Microfluidic channel sensor device and method for detecting cancer cells or circulating tumor cells using same

Abstract

The present invention relates to: a microfluidic channel sensor device; and a method for detecting cancer cells or circulating tumor cells using same. More specifically, the present invention enables efficient separation and detection of cancer cells or circulating tumor cells using time-resolved square wave voltammetry (trSWV) and a microfluidic channel sensor device having a microfluidic separation channel functionalized with a lipid-binding polymer and a detection unit including a surface-modified working electrode. This enables early detection of cancer, and furthermore, can be used for diagnosis and treatment for the management of cancer patients.

Description

Microfluidic channel sensor device and method for detecting cancer cells or circulating tumor cells using the same

[0001] The present invention relates to a detection device and a detection method for cancer cells or circulating tumor cells (CTCs), and more specifically, to a microfluidic channel sensor device with enhanced selectivity for cancer cells or circulating tumor cells and a detection method using the same.

[0002] Cancer is known today as one of the leading causes of death, accounting for approximately one-fifth of all deaths worldwide. The International Agency for Research on Cancer (IARC) estimates that there will be 21.3 million new cancer cases in 2025, of which about 1.3 million are new cases. The mortality rate among total cases is approximately 49% for both men and women (55% for men, 45% for women) (World Health Organization, 2025). Among cancer types, colon cancer is the second deadliest (National Cancer Institute, 2024), and up to 50% of initially localized colon cancers metastasize, spreading to other parts of the body, particularly regional lymph nodes, the liver, lungs, or the peritoneum. Metastatic colon cancer is characterized by the progression of an untreated or recurrent primary tumor. Treatment generally involves surgery and a combination of chemotherapy and targeted therapy. However, the prognosis for this disease has not been satisfactory to date. Therefore, research for effective diagnosis, treatment, and progression monitoring in the early stages of colorectal cancer is important.

[0003] Since survival rates decrease significantly at advanced stages, early diagnosis of metastasis is crucial for effective management. Late diagnosis of cancer often leads to metastasis via circulating tumor cells (CTCs), which exacerbates the pathology and results in high cancer-related mortality. In the early stages of metastasis, CTCs detached from the primary tumor travel through the bloodstream to distal tissues to form secondary tumors. Therefore, the early detection of CTCs is vital for improving cancer prognosis and survival rates by providing necessary prophylactic treatments. However, the extremely small number of CTCs—specifically 1 to 10 CTCs per 10 mL of whole blood—compared to billions of blood cells, and their size similar to some leukocytes, pose significant technical challenges for isolation with high sensitivity and specificity. Consequently, detecting such a small number of CTCs, along with effective isolation methods, remains a major challenge in early cancer diagnosis.

[0004] To date, various methods for isolating CTCs based on cell size, density, denaturation, electrical properties, and biological aspects have been applicable. Isolated CTCs can be detected using fluorescence, inductively coupled plasma analysis (ICP-MS), chromatography, surface-enhanced Raman spectroscopy (SERS), and electrochemical methods. However, these isolation and detection methods face limitations such as non-specific binding, complex procedures, high costs, low CTC capture efficiency, low sensitivity, and the potential for leukocyte contamination. Recently, cell surface biomarker-based methods, including immunocytochemistry (ICC), immunomagnetic, and immunoaffinity-based capture methods, have been primarily used for CTC isolation and detection. While these techniques have gained considerable popularity, they face challenges due to antibody dependence, variations in surface markers, and loss of CTC diversity. Currently, FDA-approved devices such as Cellsearch™ are considered the standard for CTC detection in clinical settings. However, they have limitations, such as low detection sensitivity, the need for large blood volumes, and the ability to isolate only epithelial-derived CTCs. On the other hand, microfluidic devices are emerging as a prominent alternative platform for CTC separation due to their speed, low cost, low sample requirements, continuous operation, and efficient cell processing; however, they still face challenges related to clogging, low purity, and complex analysis.

[0005] The objective of the present invention is to provide an apparatus capable of separating and detecting cancer cells or circulating tumor cells more efficiently and selectively, and a method for manufacturing the same.

[0006] Another objective of the present invention is to provide a method for detecting cancer cells or circulating tumor cells using the detection device described above.

[0007] To achieve the above objective, the present invention provides a microfluidic channel sensor device comprising: a microfluidic separation channel provided in a first direction through which a sample containing one or more cells selected from cancer cells or circulating tumor cells (CTCs) moves; and a detection unit located at the first direction end of the microfluidic separation channel and comprising a working electrode, a counter electrode, and a reference electrode, wherein the working electrode is surface-modified with one or more selected from the group consisting of carbon nanomaterials, conductive polymers, and cancer-specific aptamers to selectively detect cancer cells or circulating tumor cells.

[0008] The present invention provides a method for manufacturing a microfluidic channel sensor device for detecting cancer cells or circulating tumor cells, comprising the steps of: screen printing an ink mixed with carbon and silver onto a substrate to manufacture a sensor device comprising a microfluidic separation channel through which a sample moves, a working electrode, a counter electrode, and a reference electrode to detect cells; and modifying the separation channel and the working electrode.

[0009] In addition, the present invention provides a method for detecting cancer cells or circulating tumor cells, comprising the step of injecting a sample containing cancer cells or circulating tumor cells into a microfluidic channel sensor device according to the above and applying an alternating voltage.

[0010] The microfluidic channel sensor device according to the present invention overcomes the limitations of conventional approaches, such as low sensitivity and leukocyte contamination. More specifically, efficient separation and detection of cancer cells or circulating tumor cells can be achieved by using a microfluidic channel sensor device having a detection unit comprising a microfluidic separation channel functionalized with a lipid-binding polymer and a surface-modified working electrode.

[0011] The microfluidic channel sensor device and the method for detecting cancer cells or circulating tumor cells using time-resolved square wave voltammetry (trSWV) according to the present invention enable the early detection of cancer with excellent selectivity and detection sensitivity, and furthermore, can be utilized for diagnosis and treatment for cancer patient management.

[0012] FIG. 1(a) is a bare screen-printed carbon electrode (SPCE), (b) is a multiwalled carbon nanotube (mWCNTs), (c) is a multiwalled carbon nanotube / conductive poly-2,2:5,2-terthiophene-3-p-benzoic acid (mWCNTs / pTBA), and (d) is a scanning electron image (SEM) of a multiwalled carbon nanotube / conductive poly-2,2:5,2-terthiophene-3-p-benzoic acid / W3 aptamer (mWCNTs / pTBA / W3-Apt); and (e) is the working electrode modification and peak deconvolution of (f) S2p, (g) C1s, (h) N1s, and (i) P2p for (i) mWCNTs, (ii) mWCNTs / pTBA, and (iii) mWCNTs / pTBA-W3 Apt-modified electrodes. This shows the X-ray photoelectron spectroscopy (XPS) irradiation spectrum.

