KR20140105980A - Method for Analysing Glycan of Cell Surface - Google Patents

Abstract

The present invention relates to a method for simply analyzing a sugar chain on the surface of a cell in high sensitivity only by fixing, on a substrate without additionally labelling or transforming, a live cell or a tissue cell and, more specifically, to a method for analyzing a sugar chain on the surface of a cell which is characterized by comprising the steps of: (A) fixing a cell on the surface of a substrate; (B) treating lectin binding with biotin to the cell fixed in the (A) step; (C) treating a quantum dot binding with a living particle specifically binding with the biotin on the surface after treating lectin; and (D) detecting phosphor of the quantum dot.

Description

METHOD FOR ANALYZING Glycan of Cell Surface [0002]

The present invention relates to a method for efficiently analyzing oligosaccharides on a cell surface.

The carbohydrate or sugar chain structure is called glycan or oligosaccharide. Examples of sugars constituting sugar chains include fucose, sialic acid, galactose, glucose, mannose, N-acetylglucosamine, GlcNAc, N- Various sugars are known, such as N-acetylgalactosamine (GalNAc). Glycosylation, which translocates sugar chains to proteins, plays an important role in intracellular, intercellular signaling and cancer metastasis. In fact, abnormal changes in glycosylation, which directly affect the activity of cancer cells such as cell proliferation, adhesion, differentiation, invasion, angiogenesis, and immunity, are observed in cancer cells. Therefore, the change in oligosaccharide on the cell surface can be utilized as an important biomarker for early diagnosis, metastasis, and target treatment of cancer.

In view of their biochemical roles, the identification and qualitative / quantitative analysis of oligosaccharides on the cell surface is a key element in understanding the various interactions in vivo by these. As a conventional method for analyzing sugar chains of known glycoprotein, there is known a method in which a glycoprotein is extracted with a lectin-affinity column, and then hydrolysis of the sugar portion of the glycoprotein is carried out by HPLC, FAC (frontal affinity chromatography), capillary electrophoresis (Capillary Electrophoresis-Mass Spectrometry), Surface Plasmon Resonance Spectroscopy and the like. However, these methods have a disadvantage in that the preprocessing process is complicated, time consuming, and requires a large number of cells.

In order to solve such a problem, an oligosaccharide analysis method using a microchip produced using oligosaccharide and lectin has recently been used. The oligosaccharide microchip is a method of analyzing oligosaccharides using microarrayed oligosaccharides, glycan binding proteins (GBPs) and antibodies specific thereto. The above assay method must be accompanied by a process of separating and chemically transforming the oligosaccharides on the cell surface in order to immobilize the oligosaccharide on the surface of the microarray, and the artificially synthesized oligosaccharide is not sufficient to cover the natural oligosaccharide expressed on the cell surface .

The use of lectin microarrays enables rapid and simple analysis of complex oligosaccharide structures without separating oligosaccharides from cell surface glycoproteins. The separation of sugar isomers, which is impossible by methods such as mass spectrometry, There is an advantage that it is possible. However, the fundamental problem that it is necessary to label the living cells with fluorescence or the like is necessary for detection, and the problem that the accessibility of the cells to the lectin immobilized on the solid surface is limited and the uniform patterning of the lectin in the microarray is difficult do.

It is an object of the present invention to provide a method for efficiently analyzing oligosaccharides without modifying the oligosaccharide to separate or label the oligosaccharides on the cell surface.

It is another object of the present invention to provide a highly sensitive assay method which is effective and reproducible even at a low concentration of oligosaccharides by a microarray in which living cells are uniformly patterned.

According to an aspect of the present invention, there is provided a method for manufacturing a semiconductor device, comprising: (A) immobilizing a cell on a surface of a substrate; (B) treating the immobilized cells with biotin-bound lectin in step (A); (C) treating a quantum dot to which a biomolecule that specifically binds biotin is bound to the surface after treatment of the lectin; And (D) detecting fluorescence of the quantum dot; The present invention relates to a method for analyzing an oligosaccharide on a cell surface.

The substrate may be a glass or silica material or an extracellular matrix (ECM) protein capable of adhering cells, a poly-L-lysine or a polyallylamine hydrochloride to facilitate cell immobilization. hydrochloride).

Cells to be analyzed can be immobilized as living cells, but tissue cells obtained from tissue biopsy resection can be directly immobilized and analyzed.

