TWI743676B - 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 system bioelectronic device and standard-free quantitative detection method for rare cells

The present invention relates to a method for capturing, releasing and sensing rare cells; more particularly, it relates to a method for 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 activities, electrical stimulation (ES) for medical treatment, and drug or cell release for closed feedback control Electrical trigger response. So far, many bio-electronic devices have been used in the interface between artificial electronic devices and life systems and in actual clinical systems. Therefore, it is well known that this new type of medical device can be implanted into the human body to precisely adjust the electrical signal pattern of the peripheral nervous system, or be used as a non-invasive method for in vitro liquid biopsy (liquid biopsy). Most research in bioelectronic devices is achieved by integrating bioelectric interface (BEI) into electrodes and/or electronic devices. Electrodes and/or electronic devices are promising intermediate layers, and these intermediate layers are unique Ground use of 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 choice of organic □-conjugated materials (such as conductive polymers and carbon materials), because organic BEI materials are chemically modified and have low impedance without oxide interfaces in aqueous electrolytes. , Low-temperature processing and mass production have significant advantages.

One of the emerging applications of bioelectronic devices is the separation of circulating tumor cells (CTC). Circulating tumor cells are cancer cells that detach from the primary tumor or metastasis site, avoid immune surveillance, and then circulate in the peripheral blood and have the ability to form distant metastases. Compared with traditional methods, there are differences in the diagnosis of patient’s tumor incidence, recurrence and metastasis [for example, fresh tissue biopsy, positron emission tomography (PET), computer tomography (CT) and magnetic resonance imaging ( MRI)]. It is understandable that "liquid biopsy for CTC sensing" does not have the side effects of radiotherapy and can be performed normally in patients at all disease sites. Although tumor liquid biopsy can be easily used to collect CTCs, because the concentration of CTCs in a large number of blood cells (red blood cells and white blood cells) is extremely low, it is technically challenging to identify CTCs in a patient's blood sample. In order to meet this unmet need for the separation of rare cells, many technologies have been developed that use physical (e.g. size, density, charge and deformability) and biological properties [e.g., epithelial Surface binding of cell adhesion molecule antibodies (EpCAM antibody)]. Specifically, this unique concept of cell affinity substrate uses the synergistic effect of nanomaterials and CTC marker biofixation to enhance the CTC capture efficiency in the liquid biopsy process. In addition to seeking high cell capture yield and specificity from human blood on the chip, the vertical integration of CTC's downstream characteristics is bound to be used in future CTC chips, such as liquid biopsy (for example, a single CTC genotyping can be better understood Potential disease mechanisms, and rapid purification of CTC from primary culture to secondary culture) to achieve various clinical applications.

Recently, conductive polymers [ECPs; for example: polypyrrole or poly(3,4-ethylenedioxythiophene) (PEDOT)] due to their excellent electrical transport and electrochemical charge and discharge characteristics, inherent biocompatibility and high manufacturing Flexible and widely used in organic BEI. When biochemical and topographical cues (biochemical and topographical cues) are introduced into cells at the same time, various cell activities (such as adhesion, diffusion morphology, proliferation and differentiation) can be manipulated on the three-dimensional BEI, thereby expanding its role in cell and tissue regulation. Efficacy, as well as in diagnostic and therapeutic applications in non-invasive blood biopsy. In previous research, a three-dimensional PEDOT-based BEI has been developed. The carboxylic acid grafted PEDOT (PEDOTAc) nanostructure was prepared by electrochemical and chemical oxidation polymerization methods, and then combined with EpCAM antibody. Therefore, these three-dimensional BEIs can be used as NanoVelcro cell affinity wafers to enhance CTC capture performance. However, it is still necessary to further study the nanostructure characteristics and functional integration of the three-dimensional BEI to dynamically control the biological response in the device, and to develop the next generation of bioelectronic technology for point-of-care cancer.

The content disclosed in the above background description paragraph is only for improving the understanding of the background technology of the present invention. Therefore, the above content contains prior art that does not constitute an obstacle to 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 preparing high-quality and large-area PEDOT nanofiber coatings, which not only integrates three-dimensional BEI into bioelectronics for rare cell separation, but also It is proved that the integrated conductive polymer system (CP-based) organic electrochemical transistors (OECT) are used for operation to further provide the downstream label-free sensing of cancer cells. In short, the present invention first integrates a three-dimensional PEDOT-based BEI device with a PLL- g- PEG-biotin coating to study the biochemical and surface morphology capture of circulating tumor cells Efficiency, and explored their dynamic and sequential control of the capture/release performance of circulating tumor cells through the cyclic potential of electrical stimulation. In addition, the high conductivity of the PEDOT material is used as the active channel layer to prepare the OECT device module to monitor the circulating tumor cell capture performance on the chip and identify the phenotype of cancer cells.