[0013] Figure 2 shows (a, c) 4.0 mM Fe(CN)6 for bare SPCE, mWCNTs, mWCNTs / pTBA, and mWCNTs / pTBA-W3 Apt-modified layers. 3- / 4- / 1.0 M KNO3, and (b, d) 4.0 mM Ru(NH3)6 3+ / 4+ These are the electrochemical impedance spectroscopy (EIS) spectrum and cyclic voltammetry (CV) recorded in 1.0 M KNO3.

[0014] Figure 3(a) shows the square wave voltammetry (SWV) responses of (i) blank PBS (0.1M; pH 7.4), (ii) Vero cells (normal kidney cells) and (iii) colorectal cancer cells added to PBS. (b) shows the drug concentration, (c) shows the drug incubation time with colorectal cancer cells, (d) shows the aptamer concentration, (e) shows the current response with or without the aptamer, and (f) shows the optimization of aptamer selectivity for various cell lines including colorectal cancer, lung cancer, and breast cancer, and (g) shows the results of the cytotoxicity evaluation of the detection system (mWCNTs / TBA / W3) using the MTT assay.

[0015] Figure 4 shows the optimization of (A) frequency, (B) amplitude, and (C) flow rate for successful separation detection (using Col 205, MCF-7, and A549 cell lines).

[0016] Figure 5(a) shows the reaction to various numbers of colorectal cancer cells (3 to 100 cells) and (b) a calibration plot, (c) selective isolation detection of colorectal cancer cells from various cancer cell lines (inset shows individual cancer cell line reactions), and (d) results depending on the presence or absence of an interfering substance.

[0017] Figure 6(a) shows the long-term stability of the microfluidic channel sensor, and (b) is the elution curve for peripheral blood monocytes (PBMCs) extracted from (i) without the addition of colorectal cancer cells and (ii) with the addition of colorectal cancer cells.

[0018] Figure 7(a) shows the result of analysis using time-resolved square wave voltammetry (trSWV) software, and (b) shows the time elution chromatogram.

[0019] The present invention will be described in detail below.

[0020] The terms used in this application are used merely to describe specific embodiments and are not intended to limit the invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, terms such as “comprising,” “having,” etc., are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0021] Terms such as "first," "second," etc., may be used to describe various components, but said components should not be limited by said terms. These terms are used solely for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the first component may be named the second component, and similarly, the second component may be named the first component.

[0022] Meanwhile, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0023]

[0024] The inventors developed an electrodynamic microfluidic channel sensor device screen-printed with carbon ink on a thin plastic substrate and software for signal processing based on time-resolved square wave voltammetry to overcome the disadvantages of conventional microfluidic channel devices for separation detection, and completed the present invention by confirming that cancer cells or circulating tumor cells (CTCs) can be selectively detected through a separation detection method using the same.

[0025]

[0026] The present invention provides a microfluidic channel sensor device for the selective detection of cancer cells or circulating tumor cells (CTCs).

[0027] A microfluidic channel sensor device according to the present invention may include: a microfluidic separation channel provided in a first direction through which a sample containing one or more cells selected from cancer cells or circulating tumor cells (CTCs) moves; and a detection unit located at the first direction end of the microfluidic separation channel and comprising a working electrode, a counter electrode, and a reference electrode.

[0028] The above microfluidic separation channel can effectively separate cells by applying an alternating current (AC) voltage, but does not electrochemically decompose the cells.

[0029] The state of the channel, such as the size, surface potential, or hydrophilicity of the microfluidic separation channel, can influence the movement of cancer cells or circulating tumor cells under electrodynamic forces, thereby changing the environment of the channel wall for proper cell separation.

[0030] Preferably, the microfluidic separation channel may have its inner wall coated or modified with a conductive polymer to which lipid molecules are bonded.

[0031] The above lipid molecule may be selected from phosphatidylserine or phosphatidylinositol, but is not limited thereto.

[0032] The above conductive polymer is a terthiophene-based polymer, for example, (2,2':5',2"-terthiophene)-diamine ((2,2':5',2"-terthiophene)-3',4'-diamine, DAT), poly-2,2:5,2-terthiophene-3-p-benzoic acid (poly-2,2:5,2-terthiophene-3-p-benzoic acid, pTBA), poly-5,2-5,2-terthiophene-3-carboxylic acid (poly-{5,2-5,2-terthiophene-3-carboxylic acid}, pTTCA), It may be one or more selected from the group consisting of poly-{4'-([2,2':5',2''-terthiophen]-3')-[1,1'-biphenyl]-4-carboxylic acid (TTBPA), poly-{4-(2,5-di(thiophen-2)-1H-pyrrol-1)benzoic acid (DTPBA), but is not limited thereto.

[0033] The above microfluidic separation channel can improve cell separation performance by influencing the movement of cells contained in the sample through coating or modifying the inner wall of the channel with a conductive polymer to which lipid molecules are bound as described above.

[0034]

[0035] The above microfluidic separation channel can effectively separate cancer cells or circulating tumor cells by changing the AC frequency, amplitude, or flow rate.

[0036] The control range of the AC frequency of the microfluidic separation channel for effective cell separation may be in the range of 50 to 150 kHz at an AC amplitude of 0.05, preferably 100 kHz, but is not limited thereto.

[0037] The control range of the amplitude of the microfluidic separation channel for effective cell separation may be 0.05 to 0.15 V, preferably 0.1 V, but is not limited thereto.

[0038] The fluid flow rate of the microfluidic separation channel for effective cell separation may be in the range of 0.5 to 5 μL / min, preferably 1 to 3 μL / min, more preferably 2 μL / min, but is not limited thereto.

[0039]

[0040] The working electrode of the above-mentioned detector may be surface modified by one or more selected from the group consisting of carbon nanomaterials, conductive polymers, and arm-specific aptamers.

[0041] The above carbon nanomaterial may be selected from multi-walled carbon nanotubes (mWCNT) or graphene oxide, but is not limited thereto.

[0042] The above conductive polymer is (2,2':5',2"-terthiophene)-diamine ((2,2':5',2"-terthiophene)-3',4'-diamine, DAT), poly-2,2:5,2-terthiophene-3-p-benzoic acid (poly-2,2:5,2-terthiophene-3-p-benzoic acid, pTBA), poly-5,2-5,2-terthiophene-3-carboxylic acid (poly-{5,2-5,2-terthiophene-3-carboxylic acid}, pTTCA), It may be one or more selected from the group consisting of poly-{4'-([2,2':5',2''-terthiophen]-3')-[1,1'-biphenyl]-4-carboxylic acid (TTBPA), poly-{4-(2,5-di(thiophen-2)-1H-pyrrol-1)benzoic acid (DTPBA), preferably pTBA, but is not limited thereto.

[0043] The above cancer-specific aptamers may be one or more selected from the group consisting of colorectal cancer-specific aptamers W3, anti-EpCAM (epithelial cell adhesion molecule), CA19-9, and Eph receptor A2+A3, but are not limited thereto, and specific aptamers depending on the target cancer cell may be selected and used.

[0044] Preferably, the working electrode is sequentially surface-modified with multiwalled carbon nanotubes (mWCNT), poly-2,2:5,2-terthiophene-3-p-benzoic acid (pTBA), and colorectal cancer-specific aptamer W3 to more selectively detect colorectal cancer cells or circulating tumor cells.

