KR20170000142A

KR20170000142A - Methods for Detecting Circulating Tumor Cells and Stem-like Circulating Tumor Cells Using Surface-Enhanced Raman Scattering and Systems Using Thereof

Info

Publication number: KR20170000142A
Application number: KR1020150088958A
Authority: KR
Inventors: 최정우; 조현열; 칼레드 후세인
Original assignee: 서강대학교산학협력단
Priority date: 2015-06-23
Filing date: 2015-06-23
Publication date: 2017-01-02
Also published as: KR101692052B1

Abstract

The present invention relates to a method of detecting circulating tumor stem-like cells and circulating tumor cells using a surface-enhanced Raman scattering (SERS) nanotag; and a system using the same. According to the present invention, it has been confirmed that circulating tumor stem-like cells and circulating tumor cells are able to effectively be detected when a blood sample labeled with the SERS nanotag is applied to a microfluid chip and spectral analysis is performed by Raman spectroscopy. As such, the method and system of the present invention is able to usefully be used in the field of cancer therapy in which detection of circulating tumor stem-like cells and circulating tumor cells are required.

Description

Methods for Detecting Circulating Tumor Cells and Circulating Tumor Stem-like Cells Using a Surface Enhanced Raman Scattering Method (SERS) and Systems Using the Steroid-like Circulating Tumor Cells Using Surface-Enhanced Raman Scattering Using Thereof}

The present invention relates to a method for detecting circulating tumor cells and circulating tumor stem-like cells using a surface enhanced Raman scattering method (SERS) and a system using the same.

Recent studies have shown that cancer is not a homogeneous population of rapidly proliferating cells, but a heterogeneous population of cells with a constant degree of proliferation and differentiation [1]. In some malignant tumors, it is known that cancer develops and is maintained in a small population of cells called stem-like cancer cells (SCCs) [2]. Tumor stem-like cells, like normal stem cells, are self-replicating and contribute to the formation of giant tumors by creating various cell populations that multiply and differentiate. Tumor stem cells resemble stem cells where the stem cells are usually located. In addition, tumor stem-like cells are often in a dormant state, so they may play an important role in cancer recurrence and metastasis after chemotherapy because they may not be affected by anticancer drugs targeting rapidly proliferating cells [3] . Therefore, finding and characterizing tumor stem - like cells in cancer patients is very important in the treatment of cancer patients.

Tumor cells enter the bloodstream and circulate along the circulatory system, which is called circulating tumor cells (CTCs). Tumor-like cells also enter the circulatory system through the epithelial-mesenchymal transition (EMT) pathway, which is called stem-like CTC (SCTC).

Previously, CTC and CTTC were detected using immunofluorescence [7]. This method has the disadvantage that the fluorescent material photobleaches. In addition, when a plurality of fluorescent substances are used in excess to detect a large number of cell surface markers, fluorescence spectra overlap each other, making it difficult to distinguish signals for each fluorescent substance [8]. To overcome these limitations, it is required to develop a cell detection method capable of simultaneously detecting several surface markers and having high sensitivity, specificity and reproducibility.

The gold nano probe is considered to be a good alternative to the cell detection method because it has no cytotoxicity, is water-soluble, has long-term stability and excellent biocompatibility [9-12]. This kind of nanoparticles showed the possibility of overcoming the low sensitivity which was a problem of the conventional Raman spectroscopy. Therefore, the surface-enhanced Raman scattering (SERS) As shown in Fig. In addition, SERS nanotag has the advantages of narrow spectral band, high specificity, low light discoloration, and excellent multiplexing performance [15-21]. Therefore, if we can develop a method to detect circulating tumor stem-like cells and circulating tumor cells using SERS nanotechnology, it will be very helpful in cancer treatment.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to find a method for detecting circulating tumor stem-like and circulating tumor cells using SERS nanotact. As a result, the present inventors confirmed that, when a blood sample labeled with SERS nanotact is applied to a microfluidic chip and spectra are analyzed by Raman spectroscopy, it is possible to effectively detect circulating tumor stem-like cells and circulating tumor cells, Thereby completing the present invention.

It is an object of the present invention to provide a method for detecting circulating tumor stem-like cells and circulating tumor cells using SERS nanotaxes and microfluidic chips.

It is another object of the present invention to provide a system capable of detecting circulating tumor stem-like cells and circulating tumor cells using the above method.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention there is provided a method of detecting circulating tumor cells (CTC) or stem-like circulating tumor cells (SCTC) comprising the steps of:

(a) contacting a blood sample separated from a living body with a SERS nanotag (Surface Enhanced Raman Scattering Nanotag); The SERS nanotact may be (i) a nanoparticle; (ii) a Raman reporter bonded to the surface of the nanoparticles; (iii) a tumor cell surface antigen-specific antibody bound to the surface of said nanoparticle; And (iv) a non-covalent binding partner bonded to the surface of said nanoparticle;

(b) injecting a sample solution in contact with the SERS nanotubes in a channel on the surface of a microfluidic chip;

(c) obtaining a SERS spectrum from the microfluidic chip; And

(d) analyzing the SERS spectrum to detect circulating tumor cells or circulating tumor stem-like cells.

Each step will be described in detail as follows.

(a) to a blood sample separated from a living body SERS Nanotact ( Surface Enhanced Raman Scattering Nanotag ),

In the present invention, " SERS nanotechnology " means a nanoprobe for analyzing the presence and the quantitative level of a specific biomaterial using a spectrum by SERS (Surface Enhanced Raman Scattering). Specifically, SERS nano-tack is composed of (i) nanoparticles; (ii) a Raman reporter bonded to the surface of the nanoparticles; (iii) a tumor cell surface antigen-specific antibody bound to the surface of said nanoparticle; And (iv) a non-covalent binding partner bonded to the surface of the nanoparticle.

