KR101845995B1 - Liquid crystal microdroplets for sensing of breast cancer cells, Sensing method of breast cancer cells using the same and Biosensor using the same - Google Patents
Liquid crystal microdroplets for sensing of breast cancer cells, Sensing method of breast cancer cells using the same and Biosensor using the same Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3015—Breast
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F212/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
- C08F212/02—Monomers containing only one unsaturated aliphatic radical
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The present invention relates to an LC microdroplet for breast cancer cell sensing, a method for sensing breast cancer cells using the same, and a biosensor using the same. More particularly, the present invention relates to a LC microdroplet Herceptin antibody and an antigen- To LC microdroplets capable of sensing breast cancer cells by identifying changes in the LC molecule array of LC microdroplets by binding of the LC microdroplets to the cancer cells, and a biosensor using the method.
Description
The present invention relates to LC microdroplets for sensing breast cancer cells into which Herceptin antibodies are introduced, a method for sensing breast cancer cells using the same, and a biosensor using the same.
Biosensors typically consist of a bio-recognition component, a biotransformation component and an electronic system, which include a signal amplifier, a processor and a display. Recognition components are often referred to as bio-receptors and use biomolecules from receptors or organisms designed after the biological system to interact with the analyte of interest. This interaction is measured by a bio-converter that outputs a measurable signal proportional to the presence of the target analyte in the sample. The general design objective of a biosensor is to be able to test quickly and conveniently at the point of interest or treatment where the sample is obtained.
Surface science occurs in a wide range of fields, including basic science and large-scale industrial production. Liquid crystals are particularly interesting in this regard because the effects of the surface extend deep into the bulk phase. Thus, director orientation is sensitive to interfacial properties such as the shape of the surface and its chemical composition. For most liquid crystal applications such as LCDs, the surface is processed to ensure accurate fixation of the liquid crystal. Planar fixation is accomplished by rubbing the surface, but homeotrophic fixation is achieved by attachment of the surfactant to the substrate. Similarly, these molecules can be absorbed at the interface between LC and water, changing the arrangement of LCs. Since a fixed angle is very sensitive to surfactant concentration, such a system can be used as a sensor. Fixation also affects bulk liquid crystals, and the changes can be amplified through a long range orientational order of the liquid crystals and easily observed under a polarizing microscope. Thus, the LC interface can be used as a sensor to detect various chemicals, protein bonds, viruses, bacteria, and pH measurements, including phospholipids at the LC-water interface. However, recent methods for observing these changes are still based on observations with the naked eye, and in this way small changes in fixation can not be detected.
With the rapid development of LC material technology in recent decades, many practical devices have been developed and commercialized. LC devices have been widely applied in fields such as imaging, microscopy, spectroscopy, and optical probing. A variety of LC-based sensors have been developed to analyze non-labeled polymer electrolytes, ions, molecules and biological systems. The detection principle of these LC sensors is based on the highly sensitive orientation response of the LC molecules to the microscopic changes in the surface structure.
LC materials and technologies can contribute to a combination of non-marking, highly sensitive real-time detectors without complex expensive instruments. LC materials are found to be simple, convenient, and efficient in creating sensing devices to convert various chemical and biological events into optical reactions visible to the naked eye. LC-based sensors have been found to be useful for a variety of sensing applications in ambient light conditions, including environmental contaminants, enzyme reactions, protein interactions, ligand-receptor interactions, antigen-antibody immunoassay interactions, and detection of DNA hybridization . LC-based sensing technology is simple and useful for developing portable, inexpensive screening tests to quickly evaluate samples away from a central laboratory. Moreover, due to the potential applicability in areas such as chemistry, biomedicine, and environmental science, scientists have been interested in the research and development of LCs because LCs are potentially available for real-time detection sensors.
On the other hand, there is a lot of controversy about the balance between the benefits and damage of breast cancer screening. The Coclon report explains that it is unclear whether screening mammography is harmful or harmful. The US Preventive Services Task Force's 2009 report found evidence of breast cancer screening benefits among people aged 40-70, and the group recommends screening to women aged 50-74 years every two years, but Breast X Conclusions: Congenital malignant melanoma is the most common malignant breast cancer in Korea, and it has a low diagnostic yield due to high fiber content. In addition, because of the use of X-rays, it is not possible to rule out the possibility of breast cancer in the diagnosis process.
Drugs Tamoxifen or raloxifene can be used to prevent breast cancer in people at high risk for breast cancer. For some high-risk women, removal of both breasts is another useful precaution. For people diagnosed with cancer, a number of therapies, including surgery, radiation therapy, chemotherapy, hormone therapy and target therapy, can be used. However, it is very important to develop a diagnostic device to detect cancer early so that an effective therapy can be performed to prevent the growth of breast cancer.