[0045] The above sample may be a blood or plasma sample containing one or more cells selected from cancer cells or circulating tumor cells isolated from the human body, but is not limited thereto.

[0046] The above cancer cells may be cancer cells of colorectal cancer, lung cancer, breast cancer, stomach cancer, liver cancer, etc., but are not limited thereto.

[0047]

[0048] In addition, the microfluidic channel sensor device according to the present invention has excellent long-term stability.

[0049] More specifically, the device has a high detection sensitivity of over 90% for 90 days, and can provide stable performance over a long period.

[0050]

[0051] The present invention provides a method for manufacturing a microfluidic channel sensor device for the selective detection of cancer cells or circulating tumor cells.

[0052] The above manufacturing method may include the step of manufacturing a sensor device comprising a detection unit for detecting cells, which is composed of a microfluidic separation channel through which a sample moves, a working electrode, a counter electrode, and a reference electrode, by screen printing an ink mixed with carbon and silver onto a substrate; and the step of modifying the separation channel and the working electrode.

[0053] Before the step of modifying the separation channel and the working electrode, the separation channel and the working electrode may be pre-washed by acid treatment.

[0054] The step of modifying the separation channel may include: electrochemically polymerizing a conductive monomer on the inner wall of the separation channel to form a polymer layer; and binding lipid molecules to the formed polymer layer.

[0055] As previously mentioned, the conductive monomers are (2,2':5',2"-terthiophene)-diamine ((2,2':5',2"-terthiophene)-3',4'-diamine, DAT), poly-2,2:5,2-terthiophene-3-p-benzoic acid (poly-2,2:5,2-terthiophene-3-p-benzoic acid, pTBA), poly-5,2-5,2-terthiophene-3-carboxylic acid (poly-{5,2-5,2-terthiophene-3-carboxylic acid}, pTTCA), Monomers of terthiophene-based polymers composed of poly-{4'-([2,2':5',2''-terthiophene]-3')-[1,1'-biphenyl]-4-carboxylic acid (TTBPA), and poly-{4-(2,5-di(thiophen-2)-1H-pyrrol-1)benzoic acid (DTPBA) may be selected, but are not limited thereto.

[0056] The step of bonding lipid molecules to the polymer layer formed above can be performed by activating the amine functional group of the polymer layer and the carboxylic acid functional group of the lipid molecule to form an amide bond.

[0057] The inner wall of the separation channel can be formed into a stable modified surface by the above steps.

[0058] The step of modifying the working electrode may include: treating a dispersion in which carbon nanomaterials are dispersed on the surface of the working electrode; treating a conductive polymer solution on the surface of the working electrode treated with the dispersion to form a polymer layer; and immobilizing an arm-specific aptamer on the formed polymer layer.

[0059] More specifically, the dispersion in which the carbon nanomaterial is dispersed can be prepared by adding a carbon nanomaterial selected from multi-walled carbon nanotubes (mWCNT) or graphene oxide to a surfactant and ultrasonically treating it.

[0060] The above conductive polymer solution can be prepared by adding a conductive polymer to a mixture of two or more ethers.

[0061] The above conductive polymer is (2,2':5',2"-terthiophene)-diamine ((2,2':5',2"-terthiophene)-3',4'-diamine, DAT), poly-2,2:5,2-terthiophene-3-p-benzoic acid (poly-2,2:5,2-terthiophene-3-p-benzoic acid, pTBA), poly-5,2-5,2-terthiophene-3-carboxylic acid (poly-{5,2-5,2-terthiophene-3-carboxylic acid}, pTTCA), It may be one or more selected from the group consisting of poly-{4'-([2,2':5',2''-terthiophen]-3')-[1,1'-biphenyl]-4-carboxylic acid (TTBPA), poly-{4-(2,5-di(thiophen-2)-1H-pyrrol-1)benzoic acid (DTPBA), preferably pTBA, but is not limited thereto.

[0062] The dispersion solution in which the carbon nanomaterial is dispersed and the conductive polymer solution can each be sequentially drop-coated onto the surface of the working electrode, and then further polymerized after drying at 30 to 50°C for 2 to 5 hours.

[0063] The step of immobilizing an arm-specific aptamer on the polymer layer formed above can be performed by treating the aptamer with EDC / NHS for 2 to 5 hours, preferably 3 hours.

[0064] The above cancer-specific aptamers may be one or more selected from the group consisting of colorectal cancer-specific aptamers W3, anti-EpCAM, CA19-9, and Eph receptor A2+A3, but are not limited thereto, and specific aptamers according to the target cancer cells may be selected and used.

[0065] The above aptamer may be included at a concentration of 1 to 50 nM, preferably 5 to 20 nM, and more preferably 10 nM.

[0066]

[0067] In addition, the present invention provides a method for detecting cancer cells or circulating tumor cells.

[0068] More specifically, the detection method comprises the step of injecting a sample containing cancer cells or circulating tumor cells into the microfluidic channel sensor device and applying an alternating voltage.

[0069] The above sample may have been treated with an anticancer agent.

[0070] The above anticancer agent may be selected from idarubicin, daunomycin, or doxorubicin, which are anticancer agents that selectively react with cancer cells to obtain an electrochemical detection signal of electrochemically inactive cancer cells, but are not limited thereto.

[0071] The above anticancer agent can be adsorbed onto the cells by culturing with cancer cells or circulating tumor cells for 5 to 60 minutes, preferably for 10 to 30 minutes, and more preferably for 15 to 20 minutes, so that the anticancer agent can be more effectively adsorbed onto the cells.

[0072] The above anticancer agent may be included at a concentration of 0.1 μM to 4 μM, preferably at a concentration of 0.5 to 3 μM, and more preferably at a concentration of 1 to 2 μM, in terms of sensitivity, cytotoxicity, and cost-effectiveness.

[0073] For more effective cell separation, the microfluidic channel sensor device can separate and detect the sample by applying an alternating voltage with an amplitude of 0.1 V and a frequency of 100 kHz, while simultaneously introducing a running buffer at a flow rate of 2 μL / min.

[0074] In the step of applying the above AC voltage, the AC voltage can be varied in the form of a square wave so that the square wave voltammetry method can be used. Specifically, the change in current value according to the separation time can be recorded using the square wave voltammetry method.

[0075] The above detection method may further include, after the step of applying the above alternating voltage, a step of detecting the electrochemical signal of cancer cells or circulating tumor cells using time-resolved square wave voltammetry (trSWV) and converting it into an elution chromatogram.

[0076] The step of converting to the above elution chromatogram may involve obtaining an elution curve using data recording the change in current values ​​according to separation time using square wave voltammetry and converting it into an elution chromatogram.

[0077] The above data can be analyzed by software developed in the inventor's laboratory and converted into an elution chromatogram.

