TW202144077A - Method of manufacturing 3d pedot-based bioelectronic device and label-free detection of rare cells - Google Patents

Abstract

The present invention provides amethod of manufacturing 3DPEDOT-based bioelectronic device and label-free detection of rare cells. First, the 3D PEDOT-based BEI devices are integratedwith the PLL-g-PEG-biotin coating for studying the capture efficiency of CTCs in terms of both biochemical and topographical effects, and explored their possibilities for dynamically and sequential controlling rare cells capture/release performance through the cyclic potential of ES. Additionally, the OECT device module was prepared using the high electrical conductivity of PEDOT materials as the active channel layers for rapid monitoring rare cellscapture performance on chips, as well as for identifying the cancer cell phenotypes.

Description

Preparation of three-dimensional PEDOT-based bioelectronic device and method for standard-free quantitative detection of rare cells

The present invention relates to a method of capturing, releasing and sensing rare cells; more particularly, to a method of capturing, releasing and sensing circulating tumor cells without calibration technology.

Bioelectronics is developing in biomedical applications where bioelectronic devices can provide electrical signals for monitoring biological activity, electrical stimulation (ES) for medical therapy, drug or cell release for closed feedback control electrical trigger response. So far, many bioelectronic devices have been used in the interface between artificial electronic devices and living systems and in actual clinical systems. Thus, it is known that this new class of medical devices can be implanted into the human body to precisely modulate electrical signal patterns in the peripheral nervous system, or as a non-invasive method for in vitro liquid biopsy. Most research in bioelectronic devices is accomplished by integrating bioelectric interfaces (BEIs) onto electrodes and/or electronic devices, which are promising interlayers that are unique It utilizes electrons and ions as charge carriers to couple bidirectional currents, thereby enhancing the communication between electronic devices and biological systems. From the perspective of healthcare applications, BEI relies on the selection of organic-conjugated materials (e.g., conducting polymers and carbon materials), because organic BEI materials have low impedance, oxide-free interfaces in aqueous electrolytes due to chemical modification, There are significant advantages in low temperature processing and mass production.

One of the emerging bioelectronic device applications is the isolation of circulating tumor cells (CTCs). Circulating tumor cells are cancer cells that detach from the primary tumor or metastatic site, evade immune surveillance, and then circulate in the peripheral blood with the ability to form distant metastases. There are differences in diagnosing patients with tumor incidence, recurrence and metastasis compared with traditional methods [eg, fresh tissue biopsy, positron emission tomography (PET), computed tomography (CT) and magnetic resonance imaging ( MRI)]. Understandably, "Liquid Biopsy for CTC Sensing" has no side effects of radiation therapy and can be routinely performed in patients with all disease sites. Although CTCs can be readily collected using tumor liquid biopsies, identification of CTCs in patient blood samples is technically challenging due to the extremely low concentrations of CTCs in a large number of blood cells (red and white blood cells). To meet this unmet need for the isolation of rare cells, a number of techniques have been developed that use physical (eg: size, density, charge, and deformability) and biological properties at the device interface [eg: with epithelial Surface binding of cell adhesion molecule antibodies (EpCAM antibodies)]. Specifically, the unique concept of this cell-affinity substrate utilizes the synergistic effect of nanomaterials and CTC marker bioimmobilization to enhance the CTC capture efficiency of liquid biopsy procedures. In addition to seeking high cell capture yield and specificity from human blood on a wafer, vertical integration of downstream properties of CTCs is destined for future CTC wafers, e.g. liquid biopsies (e.g. single CTC genotyping for better understanding potential disease mechanisms and rapid CTC purification from primary to subculture) for various clinical applications.

Recently, conducting polymers [ECPs; e.g., polypyrrole or poly(3,4-ethylenedioxythiophene) (PEDOT)] have been developed due to their excellent electrical transport and electrochemical charge-discharge properties, inherent biocompatibility, and high fabrication It is widely used in organic BEI due to its flexibility. When biochemical and topographical cues are introduced into cells simultaneously, a variety of cellular activities (e.g., adhesion, spreading morphology, proliferation and differentiation) can be manipulated on 3D BEI, expanding their role in cell and tissue regulation efficacy, and in diagnostic and therapeutic applications in non-invasive blood biopsy. In previous studies, three-dimensional PEDOT-based BEIs have been developed, and carboxylic acid-grafted PEDOT (PEDOTAc) nanostructures were prepared via electrochemical and chemical oxidative polymerization methods, which were then conjugated to EpCAM antibodies. Therefore, these 3D BEIs can be used as NanoVelcro cell affinity wafers to enhance CTC capture performance. However, further studies on the nanostructural characterization of 3D BEIs and their functional integration to dynamically control biological responses within the device and develop next-generation bioelectronics for point-of-care cancer are still required.

The contents disclosed in the above background description paragraphs are only for enhancing understanding of the background of the present invention. Therefore, the above contents do not constitute prior art that hinders the present invention, and should be well known to those skilled in the art.

In view of this, the present invention utilizes a novel and easy-to-use method for the preparation of high-quality and large-area PEDOT nanofiber coatings, which not only integrates 3D BEI into bioelectronics for rare cell isolation, but also Operation with integrated conductive polymer system (CP-based) organic electrochemical transistors (OECTs) is demonstrated to further provide downstream label-free sensing of cancer cells. Briefly, the present invention first integrates a three-dimensional PEDOT-based BEI device with a PLL- g- PEG-biotin coating to study the capture of circulating tumor cells in terms of biochemistry and surface topography. efficiency and their dynamic and sequential control of circulating tumor cell capture/release properties through electrical stimulation of cyclic potentials. Furthermore, an OECT device module was fabricated using the high conductivity of PEDOT material as an active channel layer to monitor the capture performance of circulating tumor cells on the wafer and identify cancer cell phenotypes.