As the nanoparticles, metals such as gold, silver, and copper may be used. According to a specific example of the present invention, gold (Au) is used, but it is not limited thereto.

The Raman reporter refers to a substance that emits a strong signal in a specific wavelength band of Raman spectrum, and can be a substance such as an aromatic compound having a benzene ring or graphene. According to a specific example of the present invention, thiophenol (TP), nile blue A (NBA), 1-naphthalenethiol (NPT), 4-mercaptopyridine ) Or 2-quinolinethiol (QTH) was used as a Raman reporter, but the present invention is not limited thereto.

A tumor cell surface antigen-specific antibody bound to the surface of the nanoparticle means an antibody that specifically binds to a protein expressed on the surface of tumor cells. Depending on the type of tumor to be detected, a protein having a high expression level can be selected in each tumor cell. Specifically, when mammary tumor cells are to be detected, MUC1, EpCAM, EGFR, HER2 or CD-133 can be selected. On the other hand, a linker is used to bind the antibody to the nanoparticles. The linker serves to link the antibody to the nanoparticle by covalent bonding.

The non-covalent binding partner is for allowing the nanoparticles to be bonded to the microfluidic chip when the nanoparticles pass through the microfluidic chip. Depending on the type of the material coated on the microfluidic chip, the non- Select a material that can bind to the microfluidic chip. According to a specific example of the present invention, the microfluidic chip was coated with streptavidin, and biotin was used as a noncovalent binding partner thereto.

In the present invention, " contacting " means placing a blood sample separated from a living body and a SERS nanotact into a single container so that a reaction can occur between the sample and the SERS nanotact. Specifically, tumor cells and the like are marked by SERS nanotact by allowing specific binding between an antibody bound to the surface of SERS nanotact and a protein antigen expressed on the surface of tumor cells in the sample.

(b) SERS Nanotec and The contacted sample solution Microfluidic chip ( microfluidic chip ) Surface channel ( channel )

In step (a), the sample solution labeled by the SESS nanotube is injected into the channel of the microfluidic chip according to a general method of analyzing the sample using the microfluidic chip.

(c) From a microfluidic chip SERS Acquiring the spectrum

In the step (b), when the sample solution injected into the channel of the microfluidic chip passes through the channel, the cells labeled with the SERS nanotact in the sample solution are captured on the surface of the microfluidic chip. As described above in step (a), non-covalent bonding occurs between the non-covalent bonding partner of SERS nanotact and the substance coated on the surface of the microfluidic chip. Thus, using a device capable of analyzing the SERS spectrum, the SERS spectrum can be obtained from the captured cells on the surface of the microfluidic chip. At this time, if the surface of the microfluidic chip is segmented and analyzed, the SERS spectrum can be obtained from each single captured cell.

(d) SERS Spectrum was analyzed to determine whether the tumor was a circulating tumor or a circulating tumor Stem-like cells Detecting step

Analysis of the spectrum of each cell obtained in the step (c) can analyze the expression level of a cell surface protein that specifically binds to the antibody according to the type of the antibody bound to the SERS nanotact. By comparing the expression level of the cell surface protein thus analyzed with the level of surface protein expression of the known circulating tumor cells or stem-like stem cells, it is possible to determine whether the analyzed cells are a circulating tumor cell, a circulating tumor stem- Of the cells. According to a specific example of the present invention, SERS nanotags were prepared using antibodies against MUC1, EpCAM, EGFR, HER2 or CD-133, and SERS spectra were analyzed using the antibodies. As a result, Like cells could be distinguished and detected. More specifically, when the signal due to the expression of CD-133 appears to be 2 to 6 times higher than the signal according to the expression of MUC1, it can be judged to be stem-like cells of the mammary gland.

According to another aspect of the present invention there is provided a system for detecting circulating tumor cells (CTC) or stem-like circulating tumor cells (SCTC) comprising:

(a) Surface Enhanced Raman Scattering Nanotag; The SERS nanotact may be (i) a nanoparticle; (ii) a Raman reporter bonded to the surface of the nanoparticles; (iii) a tumor cell surface antigen-specific antibody bound to the surface of said nanoparticle; And (iv) a non-covalent binding partner bonded to the surface of said nanoparticle;

(b) Microfluidic chip.

The system of the present invention is a system for implementing the above-described method of the present invention, and detailed description thereof has been described in the above method, so that description thereof is omitted in order to avoid excessive redundancy.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for detecting circulating tumor stem-like cells or circulating tumor cells using SERS nanotaxes and microfluidic chips, and a system using the same.

(b) Using the method of the present invention, it is possible to rapidly and accurately detect circulating tumor stem-like cells and circulating tumor cells, and it is possible to continuously cultivate circulating tumor stem-like cells even after the detection of circulating tumor stem- Further studies are possible to characterize circulating tumor stem-like cells.