Therefore, it is required to develop a device capable of replacing the mammary X-ray imaging method, which is a controversial conventional breast cancer diagnosis method, and detecting cancer at an early stage.
The present invention has been devised in order to provide a device capable of detecting cancer at an early stage, replacing a controversial conventional mammography method, and is a breast cancer cell capable of real-time detection with high sensitivity without labeling Sensing LC microdroplets, a method for sensing breast cancer cells using the same, and a biosensor using the same.
In order to solve the above problems, the present invention provides a liquid crystal display device comprising: a liquid crystal; Herceptin antibody block-fixed block copolymers; And LC microdroplets for breast cancer cell sensing comprising a surfactant.
According to a preferred embodiment of the present invention, the LC micro liquid droplet for breast cancer cell sensing of the present invention is a micelle structure formed by a surfactant, the liquid crystal being present in the micro droplet, and the hydrophobic part Is present in the microdroplet, and the Herceptin antibody binds to the hydrophilic part of the block copolymer and is exposed to the outside of the microdroplet.
According to another preferred embodiment of the present invention, the LC microdroplet liquid crystal for breast cancer cell detection of the present invention is a 4-cyano-4'-pentylbiphenyl, 4-cyano- -4'- (2-methylbutyl) biphenyl can be used, and 4-cyano-4'-pentylbiphenyl can be preferably used.
According to another preferred embodiment of the present invention, the LC microdroplet block copolymer for breast cancer cell detection of the present invention is a compound represented by the following chemical formula 1, And an average molecular weight of 5,000 to 12,000.
[Chemical Formula 1]
In the above formula (1), x is 50 to 75 and y is 25 to 35.
According to another preferred embodiment of the present invention, the LC microdroplet surfactant for breast cancer cell detection of the present invention may be sodium dodecyl sulfate or dodecyltrimethylammonium.
According to another preferred embodiment of the present invention, in the LC microdroplet for breast cancer cell sensing of the present invention, the Herceptin antibody has a radial conformation when the liquid crystal is immobilized to the antigen of the breast cancer cell, and the Herceptin antibody binds to the breast cancer cell The liquid crystal may have a bipolar conformation.
According to another preferred embodiment of the present invention, the breast cancer cell that binds to the Herceptin antibody in the LC microdroplet for breast cancer cell sensing of the present invention may be SK-BR3 cells.
According to another preferred embodiment of the present invention, in the LC microdroplet for breast cancer cell sensing according to the present invention, the antigen of the breast cancer cell is HER2 + protein.
According to another preferred embodiment of the present invention, in breast cancer cells ever sensing micro LC solution of the present invention, a liquid crystal 1.0 × 10 -1 ~ 5.0 × 10 - containing a 1 mmol, Include a 2 mmol, - a block copolymer is 1.50 × 10 -3 ~ 2.0 × 10 It may include a 1 mmol - a surface active agent 5.0 × 10 -2 ~ 3.0 × 10 .
According to another preferred embodiment of the present invention, the LC microdroplet for breast cancer cell sensing of the present invention The diameter may be in the range of 2.5 占 퐉 to 20 占 퐉, preferably 5 占 퐉 to 17.5 占 퐉, and more preferably 7.5 占 퐉 to 15 占 퐉 in diameter.
According to one preferred embodiment of the present invention, 4-cyano-4'-pentylbiphenyl; Poly (styrene-b-acrylic acid) with Herceptin antibody fixed; And LC microdroplets for breast cancer cell sensing comprising sodium dodecyl sulfate or dodecyltrimethylammonium.
Another aspect of the present invention relates to a biosensor for sensing a breast cancer cell, and it can be characterized by including the LC microdroplets of the various types described above.
According to a preferred embodiment of the present invention, the biosensor for sensing a breast cancer cell of the present invention is characterized in that the LC micro liquid droplet senses breast cancer cells in a temperature range of 18 to 35 ° C.
Another aspect of the present invention relates to a method of sensing breast cancer cells and provides a method of sensing breast cancer cells using various types of LC microdroplets as described above.
According to a preferred embodiment of the present invention, the method of sensing breast cancer cells of the present invention can be characterized in that breast cancer cells are sensed by visually observing changes in LC molecule orientation of LC microdroplets through a polarizing microscope .
According to another preferred embodiment of the present invention, the LC microdroplets and the breast cancer cells are contacted with each other at a temperature ranging from 18 to 35 ° C to sense the breast cancer cells through the change of orientation of the LC molecules.
According to another preferred embodiment of the present invention, the method for sensing breast cancer cells according to the present invention comprises contacting LC microdroplets with breast cancer cells under a pH of 7.0 to 7.8 to sense breast cancer cells through an orientation change of LC molecules . ≪ / RTI >
According to a preferred embodiment of the present invention, the method of sensing breast cancer cells of the present invention may be characterized by sensing breast cancer cells in vitro.