Specifically, the present invention provides a method for preparing a three-dimensional PEDOT-based bioelectronic device, comprising: mixing an additive solution with a solution comprising PEDOT:PSS and polyethylene oxide to obtain a ternary mixed solution; mixing the ternary The solution is electrospinned to form PEDOT-based nanofibers; the PEDOT-based nanofibers are heat-treated to form a PEDOT-based nanofiber coating; and the PEDOT-based nanofiber coating is placed on a substrate , to form a three-dimensional PEDOT-based bioelectronic device; wherein, based on the total volume of the ternary mixed solution, the solution containing PEDOT:PSS and polyethylene oxide (PEO) is 94 v/v%, and the rest are additive solutions .

In an embodiment of the present invention, the additive solution includes a (3-glycidoxypropyl)trimethoxysilane solution and a dimethylsulfoxide solution.

In a specific embodiment of the present invention, based on the total volume of the ternary mixed solution, the (3-glycidoxypropyl)trimethoxysilane solution is 0.2-6 v/v%.

In the specific embodiment of the present invention, based on the total volume of the ternary mixed solution, the dimethyl sulfite solution is 0-5.8 v/v%.

In embodiments of the present invention, the substrate comprises a single-electrode form for making electrochemical electrodes or a three-electrode form for making organic electrochemical transistors.

In an embodiment of the present invention, the heat treatment is performed at 80-150° C. for 0.5 hour.

Wherein, the method for preparing a three-dimensional PEDOT-based bioelectronic device of the present invention further comprises the step of coating the biopolymer on the PEDOT-based nanofiber coating.

In a specific embodiment of the present invention, the biopolymers include PLL- g- PEG-biotin, streptavidin (or avidin) and specific biomolecules.

In an embodiment of the present invention, the specific biomolecule is a biotinylated antibody.

In the specific embodiment of the present invention, the weight ratio of PEDOT and PSS is 1:2.5～1:6.

In a specific embodiment of the present invention, based on the total weight of the solution including PEDOT:PSS and polyethylene oxide, the weight percentage of polyethylene oxide is 1 wt % to 25 wt %.

Further, the present invention further provides a method for quantitatively detecting rare cells without standard, comprising: introducing a biological fluid sample into a three-dimensional PEDOT-based bioelectronic device obtained by the above method to capture the biological fluid sample. and providing electrical stimulation to excite a three-dimensional PEDOT-based bioelectronic device to release the captured rare cells, wherein the bioelectronic device can quantitatively monitor the difference in electrical signals in real time.

In specific embodiments of the invention, the rare cells comprise circulating tumor cells.

In a specific embodiment of the present invention, the number of tumor cells, the cell type and the cancer stage are determined through the electrical signal difference.

In an embodiment of the present invention, the electrical stimulation comprises a cyclic voltammetry method, a constant voltage method or a constant current method.

In an embodiment of the present invention, the voltage range used for cyclic voltammetry is -0.8 to 0.8V.

In a specific embodiment of the present invention, the operating voltage of the OECT is below 1 V for calibration-free detection.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. The technical features, objects and advantages of the present invention will be readily understood from these descriptions and drawings and the scope of the claims. At the same time, in order to make the above-mentioned features and advantages of the present invention more obvious and easy to understand, the following embodiments are given and described in detail with the accompanying drawings.

The advantages and features of the present invention and the methods for achieving the same will be better understood by being described in more detail with reference to the exemplary embodiments and the accompanying drawings. However, the present invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough, complete and complete to convey the scope of the invention to those of ordinary skill in the art, and the invention will only be the scope of the appended claims defined.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be more understandable that terms such as those defined in commonly used dictionaries should be construed as having meanings consistent with the meanings in the relevant art, and should not be construed in an overly formal meaning unless explicitly defined herein.

The singular forms "a," "at least one," and "the" as used herein may also include plural referents unless the context clearly dictates otherwise. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In this paper, the numerical values and parameters used to define the scope of the present invention inevitably contain standard deviations caused by individual testing methods, so they are mostly expressed in approximate numerical values. However, in the specific embodiments, the Relevant numerical values presented as precisely as possible. As used herein, "about" generally depends on the considerations of those of ordinary skill in the art to which the present invention pertains, and generally means that the actual value falls within an acceptable standard error of the mean, eg, the actual value is within a Within ±10%, ±5%, ±1%, or ±0.5% of a specified value or range.

The exemplary embodiments will be described in detail below with reference to the drawings. These embodiments, however, may be embodied in different forms and should not be construed as limiting the scope of the claims of the present invention. These embodiments are provided so that the disclosure of the present invention will be complete and clear, and those skilled in the art will be able to understand the scope of the present invention through these embodiments. Materials and Methods PEDOT Nanofiber Coating Fabrication and Surface Modification

PEDOT: PSS aqueous solution (Clevios PH1000), which is purchased from HC Starck; wherein, the weight ratio of PEDOT to PSS is 1:2.5～1:6, preferably 1:2.5, and PEDOT is poly-3,4-ethylenedioxy Thiophene [poly(3,4-ethylenedioxythiophene)], PSS is polystyrene sulfonate. Polyethylene oxide (PEO) with a molecular weight of 900,000, and (3-glycidoxypropyl)trimethoxysilane (GOPS) were purchased from Sigma-Aldrich. PLL(20)-g[3.5]-PEG(2)/PEG(3.4) -Biotin(50%) (PLL-g -PEG-biotin) and PLL(20)-g[3.5]-PEG(2 )/FITC (PLL-g-PEG-FITC) was purchased from SuSoS (wherein, 20, 2, 3.4 are molecular weight k Dalton, 3.5 is the value of lysine unit/PEG chain, the information can be known from the product itself , which will not be repeated here). Streptavidin (Streptavidin; SA; 1 mg mL ^-1 ) was purchased from Invitrogen. Biotinylated EpCAM antibody (anti-humanEpCAM)/TROP1 antibody (goat IgG) was purchased from R&D Systems. Among them, the streptavidin used in the present invention can also be replaced by Avidin to achieve the purpose. cell research