Figure 1 is a schematic diagram showing the conjugation of five different SERS nanotubes (SNTs).
FIG. 2 is a table summarizing a transmission electron microscope photograph, an ultraviolet-visible spectrum, and a dynamic light scattering size data confirming the conjugation of SERS nanotact using a transmission electron microscope, an ultraviolet-visible light spectroscopy and a dynamic light scattering method.
FIG. 3 is a chart analyzing the SERS spectrum of each SNT. (a) is a combination of 4-mercaptopyridine (MPy) and anti-EpCAM antibody, b) is a combination of 1-naphthalenethiol (NPT) and anti- HER2 antibody, c) is a combination of thiophenol Is a combination of Nile blue A (NBA) and anti-HER2, and e) is a combination of 2-quinolinethiol (QTH) and anti-CD-45.
FIG. 4 is a photograph showing breast cancer cells and breast cancer stem-like cells (red arrows) captured in a microfluidic chip.
FIG. 5 is a chart for capturing efficiency according to each cell type.
Figure 6 is a SERS mapping result and chart analyzing expression levels of surface markers based on SERS map imaging of MDA-MB-231 cells.
Figure 7 is a SERS mapping result and a plot of the expression levels of surface markers based on SERS map imaging of SK-BR-3 cells.
Figure 8 is a SERS mapping result and chart analyzing expression levels of surface markers based on SERS map imaging of MCF-7 cells.
Figure 9 is a SERS mapping result and chart analyzing expression levels of surface markers based on SERS map imaging of breast SCC cells.
FIG. 10 is a photograph showing that the breast SCC was separated from the microfluidic chip and then cultured for 48 hours.
Figure 11 is a photograph showing the morphology of undifferentiated (a) or differentiated (b) breast SCCs.
12 is a SERS mapping result and chart analyzing expression levels of surface markers based on SERS map imaging of undifferentiated breast SCC.
Figure 13 is a SERS mapping result and chart analyzing expression levels of surface markers based on SERS map imaging of differentiated breast SCC.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments.

Example

Materials and Methods

Experimental material

The gold colloid was purchased from BB International-UK and was purchased from Thiophenol (TP), nile blue A (NBA), 1-naphthalenethiol (NPT), 4-mercaptopyridine (4-mercaptopyridine, MPy), 2-quinolinethiol (QTH), ethyl dimethylaminopropyl carbodiimide (EDC), N-hydroxy sulfosuccinimide , 3-aminopropyltriethoxysilane, glutaraldehyde, streptavidin, formaldehyde, phosphate buffered saline (PBS, pH 7.4, 10 mM) were purchased from Sigma Aldrich. Mouse monoclonal antibodies specific for MUC1, EpCAM, EGFR, HER2 and CD-133 were purchased from R & D systems (USA), RPMI-1640 medium was Fresh media TM (Korea), human breast cancer stem cell culture undifferentiation media was Celprogen (Trypsin EDTA solution, 1X), fetal bovine serum (FBS), penicillin-streptomycin (0.83% saline solution to which 10,000 IU / ml penicillin sodium and 10,000 mg / ml streptomycin were added) Was made by Welgene Inc. (Korea). HS-PEG-COOH (MW 5 kDa), which is a heterofunctional linker for mPEG-SH (MW 5 kDa), Creative PEG Works, NH2-DNA and biotin- DNA for Bioneer Alu I was purchased from Sigma Aldrich, Invitrogen (USA), Streptavidin conjugated quantum dots (525, 545, 565, 625, 705 nm), BD VacutainerCPT ™ cell preparation tubes from BD Franklin Lakes Ficoll-Paque plus was purchased from GE Healthcare Inc (USA). All other chemicals used analytical grade products. All solutions were prepared with purified distilled water under a specific resistance of 418 M? Cm using a Milli-Q purification system (Branstead).

Fabrication of microfluidic chip

A microfluidic chip for capturing suspended cells was prepared. Two channels with a diameter of 5 ㎛ and one channel with a diameter of 50 ㎛ were made to capture the suspended cells on the microfluidic chip surface. A silicon-on-glass (SOG) technology is used to attach a silicon wafer to a glass wafer using an anodic method to create a precise pillar array structure on the chip surface. A microfluidic chip ). The silicon layer of the chip was lapped and chemical mechanical polishing was applied to set the filter height to 50 μm. Subsequently, a photoresist (AZ 4330, Clariant Corp., Muttenz, Switzerland) was patterned and subjected to deep reactive-ion etching for 15 minutes. A laminated glass wafer was patterned using a dry film photoresist (Ordyl BF 410, Tokyo Ohka Kogyo, Kawasaki, Japan) to form a fluidic path and sandblasted (sand blasting etching to create in / outlet holes. Finally, the cover glass wafer was connected to the patterned wafer using anodic bonding.

Streptavidin was coated on the surface of the prepared microfluid chip glass. The microfluidic chip was washed with ethanol and distilled water and dried overnight at 60 ° C. The dried microfluidic chip was placed in a plasma chamber (Convance-MP, Femto Science, Korea) and exposed to an oxygen plasma for 5 minutes to activate the surface silanol. Next, the microfluidic chip was immersed in a solution of 3-aminopropyltriethoxysilane (10%), washed with distilled water, and spun at 110 ° C for 1 hour. The silanized microfluidic chip was immersed in 100 mM phosphate buffer (pH 8.0) containing 2% glutaraldehyde for 1 hour, then washed with PBS and dried with nitrogen. 10 μM of streptavidin was added to the microfluidic chip to react with immobilized glutaraldehyde at room temperature for 1 hour. Subsequently, in order to block the place where the streptavidin protein was not attached on the surface of the cell chip modified with glutaraldehyde, the microfluid chip was washed with PBS, and then 1% bovine serum albumin dissolved in PBS Was treated for 1 hour. Finally, the microfluidic chips were washed with PBS solution and then dried with nitrogen.