The LC microdroplets for breast cancer cell sensing of the present invention have high selectivity and sensitivity to breast cancer cells, can detect breast cancer cells in a non-labeled state, and can selectively detect breast cancer cells in vitro, including other proteins. It is possible to burn.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram showing the orientation change of an LC microdroplet due to antibody-antigen interaction.
2 shows a polarized optical microscope (POM) image showing a PS-b-PA coated 5CB microdroplet. Fig. 2 (a) shows the LC microdroplet showing a bipolar arrangement when SDS was not used (B) shows that when SDS is used, the LC microdroplets exhibit a radioactive array.
3 is a graph showing the diameter distribution of the LC microdroplets measured by a particle size analyzer.
Figure 4 (a) is a 5CB PSPA microdrop , (b) is an optical image (above) and POM image (below) showing an array of 5CB PAA microdroplets .
Figure 5 (a) is a 5CB PSPA-He microdrop , (b) is an optical image (top) and POM image (bottom) showing an array of 5CB PAA microdroplets .
6 (a) is a fluorescent image (top) and POM image (bottom) of Herceptin-
7 (a) shows the UV-Vis spectrum of Herceptin, Herceptin-
Fig. 8 shows fluorescence images (above) and POM images (lower) using different concentrations of Herceptin on the surface of a 5CB PSPA microdroplet , with 1 占 퐂 / ml for (a), 10 占 퐂 / ), And (d) 50 쨉 g / mL Herceptin.
(A) and (b) is a 5CB PSPA -Her micro-droplet and SK-BR3 optical image (left) and POM image showing the interaction of the cells (right), (c) and (d) of Figure 9 is 5CB PSPA (Left) and POM images (right) showing the interaction between Her microdroplets and KB cells, and (e) and (f) are optical images showing the interaction of 5CB PSPA-He microdroplets with FB cells Left) and POM image (right).
(A) and (b) is a 5CB PSPA -Her micro-droplet and SK-BR3 optical image (left) and POM image showing the interaction of the cells (right), (c) and (d) of Figure 10 is 5CB PSPA (Left) and POM images (right) showing the interaction between Her microdroplets and KB cells, and (e) and (f) are optical images showing the interaction of 5CB PSPA-He microdroplets with FB cells Left) and POM image (right).
11 (a) and (b) are optical images (left) and POM images (right) showing the interaction of 5CB PSPA- He micro droplets with SK-BR3 cells under 10% FBS, ) is 5CB PSPA -Her micro-droplet and the optical image showing the interaction of KB cells (left) and POM image (right) in the 10% FBS, (e) and (f) is 5CB PSPA -Her micro under 10% FBS The optical image (left) and the POM image (right) show the interaction of the droplet with the FB cells.
12 (a) and 12 (b) are optical images (left) and POM images (right) showing the interaction of 5CB PSPA- Her micro droplets with SK-BR3 cells under 10% plasma, ) it is 10% and under a plasma 5CB PSPA -Her micro-droplet and the optical image showing the interaction of KB cells (left) and POM image (right), (e) and (f) from 10% plasma under 5CB PSPA micro -Her The optical image (left) and the POM image (right) show the interaction of the droplet with the FB cells.
13 (a) and 13 (b) show optical images (left) showing the interaction of 5CB PSPA- Her microdroplets with FB cells (white), KB cells (white) and SK-BR3 cells (C) and (d) are optical images showing the interaction of 5CB PSPA- He micro-droplets with FB cells (blue), KB cells (blue) and SK-BR3 cells (white) under PBS (Left) and POM image (right).
14 (a) and 14 (b) show optical images showing the interaction of 5CB PSPA- Her micro droplets with FB cells (white), KB cells (white) and SK-BR3 cells (blue) under 10% FBS ) And POM image (right), (c) and (d) show the interaction of 5CB PSPA- Her microdroplets with FB cells (white), KB cells (white) and SK-BR3 cells (Left) and POM image (right).
(A) and (b) of FIG. 15 and SK-BR3 cells and 5CB PSPA -Her micro liquid cross-optical image showing the operation (left), and POM image (right) enemy, (c) and (d) SK- An optical image (left) and a POM image (right) showing the interaction of 5CB PAA- Her microdroplets with BR3 cells.
Hereinafter, the present invention will be described in more detail.
As described above, the conventional breast X-ray method for diagnosing breast cancer has a disadvantage in that the diagnosis rate is low due to a large amount of fibers in the case of dense breast, which is commonly found in Korean women. In particular, And because of the use of X-rays, there was a fatal problem that breast cancer was likely to occur during the diagnosis process.