Specifically, the present invention provides a method for preparing a three-dimensional PEDOT-based bioelectronic device, including: mixing an additive solution with a solution containing PEDOT:PSS and polyethylene oxide to obtain a ternary mixed solution; mixing the ternary The solution is subjected to an electrospinning process 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 the substrate , To form a three-dimensional PEDOT-based bioelectronic device; among them, 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 is additive solution .

In a specific embodiment of the present invention, the additive solution includes (3-glycidoxypropyl) trimethoxysilane solution and dimethyl sulfide solution.

In the 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 sulfide solution is 0~5.8 v/v%.

In a specific embodiment of the present invention, the substrate includes a single-electrode form to make an electrochemical electrode or a three-electrode form to make an organic electrochemical transistor.

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

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

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

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

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

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

Furthermore, the present invention further provides a standard-free quantitative detection method for rare cells, comprising: introducing a biological fluid sample into a three-dimensional PEDOT-based bioelectronic device obtained by the above-mentioned method to capture the biological fluid sample And provide electrical stimulation to stimulate the 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 a specific embodiment of the present invention, rare cells include circulating tumor cells.

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

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

In a specific embodiment of the present invention, the voltage range used by 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 non-calibration detection.

The details of one or more embodiments of the present invention will be described in the accompanying drawings and the following description. Based on these descriptions and drawings and the scope of the patent application, the technical features, objectives and advantages of the present invention will be easily understood. At the same time, in order to make the above-mentioned features and advantages of the present invention more comprehensible, the following specific embodiments are described in detail in conjunction with the accompanying drawings.

The advantages and features of the present invention and the method for achieving the same will be described in more detail with reference to exemplary embodiments and the accompanying drawings to make it easier to understand. However, the present invention can be implemented in different forms and should not be understood to be limited to the embodiments set forth herein. On the contrary, for those with ordinary knowledge in the technical field, the provided embodiments will make this disclosure more thorough, comprehensive and complete to convey the scope of the present invention, and the present invention will only be appended to the scope of the patent application. Defined.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as generally understood by those skilled in the art to which the present invention belongs. It will be more understandable that, for example, those terms defined in commonly used dictionaries should be understood as having meaning consistent with the meaning of the relevant field, and unless clearly defined herein, they will not be understood in an excessively formal meaning.

Unless clearly indicated otherwise herein, the singular forms "a", "at least one" and "the" used herein can also include plural referents. As used herein, the term "and/or" includes any and all combinations of one or more of the related listed items.

In this article, the numerical values and parameters used to define the scope of the present invention inevitably contain standard deviations due to individual test methods, so they are mostly expressed as approximate quantitative values. However, in specific embodiments, Relevant values presented as accurately as possible. In this context, "about" generally depends on the considerations of those with ordinary knowledge in the technical field to which the present invention belongs, and generally means that the actual value falls within the acceptable standard error of the average value. For example, the actual value is within an acceptable standard error of the average value. Within ±10%, ±5%, ±1%, or ±0.5% of a specific value or range.

The exemplary embodiments will be described in detail below in conjunction with the drawings. However, these embodiments may be included in different forms, and should not be construed as limiting the scope of patent application of the present invention. The provision of these embodiments makes the disclosure of the present invention complete and clear, and those who are familiar with this technology will be able to understand the scope of the present invention through these embodiments. Materials and methods PEDOT nanofiber coating production 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, 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) is purchased from SuSoS (where 20, 2, and 3.4 are molecular weight k Dalton, 3.5 is the value of lysine unit/PEG chain, the information can be obtained from the product itself , I won’t repeat it here). Streptavidin (Streptavidin; SA; 1 mg mL ^-1 ) was purchased from Invitrogen. Biotinylated EpCAM antibody (anti-humanEpCAM) / TROP1 antibody (goat IgG) were purchased from R&D Systems. Among them, the streptavidin used in the present invention can also be replaced by avidin (Avidin) to achieve the purpose. Cell Research

Breast cancer cell line (MCF7), lung cancer cell line (PC9), cervical cancer cell line (HeLa) and monocyte cell line (THP-1) were purchased from American Type Culture Collection. GlutaMAX-I, Vybrant ^® DiO cell labeling solution, DMEM and RPMI 1640 growth medium 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 coating

PEDOT: PSS film shows poor wet stability in water and high humidity environment, this is due to the existence of water-soluble PSS area, not to mention the wet instability of PEDOT: PSS nanofiber structure. In order to effectively improve the wet stability of the conductive PEDOT nanofiber coating, the crosslinking reaction with other additional crosslinking agents can also react with itself or with PEDOT:PSS, thereby enhancing the water resistance of the PEDOT:PSS nanofiber coating . In order to perform the water resistance test, the present invention prepares various quantities of PEO materials as a mixture of PEO/PEDOT: PSS and GOPS, and then uses 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 form a stable chemical cross-linking reaction between PEO and PSS materials. In addition, 0.2~6 v/v% (preferably 1 v/v%) of GOPS and 0~5.8 v/v% (preferably 5v/v%) of dimethylsulfene (DMSO) are added to PEO/PEDOT: PSS mixed solution (that is, the solution containing PEDOT: PSS and polyethylene oxide (PEO) accounts for 94 v/v%) to further obtain optimized water resistance and enhanced electrical conductivity. 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 using a syringe pump to control the spinning rate and deposition time. Connect the high-voltage power supply to a 27 gauge disposable needle to control the diameter of nanofibers; use an air blower at room temperature to keep the relative humidity below 30% to prevent nanofibers During the electrostatic spinning process, it is erected due to the negative charge of the conductive nanofibers (see Figure 1(a)). Device assembly and surface modification on PEDOT- based nanofiber coating