Breast cancer cell line (MCF7), lung cancer cell line (PC9), cervical cancer cell line (HeLa) and monocytic cell line (THP-1) were purchased from American Type Culture Collection. GlutaMAX-I, Vybrant ^® DiO cell labeling solution, DMEM and RPMI 1640 growth medium lines were purchased from Invitrogen. Fetal bovine serum (FBS) was purchased from Hyclone; Hoechst 33342 and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer were purchased from Life Technologies. Preparation of PEDOT Nanofiber Coatings

The PEDOT:PSS films show poor wet stability in water and high humidity environments, which is due to the presence of water-soluble PSS domains, not to mention the wet instability of the PEDOT:PSS nanofibrous structures. In order to effectively improve the wet stability of the conductive PEDOT nanofiber coating, the cross-linking reaction with other additional cross-linking agents can also react with itself or with PEDOT:PSS, thereby enhancing the water resistance of the PEDOT:PSS nanofiber coating . For the water resistance test, the present invention prepared various quantities of PEO materials as a mixture of PEO/PEDOT:PSS and GOPS, and then used a commercially available needle-type electrospinning system for electrospinning. The reason for using PEO is to enhance the spinnability of the PEDOT:PSS solution and to form a stable chemical cross-linking reaction between the PEO and PSS materials. In addition, 0.2-6 v/v% (preferably 1 v/v%) of GOPS and 0-5.8 v/v% (preferably 5 v/v%) of dimethylsulfoxide (DMSO) were added to the In the PEO/PEDOT:PSS mixed solution (that is, the solution containing PEDOT:PSS and polyethylene oxide (PEO) accounts for 94 v/v%), the optimized water resistance and enhanced electrical conductivity are further obtained, respectively. Can be used for electrospinning. In the electrospinning system of the present invention, the PEDOT-based nanofiber coating can be collected on the target electrode by controlling the spinning rate and deposition time using a syringe pump. Connect a high voltage power supply to a 27 gauge disposable needle to control the diameter of the nanofibers; use an air blower at room temperature to keep the relative humidity below 30% to prevent the nanofibers The conductive nanofibers are erected during silent electrospinning due to the negative charge (see Fig. 1(a)). Device assembly and surface modification on PEDOT- based nanofiber coatings

Devices assembled from BEI-coated ITO glass (single-electrode form) of the 3D PEDOT system contain cylindrical PDMS chambers with a height of about 4 mm and a diameter of about 10 mm, or mixers for PDMS microchannels. The device was treated with PLL- g ^-PEG- biotin (100 μg mL ^{– 1} in 10 mM HEPES buffer (pH 7.4)) for 1 h, and then incubated with SA (10 μg mL ^{– 1} in 1x PBS) for 1 h at room temperature . Biotinylated EpCAM antibody (anti-humanEpCAM)/TROP1 antibody (10 μg mL ^-1 in 1x PBS containing 0.1% BSA and 0.09% NaN ₃ ; 25 μL) was placed on the device and incubated at room temperature ( 60 minutes). Finally, the three-dimensional PEDOT-based BEI devices were washed several times with 1X PBS and then soaked in 1X PBS for 1 hour before cell experiments. Scanning Electron Microscope (SEM)

Using a scanning electron microscope (FEI Nova NanoSEM 200; accelerating voltage: 10 keV), the surface morphology of the PEDOT-based nanofiber coating and the circulating tumor cells on the wafer was observed. Prior to SEM testing, biological and PEDOT-based samples were dehydrated in grade-up EtOH (25%, 50%, 75% and 100%; each dehydration time: 20 min) and critical point drying with liquid carbon dioxide , then sputtered with gold (>3 nm). Cell capture / release assay

After removing 1X PBS from the BEI device of the 3D PEDOT line, 100 μl of cell suspension (10 ⁵ cells mL ⁻¹ ) was assembled onto the wafer. After 1 hour incubation at 37°C, 5% carbon dioxide, the substrates were gently washed (at least five times) with 1x PBS. Imaging and enumeration of cells were performed using a fluorescence microscope (CKX41, Olympus). And an automatic cell counter (Luna ^™ automatic cell counter, purchased from Logos Biosystems, Korea) was used for cell counting of dilution experiments and cell viability could be easily determined. Following the capture procedure described above, substrate-modified cancer cells were trypsinized and collected. Finally, cell lines collected from each device were prepared in 100 μl of cell suspension, which were then carefully transferred to counting slides on the Luna™ Automated Cell Counter. Cell viability was analyzed in triplicate. For electrically-triggered cell release studies, the three-dimensional PEDOT-based BEI device was operated using a standard three-electrode setup consisting of: a counter electrode (Pt), a reference electrode (Ag/AgCl), and a working electrode (BEI of the 3D PEDOT line, coated with ITO glass), and electrical stimulation (ES) of cyclic potential scanning from 0 to +0.5 V at a scanning rate of 100 mV/s in 1x PBS (electrical stimulation; ES) was performed 20 cycles. All experiments were repeated more than three times and showed similar results. Fluorescence images of monolayer PLL-g -PEG-FITC were observed on a three-dimensional PEDOT-based BEI device using a confocal microscope (FV1000; Olympus), and the changes in fluorescence density after electrical stimulation were monitored. Results and discussion

As shown in Fig. 1b, the PSS/PEO cross-linking reaction mechanism has been proposed by some scholars. In a strongly acidic environment, such as in the presence of PSS, the ether group in PEO is cleaved by protonation, and a hydroxyl group is formed at the end of the cleaved PEO chain. Under high temperature thermal annealing, these hydroxyl groups further react with sulfonic acid groups in PSS to form sulfonic acid esters. However, the presence of the cross-linked PEO/PEDOT:PSS mixture was insufficient to maintain the 24-hour long-term stable operation of the PEDOT-based nanofiber coating in PBS buffer (pH 7.2), so the present invention further added GOPS as another A cross-linking agent to improve the mechanical stability of the fibers and adhesion to the substrate, thereby preventing partial dissolution or delamination of the PEDOT-based nanofiber structure when immersed in an aqueous electrolyte solution. Different amounts of PEO additives (NF5, NF10, NF15, NF20, NF25) are included through the combination of the condensation reaction between PSS and PEO and the chemical reaction between GOPS and the components of the mixture; the numbers indicate the weight of the PEO components The percentages are respectively from 5 wt%, 10 wt%, 15 wt%, 20 wt%, to 25 wt%, which are based on the total weight of the solution of PEO/PEDOT:PSS) PEDOT-based nanofiber coating can be successful The diameter changes of the nanofibers were investigated by comparison with 1x PBS buffer treatment for 24 hours (Fig. 1c-1).