[0078] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0079]

[0080] Experimental Method

[0081] 1. Experimental Materials and Apparatus

[0082] Diamine-functionalized terthiophene [(2,2':5',2"-terthiophene)-3',4'-diamine, DAT] and conductive poly-2,2:5,2-terthiophene-3-p-benzoic acid [poly-2,2:5,2-terthiophene-3-p-benzoic acid, pTBA] monomers were synthesized according to a previously reported protocol. For lipid layer immobilization, brain phosphatidylserine (PS) was purchased from Avanti Research (USA).

[0083] We used a colorectal cancer-specific W3 aptamer with the sequence 5'-AGC AGC GTG GAG GAT AGG GGT CGG AGT GGG TGG TTA TGA TTG GCT CTT CTG CGC TGC-NH2-3' purchased from Bioneer Co., (South Korea). Colo 205 (colorectal adenocarcinoma), A549 (lung cancer), MCF-7 (breast adenocarcinoma), and Vero (normal kidney) cell lines were purchased from the Korean Cell Line Bank (South Korea). Dulbecco's Modified Eagle Medium, RPMI-1640 medium, fetal bovine serum (FBS), trypsin-EDTA, dimethyl sulfoxide (DMSO), L-glutamine, penicillin / streptomycin, Hank's balanced salt solution, Dulbecco's phosphate-buffered saline (for cell culture), Trypan Blue, and MTT reagent were purchased from Sigma-Aldrich (USA). Ficoll-Paque™ PLUS density gradient medium was purchased from Cytiva (USA).

[0084] Idarubicin, albumin, human PD-L1 recombinant protein, salicylic acid, ethylenediaminetetraacetic acid (EDTA), and potassium were purchased from Thermo Fisher Scientific. Multiwalled carbon nanotubes (mWCNT), chloroform, di- and tri(propylene glycol) ethyl ether, 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), N-hydroxysuccinimide (NHS), [Ru(NH3)6]Cl2, [Ru(NH3)6]Cl3, Fe(CN)6 3- , Fe(CN)6 4-Na₂HPO₄, NaH₂PO₄, glutaraldehyde, and NaCl were purchased from Sigma-Aldrich. All aqueous solutions were prepared using double distilled water in an 18 MΩ·cm resistance Milli-Q Millipore system.

[0085] Microfluidic channels with detection electrodes were fabricated using a screen printer from KTP Tech (South Korea). The cytotoxicity of the electrode materials was evaluated using a FLUOstar Omega Microplate Reader (BMG Labtech). Surface characteristics of the channels and working electrodes were analyzed using X-ray photoelectron spectroscopy (XPS) and field emission scanning electron microscopy (FE-SEM), and XPS data were analyzed using XPSPEAK41 software (at the core facility center in Dong-eui University). Impedance spectra were recorded using an electrochemical impedance spectrometer (EIS) (EG&G Princeton Applied Research, PARSTAT 2630 multimode).

[0086] For separation detection, an AC electric field was applied to the wall electrodes using a function generator (Model Protek 9340, South Korea), and a small automatic microsyringe pump (Harvard Apparatus, USA) was used to flow the buffer into the microchannels. Time-resolved square wave voltammetry (trSWV) was recorded using a three-electrode system equipped with an EmStat4S potentiometer / rectifier (Netherlands). Time-elution chromatograms were obtained using laboratory-developed trSWV software, and further analysis was performed in OriginPro 2018.

[0087]

[0088] 2. Fabrication of Detection Electrode and Microfluidic Channel

[0089] A microfluidic separation channel equipped with a detector consisting of a working electrode, a counter electrode, and a reference electrode located at the channel outlet was screen-printed using carbon and silver ink. The reference electrode (Ag / AgCl) was prepared by coating AgCl onto an Ag electrode. The separation channel has the following physical dimensions: length 50 ± 0.5 mm, width 150 ± 30 μm, height 16.97 ± 2.60 μm, and a detector electrode area of ​​0.070 cm². 2 Before surface modification, the carbon channel and working electrode were pre-washed by potential cycling between 0.0 and 0.9 V in 0.1 M HCl.

[0090] The channel walls were modified with 3 μL of 1.0 mM DAT monomer and electrochemically polymerized through potential cycling between 0.0 V and +1.0 V for two cycles. The carboxylic acid (-COOH) groups of lipids (0.025 mg / mL) were activated in 0.1 M PBS (pH 7.4) using EDC / NHS chemicals (10 mM each). The lipids were reacted for 4 hours using a shaking incubator set to 80 rpm. The activated -COOH groups formed strong amide bonds with the amine functional groups (-NH2) of the polymer layer, creating a stable modified surface on the channel walls.

[0091] To modify the working electrode surface, 1 mL of 1 mg / mL MWCNT and 1 mL of 1 wt% Triton X-100 were sonicated for 1 hour to obtain a homogeneous dispersion. A 1.0 mM TBA conductive polymer solution was prepared from a mixture of di(propylene glycol)ethyl ether and tri(propylene glycol)ethyl ether in a 1:1 volume ratio. Then, the working electrode surface was modified by drop-coating with 0.1 μL of MWCNT and TBA, followed by drying in an oven at 40°C for 3 hours and further polymerization by potentiocycling for two cycles. Finally, a colorectal cancer-specific W3 aptamer (10 nM) was immobilized on the pTBA layer via EDC / NHS treatment (3 hours), followed by acid washing by potentiocycling to improve CTC capture efficiency.

[0092]

[0093] 3. Cell Culture

[0094] Colo 205, MCF-7, A549, and Vero normal cell lines were cultured in T75 culture flasks containing RPMI-1640 medium supplemented with 10% FBS, 9 mg L-glutamine, and 1% penicillin-streptomycin in a 37°C, 5% humid CO2 incubator. To maintain a healthy cell growth environment, the culture medium was replaced every 2–3 days. Upon reaching 90% confluency, cells were collected by trypsin treatment and centrifuged at 1000 rpm for 3 minutes. After washing with Hanks' Balanced Salt Solution to remove remaining medium and serum, the cell pellet was suspended in PBS solution for further analysis. Cell counts were determined using a hemocytometer. Trypan blue-treated cell suspensions (1:1 ratio) were placed in the counting chamber, and concentrations were measured using Equation 1 below:

[0095] [Equation 1]

[0096] Concentration (cells / mL) = (Number of cells in Σ 5 cells / 5) × 10 4 × Dilution ratio

[0097]

[0098] 4. MTT cytotoxicity assay of electrode test substances on Col 205 cells

[0099] Colorectal cancer cells were placed in a 96-well microtiter plate with 100 μL of medium per well at a density of 3 × 10⁶ 3 Cells were seeded into cells / well and cultured overnight. After culture, cells were treated three times under identical conditions with mWCNTs / TBA / W3, 1% DMSO (negative control), and 1 μM idarubicin (positive control). Eighteen hours after treatment, 10 μL of MTT reagent (5 mg / mL in PBS, 7.4) was added to each well, and the plates were incubated in a dark room at 37°C for 4 hours to induce the formation of formazan crystals through mitochondrial dehydrogenase activity. Subsequently, the MTT-containing medium was carefully removed, and the crystals were dissolved in 100 μL of DMSO. Cell viability was evaluated by observing MTT reduction by measuring absorbance at 570 nm using a microplate reader. The viability of colorectal cancer cells was calculated according to Equation 2 below:

