A free-standing-agnwsaunps electrochemical biosensor

Abstract

The application discloses a kind of free-standing-AgNWs / AuNPs electrochemical biosensor, belongs to biological detection analysis technical field.The preparation method is as follows: CNTs are mixed with PDMS, then vacuum treatment obtains CNT-PDMS composite material, CNT-PDMS composite material is mixed with curing agent after curing obtains bare electrode, bare electrode is placed in electric field with silver nanowire suspension and is powered on to process, so that silver nanowire is sequentially embedded on bare electrode along the direction of electric field and obtains modified electrode, gold nanoparticles are electrodeposited on modified electrode, gold nanoparticles are modified with group with carboxyl group at end through Au-S bond, the carboxyl group at end of gold nanoparticles on modified electrode is activated, then amino-containing capture probe is connected with activated carboxyl group, and it is obtained.The electrochemical biosensor provided by the application is used for detecting IL-6, has wide linear range and extremely low detection limit.

Description

Technical Field

[0001] This invention belongs to the field of biodetection and analysis technology, specifically relating to a free-standing-AgNWs / AuNPs electrochemical biosensor. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Interleukin-6 (IL-6) exerts biological activities by accelerating cell cycle progression, inhibiting apoptosis, and promoting tumor metastasis. IL-6 is a serum marker in colorectal cancer patients used to assess non-cure status after surgical resection (10.8 ± 2.2 pg / ml). -1 ) and curative (8.3±1.0 pg ml) -1 The key biomarker for IL-6 is IL-6, with a serum level of 4.4 ± 0.8 pg / ml in normal individuals. -1 Various methods for detecting IL-6 have been studied to diagnose colorectal cancer and assess the prognostic effects of tumor resection, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunofluorescence. Electrochemical methods for detecting IL-6 typically utilize one-dimensional (1D) nanomaterials (e.g., carbon nanotubes (CNTs) and silver nanowires (AgNWs)), which offer advantages such as low cost, high sensitivity, and simplicity. However, one-dimensional nanomaterials are often randomly coated on the surface of electrochemical electrodes, leading to aggregation and distortion. Furthermore, electron transfer can only occur along the length of the one-dimensional nanomaterial. The circuitous and overlapping conductive paths of the one-dimensional nanomaterial reduce the electron transfer efficiency in the electrochemical sensor, thus requiring further improvement in the performance of electrochemical sensors for IL-6 detection. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the objective of this invention is to provide a...

[0005] The free-standing AgNWs / AuNPs electrochemical biosensor provided by this invention is used to detect interleukin-6 (IL-6) and has a detection range of 0.001 ng / ml. -1 Up to 100ng ml- 1 Wide linear range and 29.8 fg ml -1 Low detection limit.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, a method for preparing a free-standing-AgNWs / AuNPs electrochemical biosensor is disclosed. The method involves mixing carbon nanotubes (CNTs) with polydimethylsiloxane (PDMS), followed by vacuum treatment to obtain a CNTs-PDMS composite material. This CNTs-PDMS composite material is then mixed with a curing agent, filled into an electrode template, and cured to obtain a bare electrode. The bare electrode and a suspension of silver nanowires (AgNWs) are placed in an electric field and subjected to electrochemical treatment, causing the AgNWs to be orderly embedded into the bare electrode along the electric field direction to obtain a free-standing-AgNWs electrode. Gold nanoparticles are electrodeposited on the modified electrode, and the gold nanoparticles are modified with carboxyl groups at their ends via Au-S bonds. The carboxyl groups at the ends of the gold nanoparticles on the modified electrode are activated. Finally, a trapping probe containing an amino group is linked to the activated carboxyl group to obtain the desired electrode.

[0008] Free-standing AgNWs / AuNPs / MUA-MPA / EDC-NHS / anti-IL-6 electrode; the capture probe is capable of specifically binding to IL-6.

[0009] In a second aspect, a free-standing AgNWs / AuNPs electrochemical biosensor is obtained by the above-described preparation method.

[0010] A third aspect of the invention is the application of the above-described free-standing-AgNWs / AuNPs electrochemical biosensor in the detection of IL-6 or in the manufacture of a system for detecting IL-6.

[0011] A fourth aspect of the present invention is an IL-6 detection kit comprising the above-described free-standing AgNWs / AuNPs electrochemical biosensor and a buffer solution containing [Fe(CN)6]. 3- / 4- .

