Aptamer is successfully screened, the high-repeatability and high-purity synthesis can be carried out by using commercial raw materials. Moreover, aptamers are generally very stable compared to antibodies and enzymes, which is not expected to be accessible by other sensor recognition elements. Aptamers typically undergo specific structural changes upon binding to a target molecule. The aptamer-based sensor has high sensitivity and good specificity when detecting target molecules. The Aptamer is easy to modify. Since it is essentially an oligonucleotide chain, it is easy to chemically modify. In contrast, antigen-antibody is a protein, on which it is difficult to modify groups. Aptamers are generally considered chemical antibodies. It is a small section of oligonucleotide chain obtained by adopting in vitro screening SELEX technology (systematic evolution of ligands by exogenous implementation, i.e. exponential enrichment method ligand evolution technology), and is screened out from artificially synthesized DNA or RNA random library by means of oligonucleotide sequence capable of making high-affinity and strong-specificity combination with correspondent ligand target molecule.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention visualizes epithelial cell adhesion molecules which are one of cancer markers,

(2) compared with the prior art, the method has the advantages of simple preparation and quick detection.

Drawings

FIG. 1 is an image of epithelial cell adhesion molecules under a confocal laser microscope of example 3.

Detailed Description

The invention is further illustrated by the following figures and examples.

The invention discloses a cancer marker imaging method based on a laser confocal microscope, which comprises the following steps:

firstly, designing a nucleic acid aptamer sequence, and modifying biotin, namely aptamer-biotin, at the tail end;

secondly, modifying streptavidin, namely QDs-SA, on the CdSe quantum dots;

and thirdly, modifying 13nm gold nanoparticles on the cleaned ITO glass, dropwise adding aptamer, incubating for 14 hours at room temperature, adding mercaptoethanol, sealing for 30 minutes, finally adding 5 mu L100 fM cancer marker, and incubating for 1 hour at room temperature. After each step of combination, washing by using a buffer solution (50 mM Tris-HCl), and drying by nitrogen;

and fourthly, adding the aptamer-biotin obtained in the step 1 into the substrate in the step 3, incubating for 1 hour at room temperature, washing with a buffer solution (50 mM Tris-HCl), and drying with nitrogen.

And fifthly, adding the QDs-SA obtained in the step 2 into the step 4, incubating for 1 hour at room temperature, washing with a buffer solution (50 mM Tris-HCl), and drying with nitrogen.

And sixthly, setting the wavelength of 458nm excitation under a laser confocal microscope, and collecting image data within the range of 500-600 nm.

Example 1:

(1) preparation of aptamer and biotin complex:

and (3) taking 20 muL of nucleic acid aptamer with the concentration of 10 muM, adding 5 muL of biotin, shaking and mixing for 4 hours under the condition of 4 degrees, taking out, and placing for 12 hours under the condition of 4 degrees for combination. The aptamer of the end modified biotin can be obtained.

(2) Preparation of observation samples:

modifying 13nm nano-gold particles on cleaned ITO glass, dropwise adding aptamer (5 muL 5 mM), incubating for 14 hours at room temperature, adding mercaptoethanol (50 muL 10 mM) for sealing for 30 minutes, finally adding 5 muL 100 fM cancer marker, and incubating for 1 hour at room temperature. Adding aptamer-biotin (1 mu L10 mu M) obtained in the step 1, incubating for 1 hour at room temperature, finally adding QDs-SA (1 mu L8 mu M) obtained in the step 2, and incubating for 1 hour at room temperature. After each binding step, the cells were washed with buffer (50 mM Tris-HCl) and blown dry with nitrogen.

(3) Collecting image data by a laser confocal microscope:

the prepared glass slide is placed under a Leica sp5 laser confocal microscope, the 458nm excitation wavelength is set, and data are collected within the range of 500-600 nm. It can be clearly observed that epithelial cell adhesion molecules present a distinct fluorescent signal. As shown in fig. 1.

Claims

1. A cancer marker imaging method based on a laser confocal microscope is characterized in that: the method comprises the following specific steps:

(1) modifying biotin at the tail end of the nucleic acid aptamer sequence to obtain nucleic acid aptamer-biotin;

(2) modifying streptavidin SA on the quantum dot CdSe to obtain QDs-SA;

(3) modifying 13nm gold nanoparticles on cleaned ITO glass, dripping aptamer, incubating at room temperature for 14 hours, adding mercaptoethanol, sealing for 30 minutes, adding a cancer marker, cleaning with 50mM Tris-HCl buffer solution after each step of combination, and drying with nitrogen; the cancer marker is an epithelial cell adhesion molecule, EpCAM; the concentration of the epithelial cell adhesion molecules is 10aM-500nM, and the volume is 5 muL-10 muL;

(4) adding the aptamer-biotin obtained in the step (1) into the substrate in the step (3), incubating for 1 hour at room temperature, washing with 50mM Tris-HCl buffer solution, and drying with nitrogen;

(5) adding the QDs-SA obtained in the step (2) into the step (4), incubating for 1 hour at room temperature, washing with 50mM Tris-HCl buffer solution, and drying with nitrogen;

(6) under a laser confocal microscope, the set wavelength is 458nm for excitation, and image data is acquired within the range of 500-600 nm.

2. The confocal laser microscopy-based cancer marker imaging method of claim 1, wherein: the volume of the aptamer-biotin in the step (3) is 1 muL-5 muL, and the concentration is 5 muM-10 muM.

3. The confocal laser microscopy-based cancer marker imaging method of claim 1, wherein: the volume of the QDs-SA in the step (3) is 1 muL-5 muL, and the concentration is 5 muM-10 muM.

4. The confocal laser microscopy-based cancer marker imaging method of claim 1, wherein: the excitation wavelength of the laser confocal microscope in the step (6) is in the range of 405-488 nm.