In the case of immobilizing living cells, it is more preferable to use a microarray in which cells are immobilized on a micropattern according to the present invention in which a hydrophobic polymer pattern is formed on a substrate with a microstamp. Conventional 96- or 384-well plates have been widely used despite the many disadvantages that the amount of sample used is large and the treatment or washing of the sample is not easy. In particular, Galanina et al. Reported that the sensitivity of micropatterns in detecting oligosaccharide-protein interactions is lower than that of enzyme-linked immunosorbent assays (Lab Chip, 2003, 3, 260).

However, in the case of using the fine pattern according to the present invention, as can be seen in the following examples, the sensitivity was remarkably excellent in the fine pattern as compared with the 96-well plate, and the sample was added to the immobilized cells or washed It was also easy and more efficient. The sensitivity of MCF-7 to WGA in the 96-well plate and the fine pattern was measured by WGA concentration. The fine pattern showed similar fluorescence intensity when the WGA concentration was diluted to 1/100 of the 96-well plate. (Data not shown) Further, the number of cells per pattern was not significantly different from that of the 96-well plate, and more reliable data can be obtained because a plurality of data points can be obtained by one experiment.

The fine pattern can be easily prepared by a microcontact printing (μCP) method. That is, a microstamp having a desired shape and size can be prepared by a conventional lithography method, and the precursor of the hydrophobic polymer can be printed on a substrate using the microstamp and then cured . In the fine pattern thus manufactured, a microarray of living cells can be formed because cells are not attached to the cured portion of the hydrophobic polymer and only the portion where the hydrophobic polymer is not coated is immobilized.

Examples of the hydrophobic polymer include polydimethylsiloxane (PDMS), polyurethane (PU), and trimethyloprotane triacrylate (TMPTA), which have a contact angle to water of 80 ° or more. In the following examples, only fine patterns on which PDMS is printed have been described. However, according to the preliminary experiment, all of the above-mentioned polymers effectively formed a pattern for immobilization of living cells. In addition, polyethylene glycol (PEG), a PEG block copolymer, and bovine serum albumin (BSA), which are hydrophilic but known to prevent cell adhesion, effectively formed microarrays of living cells.

When the cell immobilized on the substrate is treated with biotin-conjugated lectin, it binds to the cell surface when an oligosaccharide that specifically binds to the lectin is present on the cell surface. Lectin is a generic term for carbohydrate-binding proteins that specifically bind to specific carbohydrate chains, and is found mainly in plants, but is also found in a variety of vertebrate animals, microorganisms, and viruses. Concanavalin A recognizing mannose, Maackia seed agglutinin (MAA) recognizing sialic acid, Ricinus communis agglutinin (RCA) recognizing N-acetylgalactosamine (GalNAc), L and wheat germ agglutinin (WGA), which recognizes A-fucose-recognizing Aleuria aurantia lectin (AAL) and N, N-diacetyl chitobios (di-GlcNAc). Of course, the present invention is not limited to the above-mentioned lectins.

It binds specifically to biotin bound to a lectin to detect lectins bound to the cell surface, and utilizes biomolecules bound to quantum dots. By binding the quantum dots directly to the lectin and measuring the fluorescence, it is possible to discriminate whether the lectin is bound or not, but in this case, due to the bonding with the quantum dots having a relatively large size, , And the fluorescence dye, which is not a quantum dot, may be less accurate due to its own photobleaching.

As a biomolecule specifically binding to biotin, strapavidine is used in the following examples, but the present invention is not limited thereto. Biomolecules known to bind specifically to biotin include avidin, neutravidin or captavidin in addition to streptavidin. Quantum dots combined with biomolecules are commercially available in various sizes such as Invitrogen and Evident Technologies, and quantum dots having a maximum optical wavelength in the range of 500 to 900 nm are preferably used.

As described above, according to the present invention, it is possible to analyze the oligosaccharide on the surface with simple and high sensitivity only by immobilizing living cells or tissue cells on a substrate without separately marking or modifying them.

Further, by analyzing the oligosaccharide on the living cell surface using the fine pattern, the oligosaccharide can be analyzed with higher sensitivity and accuracy.

The oligosaccharide analysis method of the present invention can effectively analyze oligosaccharides known as biomarkers related to various cancers and diseases, and thus can be usefully used for diagnosis, treatment, and development of new diseases.