[0012] The beneficial effects of this invention are as follows:

[0013] This invention utilizes an electric field to densely and orderly embed AgNWs into a semi-cured conductive CNT-PDMS composite material, forming an electric field-enhanced free-standing AgNWs electrode (i.e., the modified electrode described above), significantly increasing the active sites of the electrode. To overcome the challenge of rapid oxidation of AgNWs (the oxidation peak current decreased by 90.7% after 8 CV scan cycles), gold nanoparticles (AuNPs) were stably modified onto the surface of the free-standing AgNWs electrode, resulting in only a 5% reduction in the oxidation peak current. Surprisingly, the oxidation peak current was 500 times that of the bare electrode and 100 times that of the AgNWs-coated electrode. By modifying the gold nanoparticles with carboxyl-terminated groups via Au-S bonds, the activated carboxyl groups self-assembled and connected to a capture probe, thus successfully obtaining a free-standing AgNWs / AuNPs electrochemical biosensor.

[0014] Studies have shown that the electrochemical biosensor based on a free-standing AgNWs / AuNPs electrode prepared in this invention exhibits good selectivity, reproducibility, and stability for the electrochemical detection of interleukin-6 (IL-6). This biosensor demonstrates high sensitivity for IL-6 detection, showing a range from 0.001 ng / ml. -1 Up to 100ng / ml -1 Wide linear range and 29.8 fg ml -1 Its low detection limit enables dynamic detection of IL-6 in real samples. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 The present invention relates to the design and fabrication of a free-standing-AgNWs / AuNPs electrochemical biosensor; (A) the fabrication process of the free-standing-AgNWs / AuNPs electrochemical biosensor; (B) I. an experimental setup for fabricating free-standing-AgNWs electrodes using an electric field; II. AgNWs uniformly dispersed in isopropanol (IPA); III-V. under an alternating electric field (E = 160 kV m). -1 At 10 kHz and amplification factor of 40×, AgNWs are arranged, migrated and connected in an orderly manner to form AgNWs bundles;

[0017] Figure 2The electrochemical performance of AgNWs electrodes induced by electric field and AuNPs in the embodiments of the present invention is shown below: (A) CV curves of bare CNT-PDMS electrode, AgNWs-coating electrode, Free-standing AgNWs electrode (treated with electric field), AgNWs-coating / AuNPs electrode, and Free-standing AgNW / AuNPs electrode after electrochemical response stabilization; (B) CV curves of bare CNT-PDMS electrode, AgNWs-coating electrode, Free-standing AgNWs electrode, AgNWs-coating / AuNPs electrode, and Free-standing AgNW / AuNPs electrode. (C) Histogram of CV oxidation peak current of AgNWs / AuNPs electrode; (D) Redox peak current of bare CNT-PDMS electrode, AgNWs-coating / AuNPs electrode and free-standing-AgNWs / AuNPs electrode at different scan rates; (E) Microscopic effective surface area of ​​bare CNTs-PDMS electrode, AgNWs-coating / AuNPs electrode and free-standing-AgNWs / AuNPs electrode according to Randles–Sevcik equation;

[0018] Figure 3 The CNT-PDMS bare electrode, AgNWs-coating electrode, Free-standing-AgNWs electrode, AgNWs-coating / AuNPs electrode, and [other electrode types] mentioned in the embodiments of the present invention are...

[0019] CV oxidation peak potential of free-standing-AgNWs / AuNPs electrode;

[0020] Figure 4 The effective surface area of ​​the CNT-PDMS electrode (a), AgNWs-coating / AuNPs electrode (b), and Free-standing-AgNWs / AuNPs electrode (c) in the embodiments of the present invention is shown below.

[0021] Figure 5 CV and EIS characterization of the modified electrodes in this embodiment of the invention; (A) Curve I: CNT-PDMS bare electrode, Curve II: Free-standing AgNWs / AuNPs electrode, Curve III: Free-standing

[0022] AgNWs / AuNPs / MUA-MPA / EDC-NHS electrode, curve IV Free-standing

[0023] AgNWs / AuNPs / MUA-MPA / EDC-NHS / IL-6 anti-electrode, curve V at 5mM [Fe(CN)6] 3- / 4- CV curves of Free-standing AgNWs / AuNPs / MUA-MPA / EDC-NHS / anti-IL-6 / BSA electrode; (B) CNTs-PDMS bare electrode, Free-standing AgNWs / AuNPs electrode, Free-standing AgNWs / AuNPs / MUA-MPA / EDC-NHS electrode, Free-standing

[0024] AgNWs / AuNPs / MUA-MPA / EDC-NHS / IL-6-resistant electrodes and Free-standing