In order to better understand how the cross-linking reaction affects the water resistance and dimensional stability of the PEDOT-based nanofiber coating, the present invention conducted a further statistical analysis on the variation of the nanofiber diameter distribution based on the SEM results (Fig. 2a). As shown in Fig. 1c–l, the electrospun PEO/PEDOT:PSS nanofibers were heat-treated at 80–150 °C for at least 0.5 h to crosslink the PEDOT-based nanofibers, followed by water treatment for 24 h; The cured PEO/PEDOT:PSS nanofibers were seen to remain intact without any dimensional or structural changes, indicating improved water resistance and dimensional stability of the fibers after the chemical crosslinking step. Except for the NF5 nanofiber coating, the nanofiber structures in other nanofiber coatings (NF10, NF15, NF20, and NF25) exhibited good dimensional stability without significant damage. Since a higher content of PEO additive will lead to higher electrical resistance, the PEO weight percentage of more than 25 wt % is not used in the present invention. It is worth noting that the nanofibrous structures of NF5, NF10, and NF15 were easily partially dissolved when the diameter decreased by about 40 nm in PBS buffer; conversely, NF20 and NF25 were immersed in 1x PBS buffer for 24 hours, and PEO hydrogels showed an increase in fiber diameter in the swelling behavior.

Although many previous technical literatures mentioned that adding an appropriate amount of PEO would lead to a more than 100-fold increase in the conductivity of PEDOT:PSS, this phenomenon was not observed in the present invention after heat treatment for 12 hours at 130°C, which was attributed to the appearance of unmeasurable conductivity values (Table 1). In order to explore the conductivity changes of PEO/PEDOT:PSS nanofiber coatings, the present invention used transmission electron microscopy (TEM), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) on PEO/PEDOT:PSS mixtures The phase separation was characterized. In this example, since NF10 has the best dimensional stability and conductivity, TEM image analysis was first performed on the as-spun and thermally annealed NF10 samples to determine the mechanism of the decrease in conductivity. , as shown in Figures 3a-3b. The needle-type silent electrospinning process likely resulted in the formation of the core-shell nanofiber structure of the PEO/PEDOT:PSS of NF10, suggesting that the geometry of the structure is composed of a non-smooth and PEDOT-rich nanofiber core , the inner core is surrounded by a thin PSS-rich layer. However, under appropriate thermal crosslinking conditions, thermally-induced phase separation of both PEDOT-rich and PSS-rich regions can be achieved, resulting in smooth and PEDOT-rich nanofiber cores that are surrounded by thick and PSS-rich cores. layer surrounding, resulting in higher contact resistance measured by the four-point probe. To confirm the demonstration of the present invention, the present invention adopts the tapping mode of atomic force microscope (Fig. 3c-f), by measuring the morphology and phase diagram ^{of NF10 (area: 1x1 μm 2 ) coated on ITO glass substrate} to get the reason for the decrease in conductivity. Although the as-spun and thermally annealed conditions of NF10 have similar topographical features, the phase diagrams show some changes. In the phase diagram, hard domains are shown as bright regions, which the present invention assigns to PEDOT-PSS (PEDOT-rich) regions; while dark regions are shown as soft structures, which belong to excess PSS and/or PEO regions. The present inventors observe that thermal annealing treatment induces phase separation characterized by segregation of PSS and/or PEO regions surrounded by a continuous PEDOT-PSS phase to a greater degree than that of just spinning on the phase diagram images The resulting nanofiber coating is high. Table 1. Electrospinning parameters, dimensions and electrical properties of PEDOT-based nanofiber coatings sample Flow rate (ml hr ^-1 ) Applied voltage (kV) Chip resistance (Ω sq ^-1 ) Coating thickness (nm) Conductivity (S cm ^-1 ) Resistance (M) Fiber Fragments Fiber diameter (nm) Single Fiber Conductivity (mS cm ^-1 ) NF5 1 25 0.23±0.11 660 ± 217 87.9±3.6 0.22 8375 158 ± 30 2.8 ± 0.8 NF10 0.6 20 0.27±0.01 969 ± 169 38.6 ± 6.2 0.27 11750 162 ± 29 1.5 ± 0.4 NF15 0.65 twenty four 0.33±0.12 1195 ± 72 28.8±1.7 1.50 5750 144 ± 30 0.7 ± 0.3 NF20 0.85 twenty four 0.21±0.09 4867±68 13.7 ± 3.5 1.20 57000 120 ± 22 0.13±0.04 NF25 0.78 twenty three 0.27±0.01 3043±20 12.7 ± 2.7 5.16 12250 188 ± 10 0.06±0.01