[0100] [Equation 2]

[0101] Cell viability (%) = (570 nm absorbance of treated cells / 570 nm absorbance of untreated cells) × 100

[0102]

[0103] 5. Preparation of blood samples and separation and detection of CTCs

[0104] To isolate peripheral blood mononuclear cells (PBMCs) containing CTCs, CTCs were concentrated from blood using Ficoll density gradient medium. 3 mL of Ficoll-Paque Plus was placed in a 15 mL centrifuge tube, 7.5 mL of blood was layered on top, and the tube was centrifuged at 1000×g for 10 minutes at room temperature. PBMCs located in the particulate matter between the plasma and red blood cells were carefully collected and transferred to a new tube. The PBMCs were washed twice by centrifuging 10 mL of 0.1 M PBS (pH 7.4) at 250×g for 10 minutes to remove residual medium and debris, and then resuspended in 1 mL of PBS (pH 7.4). Blood samples from healthy donors and colorectal cancer patients at a university hospital were processed using this method. Samples were collected in Na₂EDTA / cell preservation solution (CellSave Tubes) to stabilize CTCs and prevent degradation. All colorectal cancer patients provided informed consent, and this study complied with the Declaration of Helsinki and was approved by the Institutional Review Board (IRB No. XYZ) of Dong-A University Hospital.

[0105] To generate electrochemical signals in electrochemically inactive cells, cultured cancer cells, PBMCs containing cancer cells, and CTC-enriched PBMCs were each placed in 1 mL of 0.1 M PBS (pH 7.4) with 1 μM idarubicin (IDA) and incubated at room temperature for 15 minutes. After incubation, the cell suspension was centrifuged at 1000 rpm for 5 minutes and washed three times with PBS to remove unbound IDA. Finally, the IDA-bound cell suspension was used for trSWV measurement. 10 μL of the prepared sample was injected into the channel through a Y-connector, while 500 μL of 0.1 M PBS (pH 7.4) running buffer was introduced using a micropump at a flow rate of 2 μL / min. This setup ensures efficient separation by allowing cells to move through the channel while simultaneously applying an AC electric field with an amplitude of 0.1 V and a frequency of 100 kHz. A multiple potential sweep was performed within the range of -0.55 V to -0.75 V to obtain a time-resolution voltammetry, and each individual potential sweep took approximately 2.0 seconds.

[0106]

[0107] 6. trSWV Analysis Procedure

[0108] After the experiment was completed, electrochemical data were collected in a spreadsheet. Then, the time-dependent current was taken as the y-axis value and the current according to peak potential as the x-axis value for the trSWV with a change of approximately 2 seconds. The formatted trSWV data was analyzed using in-house laboratory software developed in Python to convert it into an elution chromatogram. First, the baseline of the voltammetry in the software was subtracted to align it to a single plane; then, the peak current of the voltammetry with the baseline subtracted was calculated within a specific potential range and recorded for the corresponding time to simultaneously generate the elution chromatogram. The peak area of ​​the chromatogram was calculated using the commercially available OriginPro 2018.

[0109]

[0110] <Experimental Results>

[0111] 1. Verification of the morphology and characteristics of the modified microfluidic channel sensor

[0112] As in experimental method 2 above, a screen-printed microfluidic channel device was fabricated using carbon and silver ink, pre-washed with acid treatment, and then the working electrode of the detection portion was sequentially modified with mWCNT, pTBA, and colorectal cancer-specific aptamer W3, and the stepwise modification was confirmed by SEM analysis (Fig. 1).

[0113] Referring to Fig. 1, the working electrode surface appeared as a typical granular form of a bare electrode printed with carbon ink (Fig. 1(a)), was uniformly covered with wire-shaped mWCNTs (Fig. 1(b)), and an aggregated polymer network of the mWCNTs / pTBA layer was formed by the electrochemical polymerization of TBA monomers (Fig. 1(c)). Then, aptamers were finally immobilized on the mWCNTs / pTBA layer, changing the composite surface into a thin, cloudy layer (Fig. 1(d)).

[0114] To confirm the chemical composition, XPS analysis was performed on each layer of mWCNTs, mWCNTs / pTBA, and mWCNTs / pTBA-Apt. The analytical spectra revealed C 1s (283.5), S 2p (163.1), N 1s (399.3), O 1s (500 eV), and P 2p (132.6 eV) (Fig. 1(e)). While the C 1s peak was observed in the SPCE / mWCNTs layer, O 1s and S 2p peaks additionally appeared in mWCNTs / pTBA, and new peaks of N 1s and P 2p appeared in the mWCNTs / pTBA-Apt composite electrode. Indeed, this indicates successful electrode surface modification with mWCNTs, conductive polyTBA, and colorectal cancer-specific aptamers. Deconvolution of the S 2p spectrum for the composite electrode was decomposed into two peaks at 163 and 164.2 eV (CS coupling from the TBA backbone), which were slightly shifted for the final modified layer and showed reduced peak intensity (Fig. 1(f)). The C 1s spectrum deconvolved for the mWCNTs layer showed CC sp at 283.4 eV. 2 Binding and CC sp at 284.4 eV 3Two peaks are shown due to carbon-carbon bonds containing bonds (Fig. 1(g)). These peaks shifted slightly at the polymer composite electrode, and additional peaks appeared from the functionalized CP at 285.4 (CO) and 288.2 eV (C=O). In the final layer containing the aptamer, the formation of amide bonds between the -COOH groups of pTBA and the amine groups of the W3 aptamer was clearly confirmed in the deconvolved N 1s spectrum (Fig. 1(h)), and CN, NH, and NC=O bonds appeared at binding energies (BE) of 398.6, 399.4, and 400.8 eV, which appear to be derived from the amide bonds of pTBA-Apt. The successful attachment of the aptamer to the probe surface was verified by the deconvolved P 2p spectrum (Fig. 1(i)). In this case, two peaks at 132.2 and 133 eV representing phosphate atoms are defined as markers of the presence of aptamers.

[0115]

[0116] 2. Verification of the electrochemical characteristics of the sensor probe

[0117] 4.0 mM [Fe(CN)6] each using 1.0 M KNO3 as the supporting electrolyte 3- / 4- and [Ru(NH3)6] 2+ / 3+ The electrochemical characteristics of each electrode layer were investigated through electrochemical impedance spectroscopy (EIS) experiments.