The device assembled from three-dimensional PEDOT-based BEI-coated ITO glass (single electrode form) contains a cylindrical PDMS chamber with a height of about 4 mm and a diameter of about 10 mm, or a mixer for PDMS microchannels. The device was treated with PLL- g ^-PEG- Biotin (100μgmL ^-1 in 10 mM HEPES buffer (pH 7.4)) for 1 hour, and then incubated with SA (10μgmL ^{- 1} in 1x PBS) for 1 hour at room temperature . Place the biotinylated EpCAM antibody (anti-humanEpCAM)/TROP1 antibody (10 μgmL ^-1 in 1x PBS, which contains 0.1% BSA and 0.09% NaN ₃ ; 25 μL) on the device, and then incubate at room temperature ( 60 minutes). Finally, before the cell experiment, the BEI device of the three-dimensional PEDOT system was washed several times with 1x PBS, and then soaked in 1x PBS for 1 hour. Scanning electron microscope (SEM)

A scanning electron microscope (FEI Nova NanoSEM 200; acceleration voltage: 10 keV) was used to observe the surface morphology of the PEDOT-based nanofiber coating and the circulating tumor cells on the wafer. Before the SEM test, the biological and PEDOT samples are dehydrated with EtOH (25%, 50%, 75% and 100%; each dehydration time: 20 minutes), and the liquid carbon dioxide is used for critical point drying , And then sputtered with gold (>3 nm). Cell capture / release test

After removing 1x PBS from the three-dimensional PEDOT-based BEI device, 100 μl of cell suspension (10 ⁵ cells mL ^-1 ) was assembled on the wafer. After incubating at 37°C and 5% carbon dioxide for 1 hour, gently wash the substrate with 1x PBS (at least five times). Fluorescence microscope (CKX41, Olympus) was used for imaging and counting of cells. An automatic cell counter (Luna ^TM automatic cell counter, purchased from Logos Biosystems, South Korea) is used to count the cells of the dilution experiment and the cell viability can be easily determined. According to the above capture procedure, the substrate-modified cancer cells were trypsinized and collected. Finally, the cell lines collected in each device are prepared in 100μl of cell suspension, and then carefully transferred to the counting slide of the Luna™ automatic cell counter. Cell viability was analyzed in triplicate. For the study of electrically triggered cell release, the BEI device of the three-dimensional PEDOT system is operated with a standard three-electrode setup, which includes: a counter electrode (Pt), a reference electrode (Ag/AgCl), and a working electrode (The BEI of the three-dimensional PEDOT system, which is covered with ITO glass), and the electrical stimulation (ES) of the cyclic potential is scanned in 1x PBS with a scan rate of 100 mV/s from 0 to +0.5 V. 20 cycles. All experiments were repeated more than three times and showed similar results. A confocal microscope (FV1000; Olympus) was used to observe the fluorescence image of the single-layer PLL-g -PEG-FITC on a three-dimensional PEDOT-based BEI device, and to monitor the fluorescence density change after electrical stimulation. Results and discussion

As shown in Figure 1b, the PSS/PEO cross-linking reaction mechanism has been proposed by some scholars. In a strongly acidic environment, such as the presence of PSS, the ether group in PEO is decomposed by protonation, forming a hydroxyl group at the end of the cracked 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 is not enough 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 adds GOPS as another The cross-linking agent improves the mechanical stability of the fiber and the adhesion to the substrate, thereby preventing partial dissolution or delamination of the PEDOT-based nanofiber structure when immersed in an aqueous electrolyte solution. Through the combination of the condensation reaction between PSS and PEO and the chemical reaction between GOPS and the components of the mixture, different amounts of PEO additives (NF5, NF10, NF15, NF20, NF25) are included; the number indicates the weight of the PEO component The percentages are 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). The PEDOT-based nanofiber coating can be successful The results were obtained, and the diameter change of nanofibers was studied by comparing the treatment with 1x PBS buffer for 24 hours (Figure 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 changes in the diameter distribution of the nanofibers based on the SEM results (Figure 2a). As shown in Figure 1c-1, the electrospun PEO/PEDOT:PSS nanofibers are heat-treated at 80~150℃ for at least 0.5 hours to crosslink the PEDOT-based nanofibers, and then subjected to water treatment for 24 hours; It can be seen that the cured PEO/PEDOT:PSS nanofibers remain intact without any size or structure changes, indicating that the water resistance and dimensional stability of the fibers have been improved after the chemical crosslinking step. Except for NF5 nanofiber coating, the nanofiber structure in other nanofiber coatings (NF10, NF15, NF20 and NF25) all have good dimensional stability without significant damage. Since a higher content of PEO additives will lead to higher electrical resistance, the weight percentage of PEO above 25wt% is not used in the present invention. It is worth noting that the nanofiber structures of NF5, NF10 and NF15 are easily partially dissolved when the diameter is reduced by about 40 nm in PBS buffer; conversely, after immersing NF20 and NF25 in 1x PBS buffer for 24 hours, PEO hydrogel The swelling behavior shows an increase in fiber diameter.