SERS Of Nanotech (SERS NanoTag, SNT) making

SNTs with five different combinations capable of detecting surface markers of tumor cells were constructed. Figure 1 shows a step-by-step showing the conjugation process of a gold nanoparticle (AuNP) / Raman reporter / PEG / antibody / DNA conjugate. First, to prepare an activated SERS probe, a 1-5 μM Raman Reporter solution prepared in a gold colloid rapidly mixed was added dropwise to make the volume ratio of the reporter solution: gold colloid 1: 6. Raman reporter (TP, NBA, NPT, MPy, QTH) solution was prepared by dissolving each Raman reporter in ethanol to concentration.

Different concentrations of 1-5 μM were used for each Raman reporter to match the spectral peak intensities of each SNT similarly. After 10 minutes, 10 [mu] M of thiol-PEG solution was added dropwise to the gold colloid labeled with Raman reporter. The ratio of PEG-SH molecules per gold particle labeled with 60 nm Raman Reporter was at least 30,000 so as to maintain a stable structure under various conditions and to minimize particle aggregation. To the gold colloid solution labeled with 3 ml of Rahman reporter was added 1 μM of heterogeneous functional linker HS-PEG-COOH 293 μl drop by drop. After mixing for 15 minutes, 1.6 ml of PEG-SH (10 mM) was again treated to fill the uncovered portion with the heterogeneous functional linker to create a shielded and stable particle surface. The gold nanoparticles were purified through three centrifugation (1,000 g) and PBS suspension process.

The carboxyl group (-COOH group) on the particle surface was activated for covalent conjugation. To this end, 5 ml of freshly prepared ethyldiminopropyl carbodiimide (EDC) solution (40 mg / ml) and 5 ml of sulfo-NHS (110 mg / ml) Strongly mixed for 15 minutes. Excess EDC and sulfo-NHS were removed by centrifugation three times (1,000 g), and gold nanoparticles were suspended in PBS. Mouse in gold nano-particles having an activated carboxyl group by the addition of monoclonal antibodies (11.2 nmol) and _{NH 2 -dsDNA-biotin (20 nmol} ) was reacted for 2 hours at 25 ℃. Mouse monoclonal antibodies specific for MUC1, EpCAM, EGFR, HER2 and CD-133 were used according to 5 SNTs. After the reaction mixture was stored at 4 ° C overnight, excess antibody and DNA were removed by centrifugation three times (1,000 g), and then suspended in PBS. Through this process, we were able to produce five conjugated SERS nanotacts (SNTs) with different combinations:

The combination of 4-mercaptopyridine (MPy) and anti-EpCAM antibody, 1-naphthalenethiol (NPT) and anti-EGFR antibody combination, combination of Thiophenol (TP) and anti-MUC1, Nile blue A (NBA) Combination and combination of 2-quinolinethiol (QTH) and anti-CD-133.

Fully activated SERS nanotubes (SNTs) can be detected by transmission electron microscopy (TEM), UV-vis spectroscopy, dynamic light scattering, surface enhanced Raman scattering (SERS) And analyzed its characteristics. The ultraviolet-visible spectrum was measured using a Jasco V-530 UV / VIS spectrometer. Transmission electron micrographs were taken using a JEOL transmission electron microscope (JEM1010) with an accelerating voltage of 80 kV.

Cell culture

Three types of breast cancer cell lines (MCF-7, MDA-MB-231 and SK-BR-3) and one type of breast stem-like cancer cell (SCC) were purchased from ATCC . Breast cancer cells were cultured at 37 ° C in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic (penicillin-streptomycin). Breast cancer stem cell lines were cultured in human breast cancer stem cell undifferentiation medium at 5% CO ₂ . When confluence of 80% was observed, 1 × 10 ⁵ cells / ㎖ of breast cancer cells and 1 × 10 ⁶ cells / ㎖ of breast stem cell lines were cultured and cultured for 2-3 days. Then, cells were detached from the culture dish using trypsin, and then washed twice with PBS to remove trypsin. To block nonspecific binding of antibody-conjugated gold nanoparticles to cells, the cells were suspended in the culture medium and then 1.5 ml of 1% bovine serum albumin was added and incubated for 1 hour.

Preparation of test samples

Because of the difficulty in obtaining blood samples from breast cancer patients, it is possible to use breast blood-stem cells (breast CTCs) or breast stem-like CTCs (breast cells) SCTC). First, a blood sample of a normal person was prepared by the following method: A blood sample (7.5-15 ml) of a normal person was obtained from Sogang University Medical Center (Sogang University, Seoul, Korea) Has been approved by the Institutional Review Board (IRB). Blood samples were collected using a BD VacutainerCPT cell preparation tube (BD Franklin Lakes, NJ) containing sodium heparin and polyester gel. After light mixing, fresh blood was diluted with PBS twice. 6 ml of density gradient reagent Ficoll-Paque plus (GE Healthcare Inc.) was added to the centrifuge tube, and 8 ml of diluted blood was added without mixing with Ficoll-Paque plus. Then, 30 minutes at room temperature (400 g). After centrifugation, the plasma layer was removed, and a low density pneumocyte layer containing lymphocytes and monocytes was collected, leaving the phycolate and erythrocyte sediment in the tube. The collected cells were transferred to a new tube, washed with PBS, and fixed with 3.7% formaldehyde for 10 minutes. Finally, cells were washed with PBS and counted and stored at 4 ° C for subsequent experiments.

The thus prepared leukocyte cells were suspended and mixed with a breast cancer cell line (MCF-7, SK-BR-3, MDA-MB-231) or a breast cancer stem- A test sample similar to breast CTC (breast CTC) or breast stem-like CTC (breast SCTC) could be prepared.