[0118] Figures 2(a) and (b) show the impedance spectra for bare SPCE, an mWCNTs layer, an mWCNTs / pTBA composite layer, and the SPCE / mWCNTs / pTBA-Apt final probe. The Nyquist plot corresponding to the experimental data was fitted to an equivalent circuit model of R(Q(RW))(QR). As a result, bare SPCE was found to have the highest charge-transfer resistance (Rct) value of approximately 4.7 kΩ due to low conductivity. After depositing mWCNTs on the electrode surface, electrode conductivity improved, and the Rct value [Fe(CN)6] 3- / 4- At 0.32 kΩ is [Ru(NH3)6] 2+ / 3+ It was lowered to 0.29 kΩ in solution. The lower Rct value of the SPCE / mWCNTs electrode is due to negatively charged [Fe(CN)6] 3- / 4- [Ru(NH3)6] which is more positively charged than the solution 2+ / 3+ It was observed in [Fe(CN)6]. This is consistent with the fact that repulsion occurs because mWCNTs have a negatively charged surface. The Rct value of the mWCNTs / pTBA composite electrode is [Fe(CN)6] 3- / 4- and [Ru(NH3)6] 2+ / 3+ In solution, they increased to 1.75 and 1.64 kΩ, respectively. For the final probe, the Rct value was [Fe(CN)6] 3- / 4- While 2.4 kΩ was observed in solution, [Ru(NH3)6] 2+ / 3+ An Rct value of 1.28 kΩ was obtained in solution. This is because a more negative surface is formed after immobilizing the aptamer on the negatively charged mWCNTs / pTBA layer.

[0119] Then, cyclic voltammetry (CV) experiments were performed under the same experimental conditions to cross-check the impedance results. 4.0 mM [Fe(CN)6] prepared by separation in 1.0 M KNO3 3- / 4- and 4.0 mM [Ru(NH3)6] 2+ / 3+The electrochemical properties of SPCE, SPCE / mWCNTs, SPCE / mWCNTs / pTBA, and SPCE / mWCNTs / pTBA-Apt layers were investigated using the solutions (Figs. 2(c) and (d)). Depositing mWCNTs on the SPCE surface improves electrode conductivity, resulting in a significant increase in response current in both solutions. After further modification with pTBA, the peak current of SPCE / mWCNTs / pTBA [Fe(CN)6] 3- / 4- or [Ru(NH3)6] 2+ / 3+ It decreased in solution. However, it is still higher compared to the peak current of bare SPCE. This phenomenon demonstrates that conductive pTBA does not have high conductivity. In the case of the aptamer-immobilized SPCE / mWCNTs / pTBA layer, negatively charged [Fe(CN)6] 3- / 4- While the peak current in solution decreased further, the positively charged [Ru(NH3)6] 2+ / 3+ It was observed to have increased in solution. In general, the CV results confirmed the impedance results and demonstrated the successful modification of the electrode.

[0120]

[0121] 3. Optimization of Detection Parameters

[0122] To detect circulating tumor cells (CTCs), the interactions between anticancer agents, cancer cells, and normal cells were observed. To obtain electrochemical detection signals from electrochemically inactive cancer cells, idarubicin (IDA), an anticancer agent capable of generating a reversible redox current that selectively reacts with cancer cells, was co-cultured with Vero cells (normal kidney cells) and cultured colon cancer cells. The redox peaks of idarubicin adsorbed to the cell membranes of colon cancer cells were monitored using square wave voltammetry (SWV). Referring to Figure 3(a), no detectable reaction was observed in blank PBS (pH 7.4) or Vero cells (i, ii), whereas IDA-treated colon cancer cells exhibited distinct peaks due to a reduction in IDA molecules bound to their surface (iii).

[0123] To optimize drug concentration for optimal electrochemical signal generation, IDA adsorbed onto colorectal cancer cells was investigated in a concentration range of 0.1 μM to 4.0 μM. The results showed an increased current response from 0.1 to 1.0 μM, after which the current response increased less linearly (Fig. 3(b)). Considering sensitivity, cytotoxicity, and cost-effectiveness, 1.0 μM was selected for subsequent experiments.

[0124] The culture time required for idarubicin to adsorb to colorectal cancer cells was optimized by culturing the drug and cells over a range of 0 to 60 minutes. The peak current increased rapidly as the culture time increased up to 15 minutes (Fig. 3(c)); however, no increase was observed after 15 minutes as the active adsorption site became saturated. Therefore, the efficacy did not improve even with long-term culture.

[0125] Subsequently, aptamer concentrations were studied from 1.0 to 50.0 nM (Fig. 3(d)). The peak current increased as the concentration increased from 1.0 to 10.0 nM; however, no significant increase was observed beyond 10.0 nM, indicating that the reaction had reached a steady state. Therefore, 10.0 nM aptamer was selected for the final fabrication.

[0126] All additional experiments were performed with 1.0 μM idarubicin, a 15-minute incubation time, and 10.0 nM W3 aptamer. The effect of the aptamer's presence on the current response was evaluated. As shown in Figure 3(e), the aptamer nearly doubled the current response.

[0127] To evaluate the selectivity and specificity of the W3 aptamer, 1 μM idarubicin was crosslinked with 10 nM W3 aptamer using 0.3% glutaraldehyde as a crosslinking agent in 0.1 M PBS (pH 7.4). The formed aptamer-glutaraldehyde-idarubicin complex was cultured separately from colorectal, breast, and lung cancer cells at room temperature (RT) for 15 minutes. After washing the cells three times with PBS (pH 7.4) to remove unbound complexes, no peak current response was observed in specific tests targeting lung and breast cancer cell lines, whereas a significant increase in current was observed in colorectal cancer cells (Fig. 3(e)), confirming the selectivity of the W3 aptamer.

[0128]

[0129] The cytotoxicity of the detection system (mWCNTs / TBA / W3) was analyzed by an MTT assay using the colorectal cancer cell line Colo 205 (Fig. 3(g)). Cell viability was 93.1 ± 5% (n = 3), indicating minimal toxicity of the detection substance. These results were further supported by a viability of 96.0 ± 4.79% in the negative control group (cells treated with 10% DMSO) and 15.7 ± 1.0% in the positive control group (cells treated with 1 μM idarubicin). These results demonstrate that the detection platform possesses high compatibility and is suitable for additional biomedical applications.

[0130]

[0131] 4. Optimization of Separation Parameters

[0132] The AC field induces electrodynamic movement of target species, causing them to align and displace differently within the channel depending on their mass and charge, thereby improving separation precision. Separation parameters were optimized in terms of AC frequency, amplitude, and flow rate to efficiently separate lung cancer (A549), colorectal cancer (Colo 205), and breast cancer cell lines (MCF-7). At a constant flow rate, separation efficiency is primarily influenced by AC frequency, which was confirmed through subsequent trSWV experiments with and without the application of the AC field.

[0133] Referring to Fig. 4(A), the AC frequency is controlled in the range between 50.0 and 150.0 kHz at an AC amplitude of 0.05 V. When only buffer flow is activated without AC application, an unseparated broad band of A549, Colo 205, and MCF-7 cells is observed, as shown in Fig. 4(A)(i). At 50.0 kHz, the peaks of A549, Colo 205, and MCF-7 appear at 180.0 ± 120.0 s, 354.0 ± 200.0 s, and 390.0 ± 100.0 s, respectively (Fig. 4(A)(ii)), but the peaks frequently overlap, indicating that this is not an efficient frequency for separating different cancer cells. As the frequency increased to 100.0 kHz, the Colo 205 peak appeared at 506.0 ± 250.0 s, and the inter-peak distance between A549, Colo 205, and MCF-7 increased, resulting in the complete separation of the three peaks (Fig. 4(A)(iii)). Furthermore, the inter-peak distance was optimized to eliminate the possibility of peak overlap. As the frequency increased to 150.0 kHz, peaks appeared at 286.0 ± 134.0 s, 630.0 ± 220.0 s, and 780.0 ± 98.0 s (Fig. 4(A)(iv)), where the Colo 205 and MCF-7 peaks had a possibility of overlapping. Consequently, the separation of the three peaks at 150.0 kHz was less efficient than at 100.0 kHz. Finally, a frequency of 100.0 kHz was considered as the optimized frequency for the separate detection of colorectal cancer cells and CTCs.