Although many previous technical documents mentioned that adding an appropriate amount of PEO will cause the conductivity of PEDOT:PSS to increase by more than a hundred times, this phenomenon is not observed in the present invention after heat treatment for 12 hours at 130°C, which is due to the occurrence of unmeasured The conductivity value (Table 1). In order to explore the electrical conductivity change of PEO/PEDOT:PSS nanofiber coating, the present invention uses transmission electron microscope (TEM), atomic force microscope (AFM) and X-ray photoelectron spectroscopy (XPS) to compare PEO/PEDOT:PSS mixture The phase separation for characteristic analysis. In this example, because NF10 has the best dimensional stability and conductivity, the as-spun and thermally annealed NF10 samples were first subjected to TEM image analysis to determine the mechanism of conductivity reduction , As shown in Figure 3a-3b. The needle-type static electrospinning process is likely to lead to the formation of the NF10 PEO/PEDOT: PSS core-shell nanofiber structure, which indicates that the geometric feature of the structure is composed of a non-smooth and PEDOT-rich nanofiber core. Yes, the core is surrounded by a thin layer rich in PSS. However, under proper thermal crosslinking conditions, the thermally induced phase separation of the PEDOT-rich and PSS-rich regions can be achieved at the same time, thereby forming a smooth and PEDOT-rich nanofiber core that is thick and rich in PSS Surrounded by layers, resulting in a higher contact resistance measured by the four-point probe. In order to confirm the argument of the present invention, the present invention uses the tapping mode of the atomic force microscope (Figure 3c-f) to measure the morphology and phase diagram ^{of NF10 (area: 1x1μm 2) coated on an ITO glass substrate} To get the reason for the decrease in conductivity. Although the as-spinning and thermal annealing conditions of NF10 have similar morphological characteristics, the phase diagram shows some changes. In the phase diagram, the hard structure domains are shown as bright areas, which are classified as PEDOT-PSS (PEDOT-rich) areas in the present invention; while the dark areas are represented as soft structures, which belong to excessive PSS and/or PEO areas. The present invention observes that the thermal annealing treatment induces phase separation, which is characterized by isolating the PSS and/or PEO region surrounded by the continuous PEDOT-PSS phase, and the degree of change is greater than that of the freshly spun yarn on the phase image image. The nanofiber coatings come out high. Table 1. Electrospinning parameters, dimensions and electrical properties of PEDOT-based nanofiber coatings sample Flow rate (ml hr ^-1 ) Applied voltage (kV) Sheet 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