Conjugated SERS Nanotec The label of the used cell ( labeling )

Cells in the test samples prepared by the above method were labeled using conjugated SERS nanotags. 10 μM of conjugated SERS nanotact was added to a test sample containing 100 breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231) or breast cancer stem- Cells were labeled by continued treatment for 30 minutes at room temperature. PBS to remove unlabeled conjugated SERS nanotags and then suspended in 2 ml of the culture.

Cell surface Marker For analysis SERS Mapping ( mapping )

For the SERS mapping, the test sample labeled with the above method was injected into the channel of the microfluidic chip fabricated by the above method at a rate of 10 μl / min and treated at room temperature for 1 hour to allow the cells to adhere to the chip surface. Cell chips were washed with culture medium to remove debris or unattached cells.

For SERS mapping, NTEGRA spectra (AFM-Raman spectrometer, NT-MDT, Russia) equipped with a liquid nitrogen cooled CCD detector and an optical inverted microscope (Olympus IX71) were used. The SERS mapping was performed by dividing the area of the chip to be analyzed, and the SERS results were recorded for each cell. The SERS mapping was recorded using a 785 nm NIR wavelength laser with a laser power of 3 mW on the sample plane. SERS results were analyzed using Nova software.

Cell surface Marker Fluorescent labeling and fluorescence microscopy analysis

Cell surface markers were labeled with quantum dots (QD) for fluorescence microscopy imaging. Different surface markers (EpCAM, MCU1, EGFR, HER2, CD-133) were labeled with streptavidin-conjugated quantum dots with emission wavelengths of 525, 545, 565, 625 and 705 nm, respectively.

For this purpose, biotinylated antibody specific to each cell surface marker was used as a medium. Antibodies and quantum dots were mixed at a molecular ratio of 2.5: 1 to prepare antibody-quantum dot conjugates and then incubated for 2 hours at room temperature and under dark conditions. The antibody-quantum dot conjugate was purified by centrifugation (3,000 g, 10 min) and then suspended in PBS.

(MCF-7, SK-BR-3, MDA-MB-231, breast SCC) with 1 pM antibody-QD complex conjugate prepared by the above method and then mixed continuously at room temperature for 30 minutes Cells were labeled. Cells were washed three times with PBS and fluorescence signals were measured.

The cells labeled with Qdots were transferred to a 96-well plate and observed under a fluorescence microscope. Fluorescence images were taken at 400x magnification using a Nikon ECLIPSE-Ti microscope and analyzed using NIS-Elements-BR-3.2 software.

Experiment result

Conjugated SERS Nanotact (SNT) Feature Analysis

Five SNTs were fabricated with five different combinations. SNT was prepared using 60 nm sized gold nanoparticles and Raman reporter, heterofunctional linker SH-PEG-COOH, antibody and biotinylated dsDNA (biotinylated dsDNA) (Fig. 1). FIG. 2 (a) shows a transmission electron micrograph of gold nanoparticles (AuNP), Raman reporter labeled AuNP and PEG-antibody / dsDNA conjugated AuNP that were untreated. The size of the center particle of the gold colloid was 60 nm, and the PEG coating was observed as a thin white layer in the transmission electron microscope photograph.

Localized Surface Plasmon Absorbance of SNT was analyzed. UV / Vis spectroscopy was used. Fig. 2 (b) shows the ultraviolet-visible spectrum of AuNP, Raman reporter labeled AuNP, PEG-antibody / dsDNA conjugated AuNP, which were untreated. The spectrum of pure AuNP is known to exhibit maximum absorbance at 530 nm by plasmon resonance [22]. The Raman reporter labeled AuNP showed a slightly reduced maximum absorbance peak, which is presumably the result of a slight agglomeration of the gold nanoparticles by the Raman reporter. The maximum absorbance peak of PEG-antibody / dsDNA conjugated AuNP was further reduced and a red shift of about 6 nm was observed. This phenomenon is presumed to be caused by a decrease in AuNP concentration in the PEG coating and washing step.

Dynamic light scattering (DLS) of SNT was analyzed. FIG. 2 (c) shows the results of analysis of the dynamic light scattering size of AuNP, Raman reporter labeled AuNP, PEG-antibody / dsDNA conjugated AuNP, which were not treated. When the central particle size was 60 nm, the size increased to about 5 nm on average after Raman reporter labeling, and the average size increased about 12 nm after PEG reaction.

SNT of SERS Spectrum analysis

3 shows the result of analyzing the SERS spectrum of the SNT. When the antibody was not conjugated and only Raman reporter molecules were present, the SERS signal was stronger than the antibody conjugated SNT. It is known that the SERS signal intensity of Raman reporter gradually decreases as the coating layer thickness increases due to scattering shielding effects [23], but since the antibody conjugated SNT still shows a strong SERS signal intensity, Of SERS imaging studies.

SERS spectra of 1 SNT-thiophenol (thiophenol, TP) of the 1575 cm ^- to the mode in which the a1 thiophenol molecule naetneunde displayed a single peak (Fig. 3 c), from ¹ [24]. In the case of 1-naphthalenethiol (NPT) bound to AuNP of SNT-2, a strong band was observed at 1381 cm ^-1 (FIG. 3 b) stretching) [25]. Nile blue A (NBA) of SNT-32 exhibited a strong peak at 1492 cm ^-1 (FIG. 3 d), consistent with the aromatic ring stretching phenomenon [26]. The SERS spectrum of 2-quinolinethiol (QTH) of SNT-4 showed a strong peak at 1369 cm ^-1 (Fig. 3e), indicating that the υ (CC) aromatic ring vibration ) vibration [27]. 4-mercaptopyridine (MPy) of SNT-5 showed a single peak at 1096 cm ^-1 (FIG. 3 a), corresponding to the aromatic ring vibration mode [ 28].