[0134] To achieve maximum separation detection of cancer cells, the AC amplitude was also optimized at a fixed frequency and flow rate. Referring to Figure 4(B), at an amplitude of 0.05 V, peak areas of 69.85 nC, 72.145 nC, and 74.28 nC were achieved for A549, Colo 205, and MCF-7 cells, respectively. As the AC amplitude was increased to 0.1 V, the peak areas for the three cancer cell lines increased to 72.14 nC, 78.36 nC, and 77.25 nC. However, further increases in amplitude did not have a significant effect on the peak area. Finally, an amplitude of 0.1 V was considered the optimal amplitude for subsequent separation detection experiments.

[0135] Since separation efficiency is affected by the fluid flow rate in electrodynamic microfluidic channels, the flow rate was optimized as shown in Fig. 4(C). Flow rates were tested from 0.5 to 5.0 μL / min, and at 0.5 μL / min, separated peaks appeared at 356 ± 120 s, 678 ± 192 s, and 918 ± 85 s for A549, Colo 205, and MCF-7 cells, respectively. With increasing flow rate, the distance between peaks also increased, with peaks appearing at 188 ± 102 s, 528 ± 250 s, and 780 ± 138 s for the three cells at a flow rate of 2.0 μL / min. When the flow rate was 5.0 μL / min, peaks appeared for A549, Colo 205, and MCF-7 cells at 108 ± 88 s, 222 ± 174 s, and 538 ± 154 s, respectively, and peak overlap was observed. Since the distance between peaks was longest and there was no peak overlap, a flow rate of 2.0 μL / min was considered optimized for the separation and detection of cancer cells.

[0136]

[0137] 5. Detection of colorectal cancer cells and interference effect

[0138] The analytical performance of the electrodynamic microfluidic channel sensor was evaluated by a calibration curve using cultured colorectal cancer cells added to healthy donor blood. A sample series containing up to 100 colorectal cancer cells was analyzed. Referring to Fig. 5(a), the increased current response with increasing cell count is due to the accumulated electrochemical signal generated by the idarubicin-modified cancer cells. As shown in Fig. 5(b), the generated calibration curve demonstrates a clear correlation between the number of cancer cells and the measured response peak area (nC).

[0139] The sensor exhibited an excellent linear response (R² = 0.991) over a range of 3 to 100 cells. This isolation detection platform demonstrated superior detection capability with a lower limit of detection (LOD) of 3 cells. This calibration curve helps estimate the number of circulating tumor cells (CTCs) in patient blood samples. The satisfactory performance demonstrates the successful modification of electrodynamic microfluidic channels using time-resolution square wave voltammetry as the detection method. The potential of this device for early, rapid, and sensitive CTC detection in complex biological samples is highlighted.

[0140] To successfully isolate and detect colorectal cancer cells from a complex environment, various cancer cells were added to healthy donor blood. A mixture containing lung, colorectal, and breast cancer cells (10 cells each) was treated with idarubicin using the same procedure. The raw data was processed in a time-resolved format by trSWV software developed in the laboratory. All cancer cell lines used for the isolation experiment have varying sizes. The approximate size of lung cancer cell line A549 is about 7–10 μm, colorectal cancer cell line is about 11.0–12.96 μm, and MCF-7 breast cancer cell line is about 9.5–17 μm. In other words, all cells exhibit different motility within the channel, and when the data is converted into a time-resolved chromatogram, three characteristic peaks appear. As shown in Fig. 5(c), this exhibits three characteristic electrochemical peaks at 265 seconds (peak area: 170.0 nC), 600 seconds (peak area: 188.8 nC), and 880 seconds (peak area: 182.7 nC), corresponding to lung cancer cells, colorectal cancer cells, and breast cancer cells, respectively. As can be seen in the inset of Fig. 5(c), the difference in migration time is attributed to differences in cell size, which affects migration through microfluidic channels. Through a size-based separation mechanism, colorectal cancer cells can be effectively separated from other cell types. This confirms the potential of electrodynamic microfluidic channel sensors for the selective separation and detection of specific cancer cell types in complex biological samples.

[0141] To evaluate potential interference in detection performance, EDTA, albumin, PD-L1, and K were tested on healthy donor blood. +Colorectal cancer cells were added along with various interfering substances, including salicylic acid. Despite the presence of these substances, the electrodynamic microfluidic channel successfully detected the cancer cells, as shown in Fig. 5(d)(i). In comparison, the current response was slightly higher in the absence of interfering substances, as shown in Fig. 5(d)(ii). This confirmed the robustness and reliability of the sensor in overcoming interference while maintaining accurate detection in complex biological samples.

[0142]

[0143] 6. Sensor Stability and Actual Sample Analysis

[0144] The long-term stability of the electrodynamic microfluidic channel sensor was evaluated for 90 days using colon cancer cells (3 cells). As shown in Fig. 6(a), stability evaluations were performed every 10 days, and despite a slight degradation in current response, the sensor maintained stable performance for 3 months with satisfactory sensitivity of >90% of the initial response for up to 90 days. To ensure long-term preservation, the channel sensor was stored at room temperature in a sealed container under an N2 gas atmosphere. The aptamer used in the sensor offers advantages such as thermal stability and extended shelf life, which contribute to the consistent performance of the sensor.

[0145] To verify the validity of the time-resolved SWV (trSWV) detection method in electrodynamic microfluidic channels, experiments were conducted with and without the addition of colorectal cancer cells to pre-treated blood samples from healthy donors. As a result, as shown in Figure 6(b)(i), no electrochemical signal was detected in the healthy donor blood after IDA treatment. On the other hand, when IDA-treated cancer cells were introduced, a characteristic electrochemical signal was observed as shown in 6(b)(ii), confirming that colorectal cancer cells were successfully detected by the microfluidic channel sensor.