The AFM tapping mode image is usually unable to directly observe the morphological changes, because the phase image signal will be affected by factors such as surface force, tip indentation and volume characteristics. In order to confirm the hypothesis of the present invention regarding the correlation between the PEO/PEDOT:PSS phase separation and the electrical conductivity change, the present invention carried out the XPS test of NF10 to determine the internal composition change of the surface during the thermal annealing process. The XPS full spectrum analysis spectrum (survey spectra) and the high-resolution core-level spectrum (S _2p ) of NF10 before and after thermal annealing are shown in Figure 4. These XPS full-spectrum analysis results are very consistent with all the blending components in the PEO / PEDOT: PSS solution. Among them, the core level line (O _1s , C _1s , S _2p and Si) on the surface of the nanofiber _2p ) is only a slight change. In addition, Table 2 summarizes the chemical composition of NF10 analyzed based on XPS results. The present invention found that more Si features were observed on the surface of the thermally annealed nanofiber coatings, resulting in Si _2p core-level features. Then, the samples just spun out showed that a small amount of insulating GOPS was extruded. To the PSS-rich shell, resulting in a decrease in conductivity. The deconvolution process of the S( _2p ) core energy level line of XPS is performed to monitor the change of the composition ratio of PSS/PEDOT during the thermal annealing process. The XPS full spectrum analysis spectra of both samples showed high intensity between 168.0 and 168.9 eV, which corresponds to the spin split component of the sulfur atom of the PSS chain. 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 (Figure 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 areas on the surface. It can be calculated that the composition ratio of PSS to PEDOT of the freshly spun sample is 2.09, and the composition ratio of PSS to PEDOT after thermal annealing at 130°C for 12 hours has increased to 2.56, which shows that a small amount of insulating PSS has been combined with enriched PEDOT. The core structure of the nanofiber is separated, resulting in a significant decrease in conductivity. Table 2. The atomic percentage of the core energy level lines (O _1s , C _1s , S _2p and Si _2p ) on the surface of the nanofiber coating (from XPS data, calculated using Shirley method); PEDOT, PSS, and the ratio of PSS to PEDOT Atomic percentage (peak deconvolution _{of S 2p data from XPS)} sample Atomic percentage from XPS full spectrum analysis data C _1s O _1s S _2p 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 percentage from XPS (S _2p ) data PEDOT PSS The 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 yet fully understood, the preferential formation of thick PSS and/or PEO insulating layers has become one of the key points for the decrease in conductivity and may be the source From the thermal annealing process. In order to prove that the PEDOT-based nanofiber coating of the present invention can be used in BEI applications, the present invention first tested the electrochemical performance of the PEDOT-based nanofiber coating device (NF5, NF10, NF15, NF20, NF25) (Figure 5a-5b) ). Cyclic voltammetry (CV) and electrochemical impedance spectrum (EIS) are performed using a standard three-electrode system with a PEDOT-based nanofiber coating device. First, use CV (voltage sweep from -0.8 to 0.5 V) 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) (Figure 5a). NF5 (0.78 mC/cm ² ) and NF10 (0.36 mC/cm ² ) have higher CCDs, pointing out that their characteristics are higher than other structures (NF15, NF20, NF25; respectively, 0.19 mC/cm ² , 0.06 mC/cm ² , 0.05mC/cm ² ) has a higher PEDOT content, and compared with ITO electrode (0.21 mC/cm ² ), it can effectively reduce (dark sky blue) and oxidized (light gray blue) states to 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 the PEDOT-based nanofiber coating will increase moderately at all frequencies, which indicates that high PEDOT: PSS content will be easier to perform Ion exchange and doping/dedoping process during the cycle potential of ES. Previous studies have pointed out that the electrochemical polarization of the substrate will cause local pH changes, which will cause the monolayer of PLL- g -PEG to dissolve from the electrode, thereby allowing the recovery of the cell sheet and the extension of the neurites in the neurons. Therefore, the present invention expects to integrate a three-dimensional PEDOT-based nanofiber BEI device with PLL- g- PEG-biotin to demonstrate the efficiency of capturing circulating tumor cells and electrically triggering cell release.

The key feature of capturing/releasing circulating tumor cells is the dynamic control of the PLL- g- PEG-biotin coating on the BEI device of the three-dimensional PEDOT system. In order to prove the concept of operating circulating tumor cells on a chip, FITC-PLL- g -PEG was first coated on the surface of the NF10 device, and then 20 CV scanning cycles were used for electrical stimulation (ES) (voltage from -0.8 to 0.5) V) Reduce the fluorescence intensity with electrical triggering (Figure 6a-6c). As shown in Figure 6a, after soaking in FITC-PLL-g -PEG solution (100μgmL ^-1 in 10 mM HEPES buffer) for one hour, NF10 will reach saturation adsorption; the nanofiber structure is surrounded by a uniform fluorescent coating . Since the prior literature technology has known that even with a potential range of -1 to 1 V, the observed cell function and activity are negligible. Therefore, it is believed that the hole transportation driven by the oxidation of the PEDOT:PSS backbone will not only lead to the desorption of FITC-PLL-g -PEG from the surface of PEDOTAc, but also ensure the downstream characterization of circulating tumor cells. Activity and biological function (Figure 6b-6c).