[0025] AgNWs / AuNPs / MUA-MPA / EDC-NHS / IL-6 / BSA electrode at 5mM [Fe(CN)6] 3- / 4- EIS curve in;

[0026] Figure 6 The electrochemical response to the detection target IL-6 in this embodiment of the invention; (A) using a biosensor to detect different concentrations of IL-6 (0.001-100 ng / ml) diluted with PBS. -1 (A) The DPV response of the biosensor; (B) The calibration curves between the DPV peak current and IL-6 concentration (logarithmic form) showed a good linear relationship; (C) The biosensor responded to different concentrations of target IL-6 (0.001-100 ng / ml) diluted with PBS. -1 (A) CV response; (D) Specificity of the biosensor to IL-6 (interfering antigens are CEA, CTNI, and IgG); (E) Incubation of 1 ng / ml -1 Reproducibility of five biosensors for IL-6; (F) Stability of biosensors in detecting IL-6;

[0027] Figure 7 This invention provides an embodiment for dynamically monitoring IL-6 secreted by Caco-2 cells in an intestinal organ-on-a-chip (GOC); (A) a microfluidic device comprising a Caco-2 cell and a sensing system; (B) changes in current values ​​corresponding to IL-6 secreted by Caco-2 cells during different culture days (n=3); and (C) changes in IL-6 concentration corresponding to the measurement results in (B) based on I = 38.53-8.44lg c.

[0028] Figure 8 This is a DPV test of IL-6 secretion by Caco-2 cells with different culture days in this embodiment of the invention. Detailed Implementation

[0029] Given the problems of low electron transfer efficiency and low sensitivity in existing electrochemical methods for detecting IL-6, this invention proposes a free-standing-AgNWs / AuNPs electrochemical biosensor.

[0030] A typical embodiment of the present invention provides a method for preparing a free-standing AgNWs / AuNPs electrochemical biosensor. CNTs are mixed with PDMS, and then vacuum-treated to obtain a CNT-PDMS composite material. The CNT-PDMS composite material is mixed with a curing agent and filled into an electrode template and cured to obtain a bare electrode. The bare electrode and a suspension of silver nanowires (AgNWs) are placed in an electric field and subjected to electrochemical treatment, causing the AgNWs to be orderly embedded into the bare electrode along the electric field direction to obtain a modified electrode. Gold nanoparticles are electrodeposited on the modified electrode, and the gold nanoparticles are modified with carboxyl groups at their ends via Au-S bonds. The carboxyl groups at the ends of the gold nanoparticles on the modified electrode are activated. Then, a capture probe containing an amino group is linked to the activated carboxyl group to obtain the sensor. The capture probe can specifically bind to IL-6.

[0031] In some embodiments of this implementation, CNTs and PDMS are mixed using a dry mixing method; preferably, the rotation speed is 100-200 rpm and the dispersion time is 12-36 h.

[0032] In some embodiments of this implementation, the mass percentage of CNTs in the CNTs-PDMS composite material is 5–10 wt.%.

[0033] In some embodiments of this implementation, the absolute pressure of the vacuum treatment is 50–100 Pa, and the time is 20–40 min.

[0034] In some embodiments of this implementation, the curing agent is 5 to 15% of the mass of the CNTs-PDMS composite material.

[0035] In some embodiments of this implementation, the concentration of the AgNW suspension is 1.5-2.0 mg / ml. -1 .

[0036] In some embodiments of this implementation, the bare electrode serves as one electrode of the electric field during the energizing process. An AC voltage of 150–160 kV and 8–12 kHz is applied during the energizing process. The energizing process lasts for 25–35 minutes.

[0037] In some embodiments of this implementation, gold nanoparticles are deposited onto the surface of the modified electrode using electrodeposition, with the dielectric solution containing chloroauric acid. The electrodeposition method may involve depositing the gold nanoparticles onto the electrode surface using chronoamperometry (CA). This approach avoids disrupting the high orderliness of the silver nanowires during the gold nanoparticle deposition process, thereby ensuring the electrochemical performance of the electrode and consequently guaranteeing the detection performance of the free-standing AgNWs / AuNPs electrochemical biosensor.

[0038] In some embodiments of this implementation, gold nanoparticles are modified with carboxyl-terminated groups via Au-S bonds using MUA and MPA, and then the modified electrode is activated using NHS and EDC.

[0039] In some embodiments of this implementation, an amide bond is formed between the amino group and the activated carboxyl group of the capture probe through a self-assembly process, thereby attaching the capture probe to the surface of the modified electrode.