Brief Description of Drawings Fig. 1 is a diagram showing an oligosaccharide analysis process of living cells using a PDMS fine pattern. Fig.
2 is a conceptual diagram showing the principle of the oligosaccharide analysis of the cell surface of the present invention.
FIG. 3 is a microscope photograph immediately after the cells are attached to the fine pattern and after the culture according to an embodiment of the present invention. FIG.
FIG. 4 is a micrograph and fluorescence image of breast cancer cell and normal cell surface glycoprotein analyzed according to an embodiment of the present invention. FIG.
Fig. 5 is a thermogram of the results obtained by analyzing oligosaccharides on various cell line surfaces using various lectins.
6 is a graph showing the binding inhibition of WGA by N-GlcNAc addition.
FIG. 7 is a graph comparing the analysis of oligosaccharides on living cell surfaces in a 96-well plate with a fine pattern according to an embodiment of the present invention.
Fig. 8 is a photograph and a thermal map showing the results of various analyzes of oligosaccharides on the surface of breast cancer and colon cancer cell tissue.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the drawings and the embodiments are only illustrative of the contents and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

Example 1: Analysis of oligosaccharide on living cell surface

FIG. 1 is a diagram showing an oligosaccharide analysis process of living cells using a PDMS fine pattern.

1. Preparation of PDMS (poly (dimmethyl siloxane) fine pattern)

More specifically, a negative photoresist (SU-8, Microchem Co., USA) was evenly coated on a silicon wafer and spin coated at 1,000 rpm for 30 seconds to coat the photoresist with a height of 50 탆. As shown in FIG. 1, a master mold was prepared by irradiating UV with a mask through which a square pattern having a size of 100 μm and 100 μm was formed at intervals of 50 μm. Thereafter, a 10: 1 (w / w) mixture of the base of PDMS (Sylgard 184; Dow Corning, Midland, Mich.) And the curing agent was poured into the prepared master mold and cured at 65 ° C overnight, A stamp was produced.

The base and the curing agent of PDMS (Sylgard 184; Dow Corning, Midland, Mich.) Were dissolved in chloroform at a ratio of 10: 3 (w / w) and then spin-coated on the glass substrate at 2,000 rpm for 20 seconds. The microstamps prepared above were put on the PDMS spin-coated by microcontact printing, and the microstamps were transferred to the culture dish after putting the PDMS on the stamps. Thereafter, the PDMS stamp was removed after preliminarily curing at 65 DEG C for 15 minutes, and then cured at 65 DEG C for 4 hours so that a PDMS stamp-like fine pattern was printed on the culture dish.

2. Immobilization of living cells

The characteristics of the cell lines used in this experiment and related diseases are shown in Table 1.

MCF-7 cells were cultured in the presence of mammary epithelial basal (MEBM) containing bovine pituitary factor (BPE), hydrocortisone, human epidermal growth factor (hEGF), insulin and gentamicin / amphotericin- medium) medium. HCT116, HDF, ACHN, HEK293, HepG2, HeLa, and A549 were cultured in DMEM (Dulbecco's modified eagle medium) medium containing 10% fetal bovine serum and 1% penicillin streptomycin.

(50 ㎖, 4.0 × 10 ⁴ cells) were loaded onto a PDMS micropattern (1 cm × 1 cm) or a 96-well plate (well surface area 0.32 cm 2) CO ₂ environment for 24 hours and then adhered to the culture dish. Cells that did not adhere to the culture dish after incubation were washed three times with new culture medium, and then re-cultured for 12 hours to immobilize living cells.

FIG. 2 is a conceptual diagram showing the principle of the analysis method of the present invention, and FIG. 3 is a micrograph of the time immediately after the cells are attached to the fine pattern (0 hr) and after the culturing (12 hr). In the photograph, the scale bar indicates 100 μm. In FIG. 3, it can be confirmed that cells are not immobilized in the PDMS region which is hydrophobic after culturing for 12 hours, and the microarray is well formed. The number of cells per area of each pattern was found to be 14 ± 3.7 immediately after adhering and 25 (after 12 hours of incubation) the number of cells in each of the six pattern areas using a glare microscope (TE-2000, Nikon, Japan) ± 4.6, and the error that the pattern was wrongly formed per stamp was less than 5%.