In order to prove the potential use of the PEDOT-based nanofiber coating device as a liquid biopsy method for personalized cancer diagnosis, the present invention firstly used the electrospinning deposition time-dependent (NF10 deposition for 20 minutes, 15 minutes, 10 minutes, and 5 minutes). , 1 minute and 0 minute) studies detected the difference in cell capture efficiency (Figure 7a). The device configuration is based on a series of surface modification processes using PLL-g- PEG-biotin, streptavidin (SA) and biotinylated EpCAM antibody (Figure 6d-g). The single layer of PLL- g- PEG-biotin interacts with the negative charges on the surface of the PEDOT-based nanofibers through electrostatic attraction. Then, the high-affinity SA will connect the biotinylated EpCAM antibody to the monolayer PLL- g- PEG-biotin on the device, thereby providing specific binding to circulating tumor cells. The present invention cultures 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 line) 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. It is worth noting that the 1-minute electrospinning deposition time of the PEDOT-based nanofiber coating can provide an effective technology for capturing MCF7 cells, and the cell capturing yield is about 1900 cells/mm ² . Longer deposition time (5 minutes, 10 minutes, 15 minutes, 20 minutes) will slightly enhance the cell capture ability, and its cell density is about 1787 cells/mm ² , 1856 cells/mm ² , 1894 cells/mm ² and 2104 cells respectively /mm ² . In addition, when the NanoVelcro concept is applied to circulating tumor cells, it can be observed that the cell capture rate of MCF7 and PC9 cells is extremely high, while the non-specific binding capture rate of HeLa and THP-1 cells is low. The results of cell capture efficiency and cell release yield are summarized in Figure 7b; the capture density of MCF7 and PC9 cells did play a positive role, ^{showing in the range of 1750~2000 cells/mm 2} while maintaining adhesion to HeLa and THP-1 cells Resistance. Integrating PLL- g -PEG-biotin with the nanofiber coating of three-dimensional PEDOTAc will provide an optimized PEG density, which is sufficient to ensure the binding resistance of the two non-specific HeLa/THP -1 cells and a sufficient amount of biotin Used for SA binding. Therefore, it is believed that the PEDOT-based three-dimensional BEI device has the ability to integrate with other biotinylated antibodies, so that rare cells can be separated quickly. 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 that the BEI device system of the present invention has the application possibility of adapting to the downstream characterization of circulating tumor cells, the electrospinning deposition time of different NF10 devices was selected to prove the effect of dynamically controlling the capture/release of circulating tumor cells on the chip (Figure 7a). As expected, the optimized 20 CV scanning cycles (voltage sweep from -0.8 to 0.5 V) resulted in a yield of MCF7 cells released from the device’s electrospinning deposition time (10 minutes, 15 minutes, and 20 minutes) 90% (Figures 7a and 7c). However, these results indicate that during the ES scanning voltage in the range of -0.8 to 0.5 V, the electrospinning deposition time (5 minutes, 1 minute, and 0 minutes) of the BEI device does not provide a higher cell release than other deposition times. Performance, this is due to the low CCD value of the nanofiber coating and/or small changes in the local pH value during the cycle potential ES. In addition, the constant voltage method or the constant current method can also be used in this embodiment, and the effect is similar to that of the cyclic voltammetry method, and will not be repeated here.

In order to use the function of electrically triggered cell release for downstream characterization of circulating tumor cells, the present invention explores the device of the present invention under a series of low cell densities (10, 50, and 100 tumor cells are implanted in 100 μl cell suspension). Possible use for capturing/releasing MCF7 cells, which shows that the yield of cell capture is greater than 90% at different implanted tumor cell densities, and the yield of cells retained on NF10 after ES is less than 15% All have nothing to do with the number of tumor cells implanted (Figure 7d). In addition, the present invention tests the test-retest reliability of the device in three cycles of cell capture/release. Before the cell capture/release study, by repeating a series of PLL- g- PEG-biotin coatings, the cell capture and release performance of the device can restore the cell density to approximately 45,000 cells/cm ^{2 in a new cycle.} And 5000 cells/cm ² (Figure 7e). In addition, after testing, the three-dimensional PEDOTAc-based nanopillar array of the present invention can not only provide the released cells with up to about 90% cell viability (ex-chip culture on a polystyrene (TCPS) petri dish), but also The cell viability of expanding cultures can be maintained (in-chip culture on NF10 device).

Furthermore, the present invention can apply this PEDOT-based nanofiber coating to the active layer channel (channel) of an organic electrochemical transistor (OECT) (a three-electrode form) device (the size is (Width) 1.5 mm × (Length) 5 mm) (Figure 8a), except that liquid specimens of MCF7 cells (breast cancer cells) implanted with different chips can be treated within 1 hour (cell density is 0; 10 ⁴ ; 10 ⁵ ; 10 ⁶ cells/ml) for efficient capture (Figure 8b-e), electrical stimulation can also be used to dynamically control the cell-substrate interaction to achieve the effect of cancer cell release, and no calibration is performed under the operating voltage of 1V (label-free) Detect the capture signal and release signal of MCF7 cells on the component, including monitoring the electrical signal changes at different time points (T-0, T-1, T-2, T-3) (Figure 8f-h) . Wherein T-0 time point is the component without any surface treatment; T-1 time point is the component after PLL- g -PEG-biotin/SA/biotinylated EpCAM antibody coating treatment; 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 (Figure 8f) at four different time points that can be measured by the experiment is converted into a transconductance ( g _m )-V _g transfer transmission characteristic curve (Figure 8g), and _{The V g} corresponding to the maximum value of g _{m represents the shift (∆V g} ) of the gate voltage at four different time points. From the research results in Fig. 8h, it is found that the T-1 time point is treated with PLL- g- PEG-biotin/SA/biotinylated EpCAM antibody coating, and the performance can be observed compared to the T-0 time point. There is a +30 mV change in ∆Vg; after 1 hour of capturing MCF7 cells at time T-2, the effective capacitance of MCF7 cells per unit area will be affected, resulting in a +52 mV change _{that further increases ∆V g} ; After the final application of electrical stimulation to release all MCF7 cells, the results of the study found that ∆V _g will show a change close to -82 mV, which can return to the initial signal of the component without any surface treatment, which means that the OECT component can Real-time monitoring of electrical signal differences before and after surface modification, capturing circulating tumor cells, and releasing circulating tumor cells.