[0040] In some embodiments of this implementation, after the capture probe is attached, it is immersed in a bovine serum albumin solution for incubation to eliminate nonspecific binding.

[0041] A second typical embodiment of the present invention provides a free-standing-AgNWs / AuNPs electrochemical biosensor obtained by the above preparation method.

[0042] A third typical embodiment of the present invention provides an application of the above-described free-standing-AgNWs / AuNPs electrochemical biosensor in the detection of IL-6 or in the manufacture of a system for detecting IL-6.

[0043] The IL-6 detection system includes an electrochemical workstation, with the free-standing AgNWs / AuNPs electrochemical biosensor connected to the electrochemical workstation as the working electrode.

[0044] A fourth typical embodiment of the present invention provides an IL-6 detection kit, comprising the above-described free-standing-AgNWs / AuNPs electrochemical biosensor and a buffer solution, wherein the buffer solution contains [Fe(CN)6]. 3- / 4- The buffer solution is a phosphate buffer solution (PBS solution).

[0045] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0046] Example

[0047] I. Fabrication of Free-standing AgNWs / AuNPs Electrochemical Biosensors

[0048] 1. Fabrication of CNTs-PDMS bare electrodes

[0049] CNTs and PDMS at a weight ratio of 8 wt.% were dispersed by dry mixing at 150 rpm for 24 h to obtain a prepolymer CNTs-PDMS composite material. The prepolymer CNT-PDMS composite material was then vacuum-treated at approximately 80 Pa absolute pressure for 30 min. Subsequently, a PDMS curing agent was added to the CNT-PDMS composite material at a weight ratio of 1:10 to obtain a CNT-PDMS composite slurry. A cavity mold with a radius of 1.5 mm and a thickness of 0.5 mm was fabricated using 3D printing to obtain a bare CNT-PDMS electrode.

[0050] The process of fabricating bare CNT-PDMS electrodes from CNT-PDMS composite slurry using 3D printing involves: fixing the mold to a glass substrate, and then filling the cavity of the mold with the aforementioned CNT-PDMS composite slurry. Carbon fibers are embedded as wires before the CNT-PDMS is cured to connect to the electrochemical workstation during measurement. Finally, the mixed CNT-PDMS composite is cured in a drying oven at 70°C for 2 hours to obtain the bare CNT-PDMS electrode.

[0051] 2. Fabrication of Free-standing CNTs Electrodes

[0052] Dilute AgNWs to 1.7 mg / ml in isopropanol (IPA). -1 The concentration was adjusted to form an AgNWs suspension. To embed the AgNWs into the bare electrode, a semi-cured CNT-PDMS composite material was first coated onto the dry bare electrode. The CNT-PDMS bare electrode and an aluminum electrode spaced 50 mm apart served as the two ends of the electric field, as shown in the figure. Figure 1 As shown. Decane was poured into a custom-designed electric field-applied mold to dissolve the PDMS surface layer. Then, an AgNWs suspension was poured into the mold and applied at 160 kV m. -1 A modified electrode, namely a free-standing CNT electrode, was obtained by applying an AC voltage of 10 kHz for 30 minutes.

[0053] 3. Fabrication of Free-standing AgNWs / AuNPs Electrochemical Biosensors

[0054] Gold nanoparticles (AuNPs) were electroreduced on a free-standing CNT electrode for 250 seconds at a constant potential of -0.4 V using a chronoamperometry (CA) method. The dielectric solution was 2.0 × 10⁻⁶. -3 Free-standing AgNWs / AuNPs electrodes with gold nanoparticles were obtained by using M HAuCl4 and 0.5M H2SO4.

[0055] The free-standing AgNWs / AuNPs electrode was immersed in MUA-MPA solution overnight, and the carboxyl groups on the electrode were activated with 0.05M EDC-NHS solution for 1 hour.

[0056] 10 μL of anti-IL-6 capture antibody was coated onto the carboxyl-activated electrode surface. An amide bond was formed between the amino group of the anti-IL-6 capture antibody and the activated carboxyl group through a self-assembly process at 4 °C, thus attaching the anti-IL-6 capture antibody to the electrode surface. The resulting biosensor was tested with a 10 × 10⁻⁶ electrode. -3 M was washed with PBS to remove any unlinked antibodies. Furthermore, to eliminate any non-specific binding, the prepared electrode was incubated in 1% BSA for 1 hour to obtain a free-standing AgNWs / AuNPs electrochemical biosensor, prepared as follows: Figure 1 As shown in Figure A.