3. Analysis of oligosaccharides on cell surface using fine pattern

(1) General method for the analysis of oligosaccharides

2. In a microarray prepared using micropatterns, 50 nM of biotin-conjugated lectin (Vector Laboratories Inc, CA, USA) was treated at 37 ° C for 3 hours and a new culture solution was added to remove unbound lectin And washed three times. The types of lectins used in the analysis and the oligosaccharides specifically binding to each lectin are shown in Table 2 below.

Streptavidin (SA-Qdot) (Invitrogen, CA, USA) with 10 nM quantum dots was added thereto and reacted at 37 ° C in a 5% CO ₂ environment for 1 hour. The SA-Qdot is a cadmium-selenium / zinc sulfide (CdSe / ZnS) series quantum dot having a total diameter of 15 to 20 nm and 5 to 10 streptavidin (SA) There is a characteristic of emitting light. After 1 hour, unbound SA-Qdot was removed by washing three times with fresh medium. The signals on the PMDS fine pattern were observed with a fluorescence microscope equipped with a high resolution CCD camera (Coolsnap, Roper Science, USA) (TE-2000, Nikon, Japan) and each image obtained was imaged using ImagePro (Mediacybernetics, And fluorescence was analyzed.

As a control, only SA-Qdot was treated and fluorescence intensity was measured to remove the signal due to non-specific binding of SA-Qdot.

(2) Analysis of oligosaccharides on cell surface

The oligosaccharides on the cell surface were analyzed using the six lectins shown in Table 2 as micropatterns of MCF-7 and HDF cells, and the results are shown in FIG. In Fig. 4, the upper part shows a microscope image and the lower part shows a fluorescence image for each cell. For each experiment, each fine pattern was repeated three times.

In FIG. 4, MCF-7, which is a breast cancer cell, showed strong fluorescence (WGA 2 times and SBA 16 times) by WGA and SBA that specifically bind N-acetylglucosamine (N-GlcNAc) , Which is consistent with the findings that the expression of terminal N-GlcNac is higher in breast cancer cells than in normal cells (J. Biol. Chem. 2002, 277, 26103). LTL showed weak fluorescence compared to WGA or SBA, but showed significant difference. This was also consistent with previous studies showing that the expression of oligosaccharides containing fucose is high in MCF-7 cells.

5 shows the fluorescence intensities of each cell described in Table 1 as a heat map after the oligosaccharide was analyzed by the method of (1) using the lectin of Table 2. From the thermogram of FIG. 5, it can be seen that the expression of the terminal N-GlcNAc or sialic acid is high in most cancer cells. From the results of ConA, it can be seen that the expression of mannose is high only in rectal cancer (HCT116) or kidney cancer cells (ACHN). MAA has a high fluorescence intensity in rectal cancer (HCT116) and cervical cancer cells (HeLa), indicating that sialic acid a-2,3 bond is abundant on the surface of the cell.

(3) Specificity of lectin binding to cell surface oligosaccharides

In order to verify the specificity of lectin binding to cell surface oligosaccharides, the degree of inhibition of WGA binding by N-GlcNAc monomers in MCF-7 cells was measured.

(1) except that biotin-conjugated lectin was replaced with biotin-conjugated WGA (50 nM) and N-GlcNAc (0-500 mM) were mixed and dissolved in the medium and treated at 37 ° C for one hour , The oligosaccharide on the surface of MCF-7 was analyzed and the degree of inhibition was calculated by the following equation, and the result is shown in Fig.

In the above equation, FI _o represents the fluorescence intensity at the N-GlcNAc concentration of 0, and FI _inh represents the fluorescence intensity at the specific N-GlcNAc concentration.

As can be seen in FIG. 6, the fluorescence intensity was gradually decreased with increasing concentration of N-GlcNAc. It was confirmed that the fluorescence intensity observed from this was due to the specific binding with the target oligosaccharide of WGA existing on the surface of MCF-7 cells.

4. Analysis of oligosaccharides on cell surface using 96-well plate

In order to compare the efficiency of the analysis using micropatterns, living cells were immobilized on a 96-well plate, and the oligosaccharides were analyzed and compared with those of microarrays.