[0146] Furthermore, the reliability of the microfluidic channel device was evaluated using patient blood samples collected in tubes filled with CellSave EDTA to prevent coagulation and stabilize circulating tumor cells. Samples were obtained from patients of various ages, sexes, and cancer stages, including early, advanced, and metastatic, as shown in Table 1 below:

[0147] Sample Age Gender Cancer Type Cancer Stage of Progression Cell Count remark 168M Colorectal Cancer T1aN0 M0,Stage 132 62M Colorectal Cancer T2N1 M0,Stage 1 or 243 62M Colorectal Cancer Stage 244 81M Colorectal Cancer 10 Bone metastasis 57 4F Colorectal Cancer T4aN2 M18 Liver metastasis 65 6M Colorectal Cancer Tis 0 Only limited to lamina propria 76 1M Colorectal Cancer p T4N2a 58 65M Colorectal Cancer T3N0 59 47F Colorectal Cancer T4aN1 M17 Metastasis 107 6M Colorectal Cancer T4aN2 4

[0148]

[0149] Some patients developed bone or liver metastases originating from primary colorectal cancer. Samples from 10 colorectal cancer patients were analyzed using a microfluidic channel sensor, and the system successfully detected 9 samples, achieving a detection efficiency of 90%. Generally, the number of CTCs is related to cancer stage and aggressiveness, and patients with localized cancer may release fewer cells.

[0150] The experimental results indicated that CTCs were more prevalent in patients with advanced or metastatic conditions. It was estimated that approximately 3 to 10 cells were distributed within 2.5 mL of collected patient blood samples. Depending on the stage and extent of cancer progression, it can be estimated that there are approximately 9 to 30 circulating tumor cells within 7.5 mL of the patient's blood. The number of detected cells varied with each repeated test, suggesting that the distribution within the blood is not uniform. One patient's sample was not detected by this system; this is likely because the cancer was in an early stage confined to the mucosal layer, where the CTC release rate is low, and thus no undetectable signals were generated. The experimental results were consistent with information obtained from the hospital, verifying the system's performance.

[0151]

[0152] 7. trSWV Software Data Analysis

[0153] For trSWV-based detection, the existing square wave voltammetry was modified to enable the repetitive recording of voltammetry within a clear redox potential range. Multiple voltammetry reaction currents were recorded during CTC separation in an electrodynamic microfluidic channel.

[0154] trSWV data for colorectal cancer CTC detection were analyzed using a trSWV processing method developed in-laboratory according to a systematic approach. The analysis began with post-experiment data collection, followed by data preprocessing. The collected data was saved in CSV format, and recurring translocation values ​​across multiple sweeps were removed using custom Visual Basic code in Microsoft Office Excel. The data was processed using in-laboratory software developed in Python for trSWV analysis.

[0155] The software interface consists of three main sections:

[0156] Referring to Fig. 7, the voltammogram generated by the potentiostat (Fig. 7(a)(i)) is displayed in the top left window, and the baseline-subtracted voltammogram is displayed in the top right window (Fig. 7(a)(ii)). Finally, the software calculates the peak current of the baseline-subtracted voltammogram within a specific potential range and records it for the corresponding time to generate a time-resolution electrochromatogram displayed in the bottom window (Fig. 7(a)(ii)).

[0157] The time-resolved chromatograms were exported as CSV files and further analyzed using commercial graphing software (Fig. 7(b)). For CTCs coated with idarubicin, the analysis focused on the cathodic current. The time-resolved chromatograms exhibit an optimal signal-to-noise ratio, ensuring clear signal separation and precise identification of CTCs. This analysis method improves data accuracy and reliability, thereby increasing the precision of electrochemical measurements.

[0158]

[0159] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A microfluidic separation channel provided in a first direction through which a sample containing one or more cells selected from cancer cells or circulating tumor cells (CTCs) moves; and It includes a detector located at the first directional end of the microfluidic separation channel and comprising a working electrode, a counter electrode, and a reference electrode, A microfluidic channel sensor device characterized by the above-mentioned working electrode being surface-modified with one or more materials selected from the group consisting of carbon nanomaterials, conductive polymers, and cancer-specific aptamers, thereby selectively detecting cancer cells or circulating tumor cells.

2. In Paragraph 1, The above carbon nanomaterial is, A microfluidic channel sensor device characterized by being selected from multi-walled carbon nanotubes (mWCNT) or graphene oxide.

3. In Paragraph 1, The above conductive polymer is, (2,2':5',2"-terthiophene)-diamine ((2,2':5',2"-terthiophene)-3',4'-diamine, DAT), poly-2,2:5,2-terthiophene-3-p-benzoic acid (poly-2,2:5,2-terthiophene-3-p-benzoic acid, pTBA), poly-5,2-5,2-terthiophene-3-carboxylic acid (poly-{5,2-5,2-terthiophene-3-carboxylic acid}, pTTCA), A microfluidic channel sensor device characterized by comprising one or more selected from the group consisting of poly-{4'-([2,2':5',2''-terthiophen]-3')-[1,1'-biphenyl]-4-carboxylic acid (TTBPA), poly-{4-(2,5-di(thiophen-2)-1H-pyrrol-1)benzoic acid (DTPBA).

4. In Paragraph 1, The above-mentioned cancer-specific aptamer is, A microfluidic channel sensor device characterized by having one or more selected from the group consisting of colorectal cancer-specific aptamer W3, anti-EpCAM (epithelial cell adhesion molecule), CA19-9, and Eph receptor A2+A3.

5. In Paragraph 1, The above working electrode is, A microfluidic channel sensor device characterized by being sequentially surface-modified with multiwalled carbon nanotubes (mWCNT), poly-2,2:5,2-terthiophene-3-p-benzoic acid (pTBA), and colorectal cancer-specific aptamer W3.

6. In Paragraph 1, The above microfluidic channel sensor device is, A microfluidic channel sensor device characterized by having a high detection sensitivity of more than 90% for 90 days.

7. A step of manufacturing a sensor device comprising a microfluidic separation channel through which a sample moves, and a detector comprising a working electrode, a counter electrode, and a reference electrode for detecting cells, by screen printing an ink mixed with carbon and silver onto a substrate; and A method for manufacturing a microfluidic channel sensor device for detecting cancer cells or circulating tumor cells, comprising the step of modifying the separation channel and working electrode.

8. In Paragraph 7, The step of modifying the separation channel is, A step of forming a polymer layer by electrochemically polymerizing a conductive monomer on the inner wall of the separation channel; and A manufacturing method characterized by including the step of bonding lipid molecules to the polymer layer formed above.

9. In Paragraph 7, The step of modifying the above working electrode is, A step of treating a dispersion in which carbon nanomaterials are dispersed on the surface of the above-mentioned working electrode; A step of forming a polymer layer by treating a conductive polymer solution on the working electrode surface treated with the above dispersion; and A manufacturing method characterized by including the step of immobilizing an arm-specific aptamer on the polymer layer formed above.

10. A method for detecting cancer cells or circulating tumor cells, comprising the step of injecting a sample containing cancer cells or circulating tumor cells into a microfluidic channel sensor device according to any one of claims 1 to 6 and applying an alternating voltage.

11. In Paragraph 10, The above sample is, A detection method characterized by being treated with one or more anticancer agents selected from the group consisting of Idarubicin, Daunomycin, and Doxorubicin.

12. In Paragraph 10, A detection method characterized by further including, after the step of applying the above-mentioned alternating voltage, a step of detecting the electrochemical signal of cancer cells or circulating tumor cells using time-resolved square wave voltammetry (trSWV) and converting it into an elution chromatogram.

Images