Accordingly, the present invention provides a liquid crystal display device comprising: liquid crystals; Herceptin antibody block-fixed block copolymers; And a surfactant are developed to confirm the binding of breast cancer cells through the change of the orientation of the LC molecules, thereby providing a method for diagnosing breast cancer, thereby replacing the conventional breast cancer diagnosis method.
The terms used in the present invention are defined as follows.
As used herein, the term " biosensor " is an analytical device for use in detecting an analyte through interaction of biological components in a physiological environment.
In the present invention, the term " liquid crystal " refers to a material having physical properties between a liquid and a solid. Different types of liquid crystals have optical properties such as birefringence due to their different orientations when observed with a polarizing microscope Different properties appear. The liquid crystal comprises various liquid crystals known in the art and includes, for example, (i) a thermotropic liquid crystal in which a phase transition occurs according to a temperature change, (ii) a temperature and a concentration of liquid crystal molecules in a solvent Lyotropic liquid crystal in which phase transition occurs, and (iii) organic and inorganic molecules, and there is a metallotropic liquid crystal in which phase transition occurs depending on temperature and concentration as well as composition ratio of inorganic-organic molecules. The thermotropic liquid crystal includes Smectic liquid crystal, nematic liquid crystal and cholesteric liquid crystal, and the most common liquid crystal is nematic liquid crystal and can be easily commercially available. The liquid crystal usable in the present invention includes various kinds of known liquid crystals, preferably a thermotropic liquid crystal, more preferably a nematic liquid crystal.
The term " orientation " in the present invention means a physical property which depends on the molecular arrangement of the liquid crystal, for example, in the case of a nematic liquid crystal, it has a horizontal or vertical arrangement.
The term "SK-BR3" in the present invention is a human breast cancer cell line overexpressing the HER2 + (Neu / ErbB-2) gene product.
The term "fetal bovine serum (FBS)" in the present invention refers to a portion of blood that remains after natural coagulation of blood, followed by centrifugation to remove any remaining red blood cells.
The term "blood plasma" in the present invention is a pale yellow liquid component containing blood-removed proteins and serves as an extracellular matrix for blood cells in suspension.
Specifically, FIG. 1 is a schematic diagram showing that LC microdroplets according to a preferred embodiment of the present invention change orientation by antibody-antigen interaction, wherein poly (styrene-b-acrylic acid), which is an amphiphilic block copolymer, (PS-b-PA) and sodium dodecylsulfate (SDS) were used as surfactants to prepare LC microdroplets. Hydrophobic styrenic blocks hybridize with hydrophobic 4-cyano-4'-pentylbiphenyl (5CB; LC), while hydrophilic free acrylic acid blocks and Herceptin immobilized portions form the outer surface portion of the microdroplet. Herceptin was immobilized on the PA block on the 5CB microdrop surface directed to target breast cancer cells with HER2 + protein present in the cell membrane. Herceptin Introduction leads to a change in the orientation of the LC molecules from radioactive to bipolar, and these changes can be easily observed visually in a polarizing microscope.
The LC microdroplets for such breast cancer cell sensing of the present invention may comprise liquid crystals, block copolymers with Herceptin antibody immobilized, and surfactants. Preferably, the block copolymer may be a compound represented by the following formula (1).
[Chemical Formula 1]
In Formula 1, x is an integer of 50 to 75, and y is an integer of 25 to 35. The compound represented by Formula 1 may have an average molecular weight of 5,000 to 12,000, preferably 6,500 to 9,130.
Then, the LC micro-droplets The block copolymers of the present invention 1.50 × 10 -3 ~ 2.0 × 10 - 2 mmol, preferably from 2.0 × 10 -3 ~ 5.0 × 10 - 3 mmol, more preferably 2.0 × 10 -3 to 3.0 x 10 -3 mmol.
The liquid crystals present within the LC microdroplets of the present invention are 4-cyano-4'-pentylbiphenyl, 4-cyano-4'-hexylbiphenyl and 4-cyano- And biphenyl. Among them, 4-cyano-4'-pentylbiphenyl may be preferably used. And the micro LC droplets the liquid crystal of the present invention 1.0 × 10 -1 ~ 5.0 × 10 - 1 mmol, preferably from 1.0 × 10 -1 ~ 3.0 × 10 - 1 mmol, more preferably 1.0 × 10 -1 ~ 2.5 x 10 < -1 > mmol.
In addition, the LC microdroplets of the present invention can use the above surfactant as sodium dodecyl sulfate or dodecyltrimethylammonium. And the surfactant LC microdisplay LCD of the present invention is 5.0 × 10 -2 ~ 3.0 × 10 - 1 mmol, preferably from 1.0 × 10 -1 ~ 2.5 × 10 - 1 mmol, more preferably 1.5 × 10 -1 To 2.0 x 10 < -1 > mmol.