When using the above-mentioned PEDOT-based nanofiber-coated OECT device, studies have confirmed the detection performance of this device, which can perform standard-free quantitative detection of the number of circulating tumor cells that have been captured, 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 Capture of cancer cells, and then use the measured transconductance response (g _m =(GG ₀ )/G ₀ ) and gate voltage of MCF7, HeLa (cervical cancer cells) and PC9 (non-small cell lung cancer) cell lines Offset (V _g ) difference, non-standard quantitative sensing is performed, and the result confirms that the component shows a quantitative increase in electrical signal difference to the number of captured cancer cells. When the dotted line of (GG ₀ )/G ₀ (%) = 10% and V _g = 0.012 V is used as the criterion, the present invention finds that this quantitative sensing data can provide the difference in the type of cancer cells (Figure 9a-c) . Because the developed PEDOT OECT device is highly transparent, in addition to providing various electrical signal sensing, it can also use an inverted optical microscope to obtain cell optical image analysis on the device without fluorescent staining. The results (including the capture efficiency of circulating tumor cells and the coverage area (%)) (Figure 9d and Figure 8 be). In addition, this example also carried out the captured electrical signal analysis for cancer cells of different liver cancer stages ( including the use of post hepatoma HepG2 cells and primary liver cancer having resistance representative of Huh7 cells) (FIG. 9e). when the same analysis implanted cell density was 10 ^cells / ml one hour after the capture of the electric signal, may find significant The electrical signal difference between HepG2 and Huh7 cells. For example, HepG2 showed a value of (GG ₀ )/G ₀ (%) of 6.1%, which was 10% lower than the criterion; V _g was 0.021 V, slightly higher than the criterion of 0.012 V ; For the electrical signal of Huh7 cells, the value of (GG ₀ )/G ₀ (%) is 46.2%, which is much higher than 10%; V _g is 0.006 V slightly lower than the judgment standard 0.012 V. The results of this study confirm this PEDOT The OECT element has the potential for rapid detection of cancer staging of liver cancer, even when cancer cells start to deteriorate, they can be detected quickly and users can quickly strain.

All the features disclosed in the present invention can be combined in any way. The features disclosed in this specification can be replaced by features with the same, equivalent or similar purpose. Therefore, except for the emphasis placed on special statements, the feature disclosed in this specification is an embodiment in a series of equivalent or similar features.

In addition, according to the contents disclosed in this specification, those familiar with the technical field 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 The implementation mode is also included in the scope of patent application.

without

Through the detailed description and the drawings, only the drawings of the embodiments of the present invention will be more comprehensively understood; therefore, the following drawings are only used to explain the embodiments of the present invention, and do not limit the scope of patent application of the present invention; Figure 1 (A) Schematic diagram of electrospinning equipment of the present invention, (b) PEO/PEDOT: chemical structure of PSS, DMSO and GOPS blend solution, and schematic diagram of sulfonate crosslinking reaction between PEO and PSS, (c) -(g) SEM morphology of the newly spun nanofiber coating, which contains different amounts of PEO additives (NF5, NF10, NF15, NF20, NF25), (h)-(1) cross-linked nanofiber coating The layer is immersed in 1x 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 the water resistance test in the present invention (a), (b) interdigital electrode ( IDE) is used to measure the conductivity of a single fiber. The electrode has 125 sets of interdigitated fingers with a fingertip width and spacing of 20μm (DRP-G-IDEAU10, DropSens), (c) deposited on the IDE An enlarged view of the optical microscope image of the electrospun PEDOT nanofiber coating; Figure 3 is the TEM image of the (a) freshly spun and (b) thermally annealed PEDOT nanofiber coating of the present invention Image, the surface morphology (c) and (d) of the PEDOT-based nanofiber coating (freshly spun and thermally annealed, respectively) obtained by tapping mode atomic force microscopy, and the phase difference images (e) and (f) ); Figure 4 is the XPS spectrum of the newly spun and thermally annealed PEDOT nanofiber coating of the present invention. (a) XPS general full spectrum scanning, (b) XPS S2p spectrum of the sample just after spinning, (c) XPS S2p spectrum of the thermally annealed sample; Figure 5 shows the various PEDOTs on the ITO glass of the present invention in 1x PBS The electrical properties of nanofiber coatings. (a) CV: The potential is scanned from -0.8 to 0.5V at a scan rate of 100mV/s; the CCDs of NF5, NF10, NF15, NF20 and NF25 are 0.78, 0.36, 0.19, 0.06 and 0.05 mC/cm ^{2 respectively} , (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-based nanofiber coating device (NF10 device). (a) Without ES, (b) With ES, (c) Calculate the fluorescence signal frequency of NF10 device relative to the fluorescence intensity before/after ES, (d)-(h) PEDOT-PSS phase separation, PEDOT system Conceptual schematic diagram of surface modification of nanofiber coating device and its electrically triggered cell release, (d) freshly spun NF10, (e) freshly spun NF10 with non-smooth phase separation morphology, (f) Thermally annealed NF10 with a core-shell morphology, (g)-(h) binding biotinylated antibody on the device and its mechanism for capturing and releasing circulating tumor cells on the chip; Figure 7 is the (a) electrospinning mechanism of the present invention Cell capture efficiency and cell release yield on silk deposition time dependence study (NF10 deposition time 20 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute and 0 minutes), (b) MCF7, PC9, HeLa and THP-1 The cell capture efficiency and cell release yield of the cell line, (c) Under various CV scanning conditions (voltage from -0.8 to 0 V, 0 to 0.5 V, -0.8 to 0.5 V), in 1x PBS Scan rate 100 mV/S for 20 cycles, quantify the percentage of MCF7 cells remaining on the surface after ES releases cells, (d) Perform ES and implant 10, 50, and 100 cells in 100 ml cell suspension, and capture Efficiency and the percentage of cells retained on the NF10 device, (e) In multiple/sequential studies with or without ES, the cell capture/release performance of the NF10 device; Figure 8 is the present invention (a) using PEDOT Schematic diagram of uncalibrated biosensing of rice fiber coated on a three-dimensional OECT device, which can be used 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 under density (0; 10 ⁴ ; 10 ⁵ ; 10 ⁶ cells/ml), (f) I _d -V _g output characteristic curve at four different time points, (g) four different The g _m -V _g transmission characteristic curve at the time point, (h) the relative change of the gate voltage (∆V _g ) at four different time points; and Figure 9 shows the detection of different cancer cells implanted on the OECT device of the present invention As a result, (a) the transconductance change [(GG ₀ )/G ₀ (%)] and the gate of MCF7 cells at different implantation cell densities (0; 10 ⁴ ; 10 ⁵ ; 10 ^{6 cells/ml)} The electrical signal difference of the polar voltage change (∆V _g ), (b) the transconductance change of HeLa cells at different implantation cell densities (0; 10 ⁴ ; 10 ⁵ ; 10 ⁶ cells/ml) [(GG ₀ )/G ₀ (%)] and _{the electrical signal difference of the gate voltage change (∆V g} ), (c) for PC9 cells at different implant cell densities (0; 10 ⁴ ; 10 ⁵ ; 10 ⁶ cells/ A few _{The electrical signal difference of the transconductance change [(GG 0} )/G ₀ (%)] and the gate voltage change (∆V _g ) under the condition of (L), (d) for MCF7, HeLa and PC9 cells in different implanted cells Optical image analysis of coverage area ratio [Coverage Area (%)] on the active layer channel after capturing at density (0; 10 ⁴ ; 10 ⁵ ; 10 ^{6 cells/ml), (e) for HepG2 and Huh7 stem cancer cells} The electrical signal difference between the change in transconductance [(GG ₀ )/G ₀ (%)] and the change in gate voltage (∆V _g ) at the implanted cell density (10 ^{5 cells/ml).}