AFM tapping-mode images often cannot directly observe morphological changes because the phase image signal is affected by factors such as surface forces, tip indentation, and volumetric properties. To confirm the present invention's hypothesis of a correlation between PEO/PEDOT:PSS phase separation and conductivity changes, the present inventors performed XPS experiments on NF10 to determine the internal compositional changes of the surface during thermal annealing. For XPS survey spectra and high-resolution kernel-level spectra (S _2p ) of NF10 before and after thermal annealing, Figure 4 shows. These XPS full spectrum analysis results are in good agreement with all blending components in PEO/PEDOT:PSS solution, where core level lines (O _1s , C _1s , S _2p and Si) on the nanofiber surface _2p ) only with some changes. In addition, Table 2 summarizes the chemical composition of NF10 analyzed from XPS results. The present inventors found that more Si features were observed on the surface of the thermally annealed nanofiber coating, resulting in Si _2p core-level features, and then a small amount of insulating GOPS was seen squeezed from the just-spun sample. to the PSS-rich shell, resulting in reduced conductivity. A deconvolution process of the S( _2p ) kernel level lines of XPS was performed to monitor changes in the PSS/PEDOT composition ratio during thermal annealing. The XPS full spectrum analysis spectra of both samples exhibit high intensities between 168.0 and 168.9 eV, which correspond to the spin-splitting components of the sulfur atoms of the PSS chains. Between 163.9 and 167.0 eV, the present invention can see two other small peaks of the two spectra, which are assigned to the sulfur atoms of the PEDOT fragment (Fig. 4b-4c). _{The S 2p 3/2} area ratio of PSS (stronger) and PEDOT (weaker) can be used to estimate the content of PSS-rich regions on the surface. It can be calculated that the composition ratio of PSS to PEDOT is 2.09 for the freshly spun sample, while the composition ratio of PSS to PEDOT increases to 2.56 after thermal annealing at 130 °C for 12 h, which shows that a small amount of insulating PSS has been mixed with PEDOT-rich samples. The nanofiber core structure phase-separated, resulting in a significant decrease in electrical conductivity. Table 2. Atomic percentage of core energy level lines (O _1s , C _1s , S _2p and Si _2p ) on nanofiber coating surfaces (from XPS data, calculated using Shirley method); PEDOT, PSS and PSS to PEDOT ratio Atomic percentage of (peak deconvolution _{of S 2p data from XPS)} sample Atomic percentage of data from XPS full spectrum analysis C _1s O _{1s S2p} Si _2p just spun out 64.54 32.52 0.83 2.11 thermal annealing 63.11 29.88 0.97 6.04 sample Atomic percent from XPS (S _2p ) data PEDOT PSS Ratio of PSS to PEDOT just spun out 32.4 67.6 2.09 thermal annealing 28.1 71.9 2.56

Although the PEDOT-PSS phase-separation mechanism of thermally annealed PEDOT-based nanofiber coatings is not fully understood, the preferential formation of thick PSS and/or PEO insulating layers becomes one of the key points for the decrease in conductivity, and a possible source of from the thermal annealing process. In order to prove that the PEDOT-based nanofiber coating of the present invention can be applied to BEI, the present invention firstly detects the electrochemical properties of the PEDOT-based nanofiber coating devices (NF5, NF10, NF15, NF20, NF25) (Fig. 5a-5b). ). Cyclic voltammetry (CV), electrochemical impedance spectrum (EIS) were performed using a standard three-electrode system with a PEDOT-based nanofiber coating device. First, CV (voltage sweep from -0.8 to 0.5 V) was used to explore the charge capacity density (CCD) of various sizes of PEDOT-based BEI in phosphate buffered saline (PBS, 0.1 M, pH 7.2) (Fig. 5a). The higher CCD of NF5 (0.78 mC/cm ² ) and NF10 (0.36 mC/cm ² ) indicates that they are more characteristic than other structures (NF15, NF20, NF25; 0.19 mC/cm ² , 0.06 mC/cm ² , 0.06 mC/cm 2 , the apparatus 0.05mC / cm ²⁾ has a higher content of PEDOT, and compared to the ITO electrodes (0.21 mC / cm ²⁾ can be more effectively reduced (dark blue) and oxidized (blue light gray) state release cell. Further, the present invention uses the impedance (FIG. 5b) means EIS measurements in the frequency range of 1 to 10 ⁵ Hz to. With the increase of PEO content in the nanofiber coating (0.1M, pH 7.2), the electrode impedance of PEDOT-based nanofiber coating at all frequencies increases moderately, which indicates that high PEDOT:PSS content will facilitate Ion exchange and doping/dedoping processes are performed during the cycling potential of the ES. Previous studies indicated that electrochemical polarization of the substrate would lead to local pH changes that would cause the dissolution of the monolayer PLL-g -PEG from the electrode, allowing the recovery of the cell sheet and the extension of neurites in neurons. Therefore, the present invention expects to integrate a three-dimensional PEDOT-based nanofiber BEI device with PLL- g- PEG-biotin to demonstrate the efficient performance of both the capture of circulating tumor cells and the electrical-triggered cell release.

A key feature for capturing/releasing circulating tumor cells is the dynamic control of PLL- g- PEG-biotin coating on a 3D PEDOT-based BEI device. To demonstrate the concept of operating circulating tumor cells on a wafer, FITC-PLL- g -PEG was first coated on the surface of NF10 devices, followed by electrical stimulation (ES) using 20 CV scan cycles (voltages from -0.8 to 0.5 V) Decreased fluorescence intensity with electrical triggering (Figures 6a-6c). As shown in Figure 6a, after soaking in FITC-PLL-g -PEG solution (100 μgmL ^-1 in 10 mM HEPES buffer) for one hour, NF10 will reach saturation adsorption; the nanofiber structure is surrounded by a uniform fluorescent coating . As known from previous literature techniques, even with the potential range of -1 to 1 V, the observed cellular function and activity are negligible. Therefore, it is believed that the oxidation-driven hole transportation of the PEDOT:PSS backbone will not only lead to the desorption of FITC-PLL-g -PEG from the PEDOTAc surface, but also ensure that circulating tumor cells characterize cells downstream Activity and biological function (Figures 6b-6c).