As can be seen from the diameter distribution graph of the LC microdroplets measured by the particle size analyzer of Fig. 3, the diameter of the LC microdroplets of the present invention may range from 2.5 탆 to 20 탆, preferably from 5 탆 to 17.5 탆, Preferably, the diameter may range from 7.5 mu m to 15 mu m.
The above-described various LC microdroplets of the present invention can be used to sense breast cancer cells. Specifically, it is possible to visually observe the change of the orientation of the LC molecules due to the binding of the HER2 + antigen of the breast cancer cell with the Herceptin antibody of the LC microdroplet to visually observe the breast cancer cells. In order to effectively sense breast cancer cells, it is desirable to sense the LC microdroplets in contact with breast cancer cells in a temperature range of 18 to 35 ° C because the liquid crystals exhibit a nematic state in a temperature range of 18 to 35 ° C to be. Since the pH may affect the liquid crystal alignment, the LC microdroplets are contacted with the breast cancer cells at a pH of 7.0 to 7.8, preferably at a pH of 7.2 to 7.6, more preferably at a pH of 7.35 to 7.45, It is good to do.
In addition, the breast cancer cell sensing can be performed in vitro. Such extracellular conditions may include other proteins such as FBS or plasma.
The biosensor can be manufactured using the LC microdroplets of the present invention as described above.
The present invention will now be described more specifically with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, but should be construed to facilitate understanding of the present invention.
5CB PSPA Microdroplet formation
The O / W system is used for the generation of microdroplet emulsions. In previous studies, surfactants were used for droplet stability. Surfactants are used to prevent adhesion between droplets by reducing the surface tension in the emulsion. PS-b-PA acts like a surfactant due to its amphiphilic nature. The PS block can be strongly immobilized to the incorporated 5CB by penetrating the LC droplet, and the hydrophilic PA block on the surface of the LC droplet can prevent adhesion between the LC droplets.
To prepare an LC microdroplet emulsion, 10 mg (2.50 x 10 -3 mmol) of PS-b-PA was dispersed in 10 mL of Dulbecco's phosphate buffered saline (DPBS) solution placed in a 30 mL reaction vial, 0.173 mmol) of SDS was also added, and the resulting mixture was stirred at 500 rpm for 1 hour. After properly mixing PS-b-PA and SDS, the LC microdroplet emulsion was prepared by dropwise addition of 50 mg of 5CB (0.2 mmol) dissolved in solution, resulting in a homogeneous monodisperse 5CB And stirred with a homogenizer at 19000 rpm for 1 minute to obtain SDS / PSPA microdroplets . The resulting mixture is left to stand for 5 minutes to stabilize the 5CB PSPA microdroplets and remove the supernatant ( containing extremely small 5CB PSPA microdroplets , unreacted SDS and block copolymer). Stable 5CB PSPA Wash the microdroplet twice with fresh DPBS solution, then dip into the DPBS solution for the next experiment.
Herceptin fluorescence
The labeling of Herceptin was carried out using a slightly modified method in the method reported by Khan et al. [ Sensors and Actuators B: Chemical , 2014, 202 , 516-522]. Briefly, Herceptin was incubated in a reaction glass bottle to obtain a 1 mg / mL solution containing a chemical binding agent, N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (EDC) (100 mg) and N-hydroxysuccinimide Of DPBS buffer (pH = 7.4) and placed for 0 to 1 hour to activate the carboxyl group of Herceptin. Then, 1 mg of
LC Micro-droplet Combination of Herceptin on the surface
After synthesis of 5CB SDS / PSPA , excess PS-b-PA and SDS solution were removed and the LC droplets were washed twice with fresh DPBS buffer. To immobilize Herceptin, the carboxyl group of the PA chain was activated with 0.4 M EDC and 0.1 M NHS for 1 hour. Activated 5CB SDSP / PSPA The droplet was placed in 10 mL of DPBS buffer containing 50 μg / mL Herceptin and incubated at room temperature for 12 hours to obtain a Herceptin-fixed 5CB droplet. The antibody-bound LC droplets (hereinafter referred to as 5CB PSPA-Her ) were then washed with pH 8.0 PBS buffer to remove excess Herceptin and activated carboxyl groups. Finally, LC microdroplets were placed in DPBS buffer for the following detection experiments.
Herceptin-binding To polyacrylic acid LC by Droplet Produce
To evaluate the effect of the polystyrene chain on the detection of SK-BR3 cells, LC microdroplets containing polyacrylic acid (homopolymer without PAA, PS) were prepared as follows.