Claims (16)

A method for preparing a three-dimensional PEDOT-based bioelectronic device, including: Mixing an additive solution with a solution containing PEDOT: PSS and polyethylene oxide (PEO) to obtain a ternary mixed solution; The ternary mixed solution is subjected to an electrospinning process to form a PEDOT-based nanofiber; Heat 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 according to claim 1, wherein the additive solution includes a (3-glycidoxypropyl)trimethoxysilane solution and a dimethylsulfene solution. The method described in 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 described in claim 2, wherein based on the total volume of the ternary mixed solution, the volume percentage of the dimethyl sulfoxide solution is 0~5.8 v/v%. The method according to claim 1, wherein the substrate includes a single-electrode type to make an electrochemical electrode or a three-electrode type to make an organic electrochemical transistor. The method described in claim 1, wherein the heat treatment is carried out at 80 to 150°C for at least 0.5 hours. The method described in claim 1, further comprising coating a biopolymer on the PEDOT-based nanofiber coating. The method according to claim 7, wherein the biological polymer comprises PLL- g- PEG-biotin, streptavidin or avidin, and specific biomolecules. The method according to claim 8, wherein the specific biomolecule is a biotinylated antibody. The method described in claim 1, wherein the weight ratio of the PEDOT to the PSS is 1:2.5~1:6. The method described in claim 1, wherein based on the total weight of the solution containing PEDOT: PSS and PEO, the weight percentage of the PEO is 1 wt% to 25 wt%. A standard-free quantitative detection method for rare cells, including: Introducing a biological fluid sample into the three-dimensional PEDOT-based bioelectronic device obtained by any one of claims 1 to 11 to capture the rare cells in the biological fluid sample; and Provide an electrical stimulus to stimulate the three-dimensional PEDOT bioelectronic device to release the captured rare cells, Among them, the bio-electronic device can quantify and monitor the difference in electrical signals in real time. The method according to claim 12, wherein the rare cells comprise circulating tumor cells. Such as the method described in claim 13, wherein the number and type of circulating tumor cells and the stage of cancer can be determined through the difference in the electrical signal. The method according to claim 12, wherein the electrical stimulation includes a cyclic voltammetry, a constant voltage method, or a constant current method. The method described in claim 15, wherein the voltage range used by the cyclic voltammetry is -0.8 to 0.8V.

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