Based on these results, the 1575, 1381, 1492, 1096 and 1369 cm ^-1 Raman bands of SNT-1, SNT-2, SNT-3, SNT-4 and SNT-5 were assigned to MUC1, EGFR, HER2, EpCAM , And a Raman band for detecting the expression level of CD-133.

Microfluidic chip Cell Capture Efficiency Analysis

The cell capture efficiency of the microfluidic chip fabricated according to the above experimental method was analyzed. Each 100 breast cancer cell lines or breast cancer stem cell lines were labeled with SNT and then mixed with 500 μl of PBS and flowed into the channel on the chip. Incubation was carried out for 30 minutes so as to cause a reaction between streptavidin and SNT biotin on the chip surface. After washing, 5 ml of PBS was flowed in the opposite direction. After washing, the number of remaining cells was counted to calculate the capturing efficiency. The mean and error bars after repeating on three different chips were calculated, and the average value was calculated as a percentage and expressed as the capture efficiency (FIG. 5).

Of the three breast cancer cell lines, SK-BR-3 cells showed high capturing efficiency because of the high expression levels of most cell surface markers (EpCAM, EGFR, HER2). MDA-MB-231 cells expressing a relatively low amount of surface marker also showed a high capture efficiency of 89%. This is probably because the diameter of MDA-MB-231 cells is larger than that of SK-BR-3 cells, and therefore the chance of contact with streptavidin-coated columns is increased. The capture efficiency of MCF-7 and breast cancer-like stem cells was slightly lower, probably due to the smaller size of the cells.

The capture efficiencies of SK-BR-3, MDA-MB-231, MCF-7 and breast cancer-like stem cells were 91%, 90%, 84% and 75%, respectively.

Isolation of Circulating Tumor Cells ( CTC ) or Circulating Tumor Stem Cells (SCTC) and S ERS Characterization of cell surface markers using mapping

In accordance with the above-described experimental method, normal white blood cells and breast cancer cell lines were mixed with each other to prepare a test sample made similar to a breast circulating tumor cell (breast CTC) or a breast stem-like CTC (breast SCTC) The sample was labeled with SNT and then injected into the channel on the microfluidic chip at a flow rate of 10 l / min. By combining the streptavidin on the surface of the microfluidic chip with the biotin of the SNT, it is possible to obtain a breast-circulating tumor cell (breast CTC) or a breast stem-like CTC (breast SCTC) (SK-BR-3, MDA-MB-231, MCF-7) or breast cancer stem cells (SCC) The chips were washed with culture medium and then SERS mapping was performed.

Since the extent to which each SNT is attached to the cell depends on the expression level of the surface marker depending on the cell type, the relative expression level of the surface marker can be calculated according to the signal intensity of the specific Raman band selected for each SNT. 6 to 9 are the results of analysis of surface marker expression levels of MCF-7, SK-BR-3, MDA-MB-231 and breast SCTC measured by SERS mapping. The vertical scale bar to the right of each SERS map indicates the expression level of the indicated protein. The relative expression levels of each surface marker are summarized and expressed as a histogram.

In the case of MDA-MB-231 cells, the expression level of EGFR protein was the highest, and MUC1, EpCAM, CD-133 and HER2 were higher in the order of expression (Fig. 6).

In the case of SK-BR-3 cells, expression level of HER2 was the highest, followed by EpCAM, EGFR, MUC1, and CD-133, respectively (FIG. 7).

In the case of MCF-7 cells, EpCAM expression level was the highest, and EGFR, HER2, CD-133 and MUC1 were higher in the order of expression (Fig. 8).

In breast SCC, CD-133 expression level was the highest, and HER2, EGFR, EpCAM, and MUC1 were higher in the order of expression (Fig. 9).

Based on the results of the analysis, the detection accuracy of the captured cells was analyzed. The level of expression of surface markers in each cell line reported previously [29] was taken as a reference, and the number of cells whose expression level of the surface marker in the captured cells coincided with the reference was calculated and then divided by the number of captured cells The value obtained by multiplying by 100 was calculated as the detection accuracy. The detection accuracy of each cell line was 96% for SK-BR-3 cells, 95% for MCF-7 cells, 90% for MDA-MB-231 cells and 89% for breast SCCs. These results indicate that the method of the present invention can detect a specific cell with high accuracy as in the conventional detection method, and it is possible to separate the circulating tumor cells or the circulating tumor stem-like cells from blood using this method It means.

Captured Of cancer stem-like cells Isolation and Culture

To confirm whether it is possible to analyze the characteristics while continuously culturing the circulating tumor stem-like cells separated and captured by the microfluidic chip of the present invention, the captured mammary stem-like cancer cells (SCC) are separated from the microfluid chip and cultured Experiments were performed. In SNT, biotin is linked to gold nanoparticles by dsDNA, so if you cut dsDNA, you can separate cells from the microfluidic chip. Therefore, the cells were separated from the chip using a method of digesting dsDNA by treating a restriction enzyme capable of cleaving the DNA, and the separated cells were cultured in a Petri dish (FIG. 10).

We could observe the differentiation of breast SCC during culturing. The undifferentiated cells showed a round shape, and the differentiated cells showed elongated shape (Fig. 11).

SERS mapping was used to analyze surface marker expression levels of undifferentiated breast cancer cells and differentiated breast cancer cells. In the undifferentiated breast cancer cells, the expression level of CD-133 was the highest among the five surface markers and the expression level was higher in the order of EGFR, HER2, EpCAM and MUC1 (Fig. 12). In the case of the differentiated breast cancer cells, the expression level of HER2 was the highest among the five surface markers and the expression level was higher in the order of EGFR, CD-133, EpCAM and MUC1 (Fig. 13).