To prepare an LC microdroplet emulsion, 100 mg (0.324 mmol) of dodecyltrimethylammonium bromide (DTAB) was dispersed in 10 mL of a phosphate buffer saline (DPBS) solution in a 30 mL reaction vial and the mixture was stirred for 1 hour at 500 rpm Respectively. (5CB droplet) emulsion was prepared by adding 50mg (0.2mmol) of 5CB dissolved in solution dropwise to the resulting mixture and the resulting mixture was homogenized using a homogenizer at 19000rpm for 1 minute to obtain a homogeneous monodispersed LC droplet Followed by stirring. The resulting mixture was allowed to settle for 5 minutes to stabilize the 5CB DTAB droplet, and then the supernatant (with very small size LC microdroplets, unreacted DTAB) was removed. Stable 5CB DTAB The droplets were washed twice with fresh DPBS solution for the next experiment and immersed in DPBS solution. Then, the microdroplets were immersed in a 0.5 wt% PAA solution to obtain PAA-coated 5CB DTAB (hereinafter referred to as 5CB DTAB / PAA droplets) by electrostatic interaction. Thereafter, the method for fixing Herceptin to the surface of the LC droplet (hereinafter referred to as 5CB PAA- Her droplet) is similar to the method described above.
LC Microdroplet Emulsion characteristic
The prepared LC microdroplet emulsion was found to have a uniform size distribution and the LC droplets were bipolar when using the block copolymer without SDS (Figure 2 (a)), whereas in the presence of sodium dodecyl sulfate at room temperature, (Fig. 2 (b)). Based on these results, we propose that SDS, due to its amphoteric nature, can penetrate through the PA block layer and concentrate on the surface of the LC droplets. This study clearly demonstrated that LC molecules were stable in a radioactive array within a size variation of less than 20 탆 of LC microdroplets when using 50 mg (0.173 mmol) of SDS and 20 mg of PS-b-PA.
Particle size and distribution can be ascertained through a particle size analyzer (Figure 3). The average diameter of the LC microdroplets is 10.5 mu m. The orientation of the microdroplets less than 1 mu m is not easy to detect in a polarization microscope used in the experiment. They were therefore removed by centrifugation.
5CB PSPA , 5CB PSPA -Her , 5CB PAA And 5CB PAA -Her Droplet Orientation
A 5CB droplet not bound to anything dispersed in the aqueous medium shows a bipolar arrangement as reported elsewhere. The radioactive arrangement is due to the adsorption of the hydrophobic tail of the surfactant to the 5CB molecule, regardless of the effect from the properties of the hydrophilic head group. Figure 4 shows the optical (left) and POM (right) images of 5CB SDS / PSPA and 5CB DTAB / PAA showing radioactive arrays. Based on these results, surfactants (SDS and DTAB), due to their amphipathic nature, can penetrate through the polymer electrolyte (PS-b-PA and PAA) layers and can be concentrated on the surface of the LC droplets have. Thus, the presence of a surfactant affects the LC arrangement in the droplet.
FIG. 5 shows the optical (left) and POM (right) images of 5CB PSPA- Her and 5CB PAA- Her showing radioactive sequences after fixing Herceptin to the activated carboxyl groups from PS-b-PA or PAA, respectively. As a result, it was confirmed that the chemical fixation of Herceptin did not affect the arrangement of the LC droplets, and both showed radioactive forms.
LC In a microdroplet Identification of Immobilized Herceptin
In order to confirm that Herceptin was immobilized on the PA chain of the block copolymer, a fluorescence microscope was used to observe the 5CB PSPA- Herd rhd droplet as shown in FIG. The droplet clearly showed a green sphere in the fluorescence image of 5CB PSPA- Herd rhd while the rest of the area appeared black, indicating that Herceptin was successfully immobilized on the PA chain and remained a radioactive array. Figure 7 shows the UV-Vis spectra of Herceptin-
Herceptin-
SK-BR3 cells, KB cells and FB Cell and 5CB PSPA -Her Microdroplet emulsion
In order to evaluate the sensitivity of Herceptin-binding LC microdroplet emulsion in the detection of SK-BR3 cells, 5CB PSPA- Her micro liquid droplets were added to SK-BR3 cells (human breast cancer cells), KB cancer cells (epithelial cancer cells) Cells were incubated at a cell concentration of 5000 cells / mL for 3 hours at 30 ° C and then the bright field and polarized (cross-polarity) images of the LC microdroplets were observed Was recorded according to the director profile of the LC molecule. (FIGS. 9A, 9C and 9E) of the 5CB PSPA- Her droplet in the presence of SK-BR3 cells, KB cells and FB cells were incubated with 5CB PSPA- Her micro liquid 9 (c)), FB cells (Fig. 9 (e)) and SK-BR3 cells (Fig. 9 (a)). In order to evaluate the effect of these cell interactions on the alignment state of the LC microdroplets , a polarizing microscope photograph shows 5CB PSPA- He micro-droplets interacting with SK-BR3 cells, KB cells and FB cells, respectively b), (d) and (f). The POM image of the 5CB PSPA- He micro-droplet in the presence of SK-BR3 cells (Figure 9 (b)) indicates that the LC droplet orientation is converted from radioactive to bipolar. The interaction between the KB cells (Fig. 9 (d)) and the FB cells (Fig. 9 (f)) and the 5CB PSPA- Her micro-droplet emulsion did not show any orientation change from radioactive to bipolar.