In order to demonstrate the potential use of PEDOT-based nanofiber-coated devices as a liquid biopsy method for personalized cancer diagnosis, the present invention firstly deposited time-dependent electrospinning (NF10 deposition for 20 minutes, 15 minutes, 10 minutes, 5 minutes) , 1 min, and 0 min) studies examined differences in cell capture efficiency (Fig. 7a). The device configuration was based on a series of surface modification procedures using PLL-g- PEG-biotin, streptavidin (SA), and biotinylated EpCAM antibodies (Fig. 6d-g). Monolayer PLL- g- PEG-biotin interacts with negative charges on the surface of PEDOT-based nanofibers via electrostatic attraction. High-affinity SA will then link the biotinylated EpCAM antibody to the monolayer PLL- g- PEG-biotin on the device, providing specific binding to circulating tumor cells. In the present invention, MCF7 breast cancer cells (EpCAM positive cell line), PC9 non-small cell lung cancer cells (EpCAM positive cell line), HeLa cervical cancer cells (EpCAM negative cell line) and THP-1 leukemia cells ( EpCAM-negative cell lines) for 1 hour. Due to the specific cell-substrate interaction and its high degree of transparency, the present invention uses an inverted optical microscope to monitor cell capture efficiency by using bright-field phase contrast images of cell capture on a wafer. Notably, a 1-minute electrospinning deposition time of the PEDOT-based nanofiber coating has provided an efficient technique for capturing MCF7 cells with a cell capture yield of approximately 1900 cells/mm ² . Longer deposition times (5 minutes, 10 minutes, 15 minutes, 20 minutes) slightly enhanced cell capture with cell densities of approximately 1787 cells/mm ² , 1856 cells/mm ² , 1894 cells/mm ² and 2104 cells, respectively /mm ² . Furthermore, applying the NanoVelcro concept to circulating tumor cells observed extremely high cell capture rates for MCF7 and PC9 cells, while lower non-specific binding capture rates for HeLa and THP-1 cells. Results of cell capture efficiency and the cells release yields are summarized in Figure 7b; MCF7 and PC9 cell capture densities indeed play a positive role, to show in the range of 1750 to 2000 cells / mm ^2, while maintaining on HeLa and THP-1 cell adhesion resistance. Integrating PLL- g -PEG-biotin with the nanofiber coating of three-dimensional PEDOTAc will provide optimized PEG density, which is sufficient to ensure the binding resistance of two non-specific HeLa/THP-1 cells and a sufficient amount of biotin to For SA binding. Therefore, it is believed that the three-dimensional BEI device of the PEDOT line has the ability to integrate with other biotinylated antibodies, thereby enabling rapid isolation of rare cells. Even other specific biomolecules can be used in the present invention as long as they meet the requirements of the present invention in principle.

In order to verify the potential application of the BEI device system of the present invention to adapt to the downstream characterization of circulating tumor cells, the electrospinning deposition times of different NF10 devices were therefore selected to demonstrate the efficacy of dynamically controlling the capture/release of circulating tumor cells on the wafer (Fig. 7a). As expected, the optimized 20 CV scan cycles (voltage sweep from -0.8 to 0.5 V) resulted in a yield of MCF7 cells released from the device's electrospinning deposition times (10 min, 15 min and 20 min) of approximately 90% (Figures 7a and 7c). However, these results illustrate that the electrospinning deposition times (5 min, 1 min and 0 min) of the BEI device did not provide higher cell release than the other deposition times during ES scan voltages in the range of -0.8 to 0.5 V performance due to lower CCD values and/or smaller local pH changes of nanofiber coatings during cycling potential ES. In addition, a constant voltage method or a constant current method can also be used in this embodiment, and the effect thereof is also similar to that of the cyclic voltammetry method, which will not be repeated here.

In order to use the electrically triggered cell release function for downstream characterization of circulating tumor cells, the present invention explores the device of the present invention at a series of low cell densities (10, 50 and 100 tumor cells were implanted in 100 μl of cell suspension). Potential use for capture/release of MCF7 cells, showing cell capture yields greater than 90% at various densities of implanted tumor cells and retention of cell yields on NF10 after ES less than 15% Neither was related to the number of engrafted tumor cells (Fig. 7d). In addition, the present invention examined the test-retest reliability of the device for cell capture/release over three cycles. Before cell capture / release studies, through a series of repeated coating PLL- g -PEG- biotin, cells catch and release performance of the device may be about to cell density were restored in the new cycle 45,000 cells / cm ² and 5000 cells/cm ² (Fig. 7e). In addition, after testing, the nanopillar array of the three-dimensional PEDOTAc line of the present invention can not only provide the released cells with up to about 90% cell viability (ex-wafer culture on a polystyrene (TCPS) petri dish), but also Cell viability of expanding cultures (in-chip cultures on NF10 devices) can be maintained.

Furthermore, the present invention can apply the PEDOT-based nanofiber coating to the active layer channel (the size is (W) 1.5 mm x (L) 5 mm) (Fig. 8a), except that liquid samples of MCF7 cells (breast cancer cells) implanted on different wafers (cell density 0; 10 ⁴ ; 10 ⁵ ) can be tested within 1 hour; 10 ⁶ cells/ml) for high-efficiency capture (Figure 8b-e), and dynamic control of cell-substrate interactions through electrical stimulation to achieve cancer cell release effects, and no calibration at an operating voltage below 1V (label-free) to detect the capture and release signals of MCF7 cells on the device, including monitoring the electrical signal changes at different time points (T-0, T-1, T-2, T-3) (Figure 8f-h) . The T-0 time point is the element without any surface treatment; the T-1 time point is the element after PLL- g- PEG-biotin/SA/biotinylated EpCAM antibody coating; T-2 time point 1 hour after the capture of MCF7 cells (suspension cells are implanted with a density of ¹⁰⁵ cells / ml) element; T-3 time points after release of all components by electrical stimulation of MCF7 cells. Mainly, the I _d- V _g output characteristic curve (Fig. 8f) at four different time points can be measured in the experiment , which is converted into the transconductance ( g _m )-V _g transfer characteristic curve (Fig. 8g) by the formula, and _{The V g} corresponding to the maximum value of g _m represents the gate voltage shift (ΔV _g ) at four different time points. From the research results in Figure 8h, it was found that after the T-1 time point was treated with PLL- g- PEG-biotin/SA/biotinylated EpCAM antibody coating, it was observed that compared with the T-0 time point, the performance was higher. +30 mV there is a change ΔVg; T-2 time points after 1 hour capture MCF7 cells, MCF7 cells will be affected due to the effective capacitance per unit area of the resulting change of +52 mV to further enhance ΔV _g ; After the final electrical stimulation was applied to release all MCF7 cells, it was found that ΔV _g showed a change close to -82 mV, which could return to the original signal of the element without any surface treatment, which means that the OECT element can Real-time monitoring of electrical signal differences before and after surface modification, after capturing and releasing circulating tumor cells.