These results show that cancer stem-like cells isolated by the method of the present invention can be further cultured after their characteristics are analyzed by SERS mapping.

references

[1] Yi, S.-Y., Hao, Yi.-B., Nan K.J. & Fan, T.L. (2013). Cancer stem cell niche: A target for novel cancer therapeutics. Cancer Treatment Reviews, 39, 290296.

[2] Visvader, J. E. & Lindeman, J. E. (2008). Cancer stem cells in solid tumors: accumulating evidence and unresolved questions. Nature Reviews Cancer, 8, 755-768.

[3] Trumpp, A. et al. (2008). Mechanisms of disease: cancer cell tumors targeting the evil twin. Nature Clinical Practice Oncology, 5, 337-347.

[4] Gangopadhyay, S., Nandy, A., Hor, P. & Mukhopadhyay, A. (2013). Breast cancer stem cells: A novel therapeutic target. Clinical Breast Cancer, 13, 715.

[5] Gerges, N., Rak, J. & Jobado, N. (2010). New technologies for the detection of circulating tumor cells. British Medical Bulletin, 94, 49-64.

[6] Pantel, K & Alix-Panabieres, C. (2010). Circulating tumor cells in cancer patients: challenges and perspectives. Trends in Molecular Medicine, 16, 398-406.

[7] Theodoropoulos, P., A. et al. (2010). Circulating tumor cells with a putative stem cell phototype in peripheral blood of patients with breast cancer. Cancer Letters, 288, 99106.

[8] Nguyen, C., T., Nguyen, J., T., Rutledge, S., Zhang, J., Wang, Chen & Walker G., C. (2010). Detection of chronic lymphocytic leukemia cell surface markers using surface enhanced Raman scattering gold nano particles. Cancer Letters, 292, 91-97.

[9] Reinhard, B. M., Siu, M., Agarwal, H., Alivisatos, A. P. & Liphardt. (2005). Calibration of dynamic molecular rules based on plasmon coupling between gold nanoparticles. Nano Letters, 5, 2246-2252.

[10] Sonnichsen, C. & Alivisatos, A. P. (2005). Gold nanorods as novel nonbleaching plasmon-based orientation sensors for polarized single-particle microscopy. Nano Letters, 5, 301-304.

[11] Aslan, K., Lakowicz, J. R. & Geddes, C. D. (2005). Plasmon light scattering in biology and medicine: new sensing approaches, visions and perspectives. Current Opinion in Chemical Biology, 9, 538-544.

[12] Sokolov, K. et al. (2003). Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles. Cancer Research, 63, 1999-2004.

[13] Doering, W. E., Piotti, M. E., Nata, M. J. & Freeman, R. G. (2007). SERS as a foundation for nanoscale optically detected biological labels. Advancd Materials, 19, 3100-3108.

[14] Yonzon, C. R., Haynes, L., Zhang, X., Walsh, J.T., Jr., & R. Van Duyne, R. P. (2004). A glucose biosensor based on surface-enhanced Raman scattering: improved partition layer, temporal stability, reversibility, and resistance to serum protein interference. Analytical Chemistry, 76, 78-85.

[15] Qian, X. M., Zhou, X. & Nie, S. (2008). Surface-enhanced Raman nanoparticle beacons based on bioconjugated gold nanocrystals and long range plasmonic coupling. Journal of American Chemical Society, 130, 1493414935.

[16] Keren, S. et al. (2008). Noninvasive molecular imaging of small living subjects using Raman spectroscopy. Proceedings of the National Academy of Science, U. S. A. 105, 58445849.

[17] Kneipp, J., Kneipp, H., Wittig, B. & Kneipp, K. (2010). Novel optical nanosensors for probing and imaging live cells. Nanomedicine: Nanotechnology, Biology and Medicine. 6, 214226.

[18] Matschulat, A., Drescher, D. & Kneipp, J. (2010). Surface-enhanced Raman scattering hybrid nanoprobe multiplexing and imaging in biological systems. ACS Nano, 4, 32593269.

[19] Doering, W. E. & Nie, S. (2002). Single-molecule and single-nanoparticle SERS: Examining the roles of surface active sites and chemical enhancement. Journal of Physical Chemistry B, 106, 311-317.

[20] Kneipp, J., Kneipp, H. & Kneipp, K. (2008). SERSA single-molecule and nanoscale tool for bioanalytics. Chemical Society Review, 37, 10521060.

[21] Yang, J. et al. (2012). Distinguishing breast cancer cells using surface enhanced Raman scattering. Istry, 402, 1093-1100.

[22] Qian, X. et al. (2008). In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags. Nature Biotechnology, 26, 83-90.

[23] Park, H. et al. (2009). SERS imaging of HER2-overexpressed MCF7 cells using antibody-conjugated gold nanorods. Physical Chemistry Chemical Physics, 11, 74447449.

[24] Carron, K. T. & Hurley, L. G. (1991). Axial and azimuthal angle determination with surface-enhanced Raman spectroscopy: thiophenol on copper, silver, and gold metal surfaces. Journal of Physical Chemistry, 95, 9979-9984.

[25] Kim, J., H. et al. (2006). Nanoparticle Probes with Surface Enhanced Raman Spectroscopic Tags for Cellular Cancer Targeting .. Analytical Chemistry, 78, 7967-6973.