9 (c) and 9 (d)) and FB cells (Fig. 9 (e) and (f)) are effective in the interaction between SK- BR3 cells and Herceptin- (Fig. 9 (a), (b)), and can be used for detection of SK-BR3 in abnormal fluid PBS.
5CB PSPA -Her Microdroplet Emulsion and Long-term cultured SK-BR3 cells, KB cells and FB cell
To assess cytologic morphology effects on the detection of SK-BR3 cells, all cells were cultured for a long time until the cell membrane was flattened. Then, LC micro-droplets SK-BR3 cells in 30 ℃ in cancer cells and KB cells and FB 5000 cells / mL of cell concentration were incubated for 3 hours, and then I 5CB PSPA -Her micro-droplet bright field and polarized light ( Cross polarity) image was recorded according to the director profile of the LC molecules in the droplet as shown in FIG.
SK-BR3 cells, KB cells, and the presence of cells FB 5CB liquid PSPA -Her field image (Fig. 10 (a), (c) and (e)) enemy's name, as shown by a circle 5CB PSPA -Her micro liquid 10 (c), FB cells (Figure 10 (e)) and SK-BR3 cells (Figure 10 (a) to evaluate the impact on the enemy alignment, (b of the polarizing micrograph each SK-BR3 cells, KB cells and FB cells interact with 5CB PSPA -Her micro-droplet (Fig. 10), (d) and (f The POM image of the 5CB PSPA- He micro-droplet (Figure 10 (b)) in the presence of SK-BR3 cells indicates that the LC droplet orientation is converted from radioactive to bipolar. 10 (d)) and the FB cancer cells (Fig. 10 (f)) and 5CB PSPA- Her micro-droplets, No changes were seen.
10 (c) and 10 (d)) and FB cells (Fig. 10 (e) and (f)) are effective for the interaction between SK- BR3 cells and Herceptin- (Figs. 10 (a) and 10 (b)) in comparison with the interaction with the LC molecule, RTI ID = 0.0 > SK-BR3. ≪ / RTI >
FBS Or in human plasma 5CB PSPA -Her Microdroplet Emulsion and SK-BR3 cells, FB Cells and KB cells
In order to evaluate the sensitivity of Herceptin-binding LC microdroplet emulsion in the detection of SK-BR3 cells, 5CB PSPA- Her microdilutions were incubated with SK-BR3 cells, KB cancer cells and FB cells at a cell concentration of 5000 cells / For 3 hours and then the bright field and polarized images of the 5CB PSPA- He micro-droplet were recorded according to the director profile of the LC molecules in the droplet as shown in Fig. SK-BR3 cells, KB cells, and the cells present under the FB 5CB PSPA -Her droplet bright field image (Fig. 11 (a), (c) and (e)) of is as indicated by the circle 5CB PSPA -Her micro liquid And the interaction between SK-BR3 cells (FIG. 11 (a)) and KB cancer cells (FIG. 11 (c)) and fibroblasts (FIG. 11 (e)). To evaluate the effect of the interaction of these cells on the orientation state of LC microdroplets, a polarizing microscope photograph of LC microdroplets cultured with SK-BR3 cells, KB cells and FB cells (Fig. 11 (b), ) And (f)) were recorded respectively. The POM image of the LC microdroplets in the presence of SK-BR3 cells showed that the orientation of the LC droplets was changed from radioactive to polar, as indicated by the arrow in the subset of Figure 11 (b), while KB cancer cells 11 (d)) and FB cells (Fig. 11 (e)) did not show any orientation change from radioactive to bipolar.
These experiments show that the interaction of SK-BR3 cells with Herceptin-binding LC microdroplets is effective under FBS, and KB cancer cells (Fig. 11 (c), (d)) and fibroblasts (Fig. 11 (a), (b)), and can be used for the detection of SK-BR3 cells.
As shown in Fig. 12, similar results were obtained from 10% plasma. Therefore, there was no effect of 10% FBS or 10% plasma in this experiment.