When the above-mentioned PEDOT nanofiber-coated OECT element is used, research has confirmed the detection performance of this device, which can perform standard-free quantitative detection of the number of captured circulating tumor cells, and even use electrical signal sensing to help quickly identify cancer. Cell number, type and cancer stage, etc. (Figure 9). This Example evaluated the implant range of different cancer cell density of experiments, i.e. for 1 hour using a cell density of 0, 10 ^4, 10 ⁵ and 10 ⁶ cells / 1 ml cell suspension into the active layer channels The cancer cells were captured, followed by MCF7, HeLa (cervical cancer cells) and PC9 (non-small cell lung cancer) cell lines, using the measured transconductance response ( g _m = (GG ₀ )/G ₀ ) and gate voltage of Offset (V _g ) differences, standard-free quantitative sensing, and results confirm that the element exhibits quantitatively increased electrical signal differences for the number of captured cancer cells. When the dashed line of (GG ₀ )/G ₀ (%) = 10% and V _g = 0.012 V was used as the criterion, the present inventors found that the quantitative sensing data can provide differences in discriminating cancer cell types ( FIG. 9 a - c ) . Due to the high transparency of the developed PEDOT-based OECT device, in addition to providing sensing of various electrical signals, it can also obtain optical image analysis of cells on the device using an inverted optical microscope without fluorescent staining. The results (including the capture efficiency and coverage area (%) of circulating tumor cells (Figure 9d and Figure 8be) were obtained. In addition, this example also carried out electrical signal analysis for cancer cells of different liver cancer stages after capture ( Including the use of HepG2 cells representing the early stage of liver cancer and drug-resistant Huh7 cells in the late stage of liver cancer) (Fig. 9e). When the same implanted cell density was 10 ⁵ cells/ml after 1 hour of capture, it can be clearly found that The electrical signal difference between HepG2 and Huh7 cells. For example, HepG2 showed a (GG ₀ )/G ₀ (%) value of 6.1%, which was 10% lower than the criterion; V _g was 0.021 V, slightly higher than the criterion 0.012 V ; electrical signal and for Huh7 cells, rendering _{(GG 0) / G 0 (} %) 46.2% value is much higher than 10%; V _g to 0.006 V 0.012 V. thus Study results demonstrated slightly lower than the criteria of this PEDOT The OECT element has the potential to rapidly detect the cancer staging of liver cancer, and even when the cancer cells begin to deteriorate, it can be quickly detected and the user can respond quickly.

All features disclosed in this specification can be combined in any way. Features disclosed in this specification may be replaced by features of the same, equivalent or similar purpose. Accordingly, unless expressly stated otherwise, a feature disclosed in this specification is one embodiment of a series of equivalent or similar features.

In addition, according to the contents disclosed in this specification, those skilled in the art can easily make appropriate changes and modifications for different usage methods and situations according to the basic features of the present invention without departing from the spirit and scope of the present invention. Therefore, other Embodiments are also included in the scope of the patent application.