[26] Caceres, R. C., Martin, B. S. & Barbero, A. F. (2011). Surface-enhanced Raman scattering sensors based on hybrid nanoparticles. Microsensors, DOI: 10.5772 / 18735.

[27] Dey, P. et al. (2013). SERSbased detection of barcoded gold nanoparticle assemblies from within animal tissue. Journal of Raman Spectroscopy, 44, 1659-1665.

[28] Song, W. et al. (2007). Surface-enhanced Raman scattering of 4-mercaptopyridine on the surface of TiO2 nanofiberscoatedwithAgnanoparticles. Journal of Physical Chemistry C, 111, 12786-12791.

[29] Lee H.J. et al. (2013). Simultaneous capture and in situ analysis of circulating tumor cells using multiple hybrid nanoparticles. Biosens Bioelectron. 47, 508514.

Claims

A method for detecting circulating tumor cells (CTC) or stem-like circulating tumor cells (SCTC) comprising the steps of:
(a) contacting a blood sample separated from a living body with a SERS nanotag (Surface Enhanced Raman Scattering Nanotag); The SERS nanotact may be (i) a nanoparticle; (ii) a Raman reporter bonded to the surface of the nanoparticles; (iii) a tumor cell surface antigen-specific antibody bound to the surface of said nanoparticle; And (iv) a non-covalent binding partner bonded to the surface of said nanoparticle;
(b) injecting a sample solution in contact with the SERS nanotubes in a channel on the surface of a microfluidic chip;
(c) obtaining a SERS spectrum from the microfluidic chip; And
(d) analyzing the SERS spectrum to detect circulating tumor cells or circulating tumor stem-like cells.

2. The method of claim 1, wherein the circulating tumor cells or the circulating tumor stem-like cells are mammary carcinoma cells or mammary carcinoma stem-like cells.

The method of claim 1, wherein the nanoparticles of step (a) are gold.

The method of claim 1, wherein the Raman reporter in step (a) is selected from the group consisting of thiophenol (TP), nile blue A (NBA), 1-naphthalenethiol 4-mercaptopyridine (MPy) or 2-quinolinethiol (QTH).

2. The method of claim 1, wherein the tumor cell surface antigen-specific antibody of step (a) specifically binds to surface antigens of a circulating tumor cell or a circulating tumor stem-like cell.

2. The method of claim 1, wherein the tumor cell surface antigen-specific antibody of step (a) specifically binds to surface antigen of a mammary gland tumor cell or a mammary gland tumor stem-like cell.

7. The method according to claim 6, wherein the surface antigen of the mammary gland or mammary tumor stem-like cells is MUC1, EpCAM, EGFR, HER2 or CD-133.

The method of claim 1, wherein the antibody of step (a) is bound to the surface of the nanoparticle by a linker.

2. The method of claim 1, wherein the non-covalent binding partner of step (a) is biotin.

10. The method of claim 9, wherein the non-covalent partner of step (a) is bound to the surface of the nanoparticle by a linker and double helix DNA.

The method of claim 1, wherein the microfluidic chip of step (b) has a surface coated with streptavidin.

2. The method of claim 1, wherein the tumor cell surface antigen-specific antibody of step (a) comprises an antibody specifically binding to MUC1 and CD-133, and the SERS spectrum of step (d) Wherein the stem cell-like cell is judged to be a mammary tumor when the signal due to the expression of CD-133 is 2 to 6 times higher than the signal according to the expression of MUC1.

A system for detecting circulating tumor cells (CTC) or stem-like circulating tumor cells (SCTC) comprising:
(a) Surface Enhanced Raman Scattering Nanotag; The SERS nanotact may be (i) a nanoparticle; (ii) a Raman reporter bonded to the surface of the nanoparticles; (iii) a tumor cell surface antigen-specific antibody bound to the surface of said nanoparticle; And (iv) a non-covalent binding partner bonded to the surface of said nanoparticle;
(b) Microfluidic chip.

14. The system of claim 13, wherein the circulating tumor cells or the circulating tumor stem-like cells are mammary carcinoma cells or mammary carcinoma stem-like cells.

14. The system of claim 13, wherein the nanoparticles are gold.

14. The method of claim 13, wherein the Raman reporter is selected from the group consisting of thiophenol (TP), nile blue A (NBA), 1-naphthalenethiol (NPT), 4- mercaptopyridine , MPy) or 2-quinolinethiol (QTH).

14. The system of claim 13, wherein the tumor cell surface antigen-specific antibody specifically binds to surface antigens of a circulating tumor cell or a circulating tumor stem-like cell.

14. The system of claim 13, wherein said tumor cell surface antigen-specific antibody specifically binds to surface antigens of mammary gland tumor cells or mammary gland tumoral stem-like cells.

19. The system of claim 18, wherein the surface antigen of the mammary gland or mammary tumor stem-like cells is MUC1, EpCAM, EGFR, HER2 or CD-133.

14. The system of claim 13, wherein the antibody is bound to the surface of the nanoparticle by a linker.

14. The system of claim 13, wherein the non-covalent binding partner is biotin.

14. The system of claim 13, wherein the non-covalent partner is attached to the surface of the nanoparticle by a linker and double helix DNA.

14. The system of claim 13, wherein the surface of the microfluidic chip of step (b) is coated with streptavidin.

14. The method of claim 13, wherein the tumor cell surface antigen-specific antibody of step (a) comprises an antibody that specifically binds MUC1 and CD-133, and the SERS spectrum analysis shows that CD- Wherein the stem cell is judged to be a mammary gland tumor-like cell when the signal according to the expression of MUC1 is 2 to 6 times higher than the signal according to the expression of MUC1.