Selective detection of SK-BR3 by Herceptin-coupled LC droplets
The bright field image of FIG. 13 showed LC microdroplet contact with stained SK-BR3 cells and unstained control cells. The interaction of the LC-microdroplet with the SK-BR3 cells (blue) induced an array change from radioactive to bipolar as shown in the two LC microdroplets contacted with the blue stained cells, whereas the control cells (white) The arrangement of the LC droplets was not induced as shown in the subset of Figs. 13 (a) and 13 (b). These studies clearly showed that Herceptin-binding LC microdroplets were selective for effective interaction with SK-BR3 cells in the presence of KB cells or FB cells. On the one hand, to avoid the effect of stained methylene blue, a similar experiment was performed using stained control cells and unstained SK-BR3 under the same conditions. As a result, similar results were obtained as shown in Figs. 13 (c) and (d).
These results suggest that Herceptin-coupled LC microdropic emulsion can detect SK-BR3 cells in the presence of KB cancer cells and other control cells, and that the selectivity for SK-BR3 cells is affected by the presence of KB cancer cells and other control cells Of the population.
The same results were also observed in the 10% FBS solution (FIGS. 14A and 14B) and the 10% plasma solution (FIGS. 14C and 14D). Therefore, it can be said that there is no effect of other proteins in the co-culture experiment. The selectivity of the LC microdroplet emulsion for the detection of SK-BR3 cells has also been demonstrated in the presence of other control cancer cells, such as human prostate cancer cells, HER-2 low-expressing breast cancer cells and colon cancer cells, And that the selectivity shown in the electrochemical biosensor is the same.
SK-BR3 cells and 5CB PAA -Her Microdroplet emulsion
To evaluate the effect of the styrene segment in the detection of SK-BR3 cells, LC microdroplets containing PA (polyacrylic acid, homopolymer without PS) were combined with Herceptin, and then 5000 cells / mL of SK-BR3 cells at 30 < 0 > C for 3 hours at the cell concentration. Bright field and polarized (cross-polar) images of the LC microdroplets were recorded and were as shown in Figure 15 according to the director profile of the LC molecules in the droplets. The bright field image of the LC microdroplets in the presence of SK-BR3 cells was analyzed with 5CB PSPA - Her microdrop (Figure 15 (a)) and 5CB PAA - Her microdrop (Figure 15 SK-BR3 cells. However, 5CB PSPA -Her micro liquid Despite indicate the orientation change of the polarity on the radioactive, as shown in a small subset of Fig. 15 (b) by the arrow, 5CB PAA-Her micro-droplet (Herceptin - a micro-LC in combination with a fixed PAA Droplet) did not show the orientation change from radioactive to bipolar as indicated by the arrow in the subset of (d) of Fig.
Claims (11)
A block copolymer represented by the following formula (1) immobilized with a Herceptin antibody labeled with rhodamine 6G
[Chemical Formula 1]
In Formula 1, x is 50 to 75 and y is 25 to 35; And
A surfactant selected from the group consisting of sodium dodecyl sulfate and dodecyltrimethylammonium; Lt; RTI ID = 0.0 > LC < / RTI > microdroplets for breast cancer cell sensing,
The LC microdroplet for the detection of breast cancer cells having a particle diameter distribution of the LC microdroplets ranging from 2.5 mu m to 20 mu m and an average particle diameter of 10.5 mu m.
Wherein the liquid crystal is present inside the microdroplet, the hydrophobic portion of the block copolymer is present inside the microdrop, and the Herceptin antibody is exposed to the outside of the microdroplet in combination with the hydrophilic portion of the block copolymer. LC microdroplets for breast cancer cell sensing.
Poly (styrene-b-acrylic acid) to which Herceptin 6G-labeled Herceptin antibody is immobilized; And
As an LC microdroplet for breast cancer cell sensing comprising sodium dodecyl sulfate or dodecyltrimethylammonium,
The LC microdroplet for the detection of breast cancer cells having a particle diameter distribution of the LC microdroplets ranging from 2.5 mu m to 20 mu m and an average particle diameter of 10.5 mu m.
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| Detection of Antigen molecules using Antibody-anchored liquid crystal microdroplets (한국고분자학회 학술대회 발표논문집, (2015.04.), 40권 1호.) |
| Folate Ligand Anchored Liquid Crystal Microdroplets Emulsion for in Vitro Detection of KB Cancer Cells (Langmuir, (2014.08.), 30, pp 10668-10677.) |
| Herceptin-conjugated liquid crystal microdroplets for the recognition of breast cancer cells (경북대학교 석사학위논문, (2015.08.), pp 1-70.) |
| Liquid Crystal Droplet-Based Amplification of Microvesicles that are shed by Mammalian cells (Analyst, (2014), 139(10), pp 2386-2396.) |
| 구강 상피암세포를 인식하는 액정 microdroplet의 제조 (경북대학교 석사학위논문, (2014.02.), pp 1-61.) |
| 유방암 진단을 위한 CdSe/ZnS 양자점-허셉틴 합성물의 제조 및 특성 평가 (경북대학교 석사학위논문, (2012), pp 1-67.) |
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