3. Using a 96-well plate immobilized with live cells prepared in (2) instead of the micropatterns in which live cells were immobilized in (1), signals were analyzed with a Multi-Label Reader (Perkin Elmer LAS model, MA, USA) The oligosaccharides on the cell surface were analyzed by the same method as in (3). Cells were analyzed for MCF-7 cells and HDF cells, lectins were analyzed for WGA, and the results are shown in Fig. 7 together with the results obtained from the oligosaccharide analysis using microarrays.

As shown in FIG. 7, the oligosaccharide analysis using the same amount of the 96-well plate also showed that the fluorescence intensity of MCF-7, which is a breast cancer cell, was higher than that of normal cells, like the fine pattern, Showed a significance of 0.001 or less, whereas the significance of the 96-well plate was about 0.6, which was lower than that of the fine pattern. The fluorescence intensity itself was also much larger than that of the 96-well plate in terms of detection limit and reliability because the measurement value in the fine pattern was much larger than that in the case of 100 times or more of the sensitivity difference.

Example 2: Analysis of oligosaccharide on tissue cell surface

The oligosaccharides on the surface of tissue cells obtained from a tissue cell array purchased from SuperBiochips were analyzed. Tissue cells were fixed in 4% buffered neutral formalin for 24 hours according to the manufacturer's protocol and dehydrated three times for 1 hour with sequential use of 70%, 90%, 95%, 99%, 100% ethanol, For 1 hour, and then embedded in paraffin. The central portion of the paraffin containing the tissue cells was transferred to a recipient array and resected to a thickness of 4 쨉 m. Tissue samples were immobilized on silane coated slides.

The process of immersing the tissue array immobilized on the slide at 65 DEG C for 1 hour and immersing in xylene for 4 minutes was repeated 5 times to remove paraffin. Then, 100%, 95%, and 75% ethanol were sequentially used, treated for 2 minutes each for 3 minutes, and dipped in water for 5 minutes. To prevent nonspecific binding with SA-Qdot, streptavidin / biotin blocking solution (Vector Laboratories Inc, CA, USA) was treated for 15 minutes and washed with PBS buffer to prepare samples of tissue cells.

The oligosaccharides on the surface of tissue cells were analyzed using lectin and SA-Qdot to which 500 nM of biotin was bound according to the method of (3), and the results are shown in FIG. Tissue cells used in the experiment were breast or colon normal cells or cancer cells. Details of breast and colon cancer are shown in Table 3 and Table 4, respectively. SBA for breast cancer and WGA for colon cancer were analyzed using lectin.

FIG. 8 a) is a representative micrograph and fluorescence image in the analysis of oligosaccharides on breast cancer and colon cancer cell surface, and b) is an open view of the oligosaccharide analysis results of the tissue cell surfaces in Tables 3 and 4. In FIG. 8, early breast cancer showed strong fluorescence due to binding of SBA to metastatic breast cancer, and rectal cancer showed strong fluorescence in all tissues by WGA.

Claims (7)

(A) immobilizing a cell on a surface of a substrate;
(B) treating the immobilized cells with biotin-bound lectin in step (A);
(C) treating a quantum dot to which a biomolecule that specifically binds biotin is bound to the surface after treatment of the lectin; And
(D) detecting fluorescence of the quantum dot;
Wherein the cell surface analyzing method comprises the steps of:
The method according to claim 1,
Wherein the substrate is a glass or silica material or a surface modified with an extracellular matrix (ECM) protein, poly-L-lysine or polyallylamine hydrochloride Method for analyzing oligosaccharide on cell surface.
The method according to claim 1,
Wherein the cell is a living cell or a tissue cell.
The method of claim 3,
Wherein the living cells are immobilized on a micropattern formed with a pattern of a polymer having a contact angle with water of 80 ㅀ or more on a substrate by microstamping.
5. The method of claim 4,
Wherein the polymer is polydimethylsiloxane (PDMS), polyurethane (PU), or trimethyloprotane triacrylate (TMPTA).
The method of claim 3,
Wherein the living cells are immobilized on a microstructure by a microstamp to a fine pattern formed with a pattern of polyethylene glycol (PEG), PEG block copolymer or bovine serum albumin (BSA) on a substrate.
7. The method according to any one of claims 1 to 6,
Wherein the biomolecule is streptavidin, avidin, neutravidin, or captavidin. The method of claim 1, wherein the biomolecule is selected from the group consisting of streptavidin, avidin, neutravidin, and captavidin.

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