[0057] Figure 1 A illustrates the stepwise modification process of a free-standing AgNWs / AuNPs electrode to prepare a free-standing AgNWs / AuNPs electrochemical biosensor for the detection of interleukin-6 (IL-6). An alternating electric field (E = 160 kV m) was used. -1 The AgNWs were rearranged (at 10 kHz). The experimental setup and force analysis diagram of the AgNWs are shown below. Figure 1 As shown in B-Ⅰ and Ⅱ, AgNWs are rotated in the direction of the electric field and embedded in semi-cured conductive CNT-PDMS.

[0058] To visually demonstrate the dynamic rotation and migration of AgNWs under an electric field, the process was demonstrated under an optical microscope. Figure 1 B-III to V). Initially, AgNWs (small black dots) were uniformly dispersed in isopropanol (IPA), such as... Figure 1 As shown in B-Ⅲ. An electric field (10kHz, 160kV m) is applied. -1 After that, AgNWs were forced to migrate along the direction of the electric field. Figure 1 B-Ⅳ). Over time, more AgNWs connect together and are considered as bundles of silver nanowires (B-Ⅳ). Figure 1B-V). These are because AgNWs are subjected to dielectric electrophoretic forces and torques after polarization. Figure 1 B-Ⅱ), as shown in equation (1). When the orientation and position of AgNWs are fixed, some AgNWs exhibit a shape similar to that of eukaryotic flagella (B-Ⅱ), as shown in equation (1). Figure 1 B-V).

[0059]

[0060] Where η is the viscosity of the medium, Talign is the corresponding induced torque, and I is the moment of inertia of a single AgNWs.

[0061] II. Electrochemical Measurements of Freestanding-AgNWs / AuNPs Electrochemical Biosensors

[0062] Because it is necessary to compare and demonstrate the advantages of the free-standing-AgNWs / AuNPs electrochemical biosensor of this invention in electrochemical measurements, the following two electrode preparation methods are supplemented:

[0063] (1) AgNWs-coating electrode: Dilute AgNWs in isopropanol (IPA) to 1.7 mg / ml -1 The concentration of AgNWs was adjusted to form an AgNWs suspension. To embed AgNWs into a bare electrode, a semi-cured CNTs-PDMS composite material was first coated onto the dry bare electrode. The AgNWs suspension was then dropwise added to the electrode surface to obtain an AgNWs-coated electrode.

[0064] (2) AgNWs-coating / AuNPs electrode: Gold nanoparticles were electroreduced on the electrode surface of the AgNWs-coating electrode to obtain the AgNWs-coating / AuNPs electrode.

[0065] 1. Enhancement of electrochemical performance of AgNWs electrodes by electric field and AuNPs

[0066] In the presence of 5 mM [Fe(CN)6] 3- / 4- Cyclic voltammetry (CV) measurements were performed on the free-standing AgNWs / AuNPs electrode, AgNWs-coating / AuNPs electrode, free-standing AgNWs electrode, AgNWs-coating electrode, and bare CNT-PDMS electrode after the electrochemical response stabilized in 10 mM PBS.

[0067] With 50mV s -1 The CV curve is recorded at the scan rate, and the CV response is detected. The resulting CV curve is shown in the figure. Figure 2As shown in Figure A, the bare CNT-PDMS electrode does not exhibit a significant potassium ferricyanide redox peak, while the oxidation peak current of the free-standing AgNWs / AuNPs electrode is 500 times that of the bare electrode and 100 times that of the AgNWs-coated electrode. Figure 2 As shown in Figure B, after 8 CV scan cycles, the oxidation peak current of the AgNWs-coated electrode and the free-standing AgNWs electrode without AuNPs decreased to 5 μA and 20 μA, respectively. The oxidation peak current of the free-standing AgNWs / AuNPs electrode was 500 times that of the bare CNT-PDMS electrode, 100 times that of the AgNWs-coated electrode, 25 times that of the free-standing AgNWs electrode, and 1.7 times that of the AgNWs-coated / AuNPs electrode. Figure 3 As shown, the oxidation peak potential shifts to the left with electrode modification. This indicates that the parallel arrangement of free-standing AgNWs and the electroreduction of AuNPs can improve the redox response current of the electrode.

[0068] Analysis of the range of 20-200 mV s -1 The CV response at different scan rates was used to study the kinetics of three electrodes: CNT-PDMS electrode, AgNWs-coating / AuNPs electrode, and free-standing-AgNWs / AuNPs electrode. The