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Through the detailed description and the accompanying drawings, only the accompanying drawings of the embodiments of the present invention will be more fully understood; therefore, the following drawings are only used to explain the embodiments of the present invention and do not limit the scope of the invention; (a) Schematic diagram of electrospinning equipment of the present invention, (b) chemical structure of PEO/PEDOT:PSS blended solution with DMSO and GOPS, and schematic diagram of sulfonate crosslinking reaction between PEO and PSS, (c) -(g) SEM morphologies of as-spun nanofiber coatings containing different amounts of PEO additives (NF5, NF10, NF15, NF20, NF25), (h)-(1) cross-linked nanofiber coatings The layer was soaked in 1 times PBS buffer for 24 hours; Figure 2 is a statistical analysis of the average fiber diameter distribution changes of various PEDOT-based nanofiber coatings after (a) the water resistance test of the present invention, (b) interdigitated electrodes ( IDE) was used to measure the conductivity of a single fiber with 125 sets of interdigitated fingers with a fingertip width and spacing of 20 μm (DRP-G-IDEAU10, DropSens), (c) the electrodes deposited on IDE The enlarged view of the optical microscope image of the electrospun PEDOT-based nanofiber coating; Figure 3 is the TEM image of the (a) as-spun and (b) thermally annealed PEDOT-based nanofiber coating of the present invention image, surface morphologies (c) and (d), and phase contrast images (e) and (f) of PEDOT-based nanofiber coatings (as-spun and thermally annealed, respectively) obtained by tapping-mode atomic force microscopy ); Figure 4 is the XPS spectrum of the PEDOT nanofiber coating that has just been spun and thermally annealed. (a) General full spectrum scan of XPS, (b) XPS S2p spectrum of freshly spun sample, (c) XPS S2p spectrum of thermally annealed sample; Figure 5 shows various PEDOTs of the present invention on ITO glass in 1X PBS Electrical properties of nanofiber coatings. (a) CV: potential scan rate 100mV / s swept from -0.8 to 0.5V; NF5, NF10, NF15, NF20 and NF25 were 0.78,0.36,0.19,0.06 and a CCD 0.05 mC / cm ^2, (b ) Electrochemical Impedance Spectroscopy (frequency range: 1~10 ⁵ Hz); Figure 6 is a fluorescent image of the PLL-g -PEG-FITC of the present invention coated on a PEDOT nanofiber coating device (NF10 device). (a) without ES, (b) with ES, (c) fluorescence signal frequency of NF10 device relative to fluorescence intensity before/after ES, (d)-(h) PEDOT-PSS phase separation, PEDOT system Conceptual schematic of surface modification of nanofiber-coated devices and their electrically-triggered cell release, (d) as-spun NF10, (e) as-spun NF10 with matte phase-separated morphology, (f) Thermally annealed NF10 with core-shell morphology, (g)-(h) binding of biotinylated antibodies on the device and its mechanism of capturing and releasing circulating tumor cells on the wafer; Figure 7 is (a) electrospinning of the present invention Cell capture efficiency and cell release yields on silk deposition time-dependent studies (NF10 deposition times of 20, 15, 10, 5, 1, and 0 minutes), (b) MCF7, PC9, HeLa, and THP-1 Cell capture efficiency and cell release yield of cell lines, (c) under various CV scan conditions (voltage sweep from -0.8 to 0 V, 0 to 0.5 V, -0.8 to 0.5 V) in 1x PBS at Quantification of the percentage of MCF7 cells remaining on the surface after release of cells by ES for 20 cycles at a scan rate of 100 mV/s, (d) Capture after ES was performed and 10, 50 and 100 cells were implanted in 100 ml of cell suspension Efficiency and percentage of cells retained on NF10 devices, (e) Cell capture/release performance of NF10 devices in multiple/sequential studies with or without ES; Schematic diagram of calibration-free biosensing of Mifiber-coated 3D OECT device for electrical signal monitoring at four different time points during the capture/release of circulating tumor cells, (b)-(e) different implanted MCF7 cells Optical image on OECT device at density (0; 10 ⁴ ; 10 ⁵ ; 10 ⁶ cells/ml), (f) I _d- V _g output characteristic curves at four different time points, (g) four different The transmission characteristic curve of g _m -V _{g at} time points, (h) the relative change of gate voltage (ΔV _g ) at four different time points; and Fig. 9 is the detection of the present invention implanted with different cancer cells on the OECT device Results, (a) Transconductivity changes [(GG ₀ )/G ₀ (%)] and gates for MCF7 cells at different implanted cell densities (0; 10 ⁴ ; 10 ⁵ ; 10 ^{6 cells/ml)} The electrical signal difference of pole voltage change (ΔV _g ), (b) transconductance change for HeLa cells at different implanted cell densities (0; 10 ⁴ ; 10 ⁵ ; 10 ⁶ cells/ml) [(GG ₀ )/G ₀ (%)] and _{the electrical signal difference of gate voltage change (ΔV g} ), (c) for PC9 cells at different implanted cell densities (0; 10 ⁴ ; 10 ⁵ ; 10 ⁶ cells/ milli The electrical signal difference of transconductance change [(GG ₀ )/G ₀ (%)] and gate voltage change (ΔV _{g ) under the} Optical image analysis of coverage area ratio [Coverage Area (%)] on active layer channel after capture at density (0; 10 ⁴ ; 10 ⁵ ; 10 ^{6 cells/ml), (e) for HepG2 and Huh7 stem cancer cells} Electrical signal difference of transconductance change [(GG ₀ )/G ₀ (%)] and gate voltage change (ΔV _g ) at implanted cell density (10 ^{5 cells/ml).}

Claims (16)

A method for preparing a three-dimensional PEDOT-based bioelectronic device, comprising: mixing an additive solution with a solution comprising PEDOT:PSS and polyethylene oxide (PEO) to obtain a ternary mixed solution; performing an electrospinning process on the ternary mixed solution to form a PEDOT-based nanofiber; heat-treating the PEDOT-based nanofibers to form a PEDOT-based nanofiber coating; and placing the PEDOT-based nanofiber coating on a substrate to form the three-dimensional PEDOT-based bioelectronic device; Wherein, based on the total volume of the ternary mixed solution, the volume percentage of the solution containing PEDOT:PSS and PEO is 94 v/v%, and the rest is the additive solution. The method of claim 1, wherein the additive solution comprises a (3-glycidoxypropyl)trimethoxysilane solution and a dimethylsulfite solution. The method according to claim 2, wherein based on the total volume of the ternary mixed solution, the volume percentage of the (3-glycidoxypropyl)trimethoxysilane solution is 0.2-6 v/v%. The method as described in claim 2, wherein based on the total volume of the ternary mixed solution, the volume percentage of the dimethyl sulfite solution is 0-5.8 v/v%. The method of claim 1, wherein the substrate comprises a single-electrode pattern for making electrochemical electrodes or a three-electrode pattern for making organic electrochemical transistors. The method according to claim 1, wherein the heat treatment is performed at 80-150°C for at least 0.5 hour. The method according to claim 1, further comprising coating a biopolymer on the PEDOT-based nanofiber coating. The method according to claim 7, wherein the biopolymer comprises PLL- g- PEG-biotin, streptavidin or avidin, and a specific biomolecule. The method of claim 8, wherein the specific biomolecule is a biotinylated antibody. The method as described in claim 1, wherein the weight ratio of the PEDOT to the PSS is 1:2.5 to 1:6. The method according to claim 1, wherein based on the total weight of the solution comprising PEDOT:PSS and PEO, the weight percentage of the PEO is 1wt%-25wt%. A standard-free quantitative method for the detection of rare cells, comprising: introducing a biological fluid sample into the three-dimensional PEDOT-based bioelectronic device obtained by the method of any one of claims 1 to 11, to capture the rare cells in the biological fluid sample; and providing an electrical stimulus to excite the three-dimensional PEDOT-based bioelectronic device to release the trapped rare cells, Among them, the bioelectronic device can quantitatively monitor the difference of electrical signals in real time. The method of claim 12, wherein the rare cells comprise circulating tumor cells. The method as recited in claim 13, wherein the number and type of circulating tumor cells and the cancer stage can be determined through the electrical signal difference. The method of claim 12, wherein the electrical stimulation comprises a cyclic voltammetry, constant voltage method or constant current method. The method of claim 15, wherein the voltage range utilized by the cyclic voltammetry is -0.8 to 0.8V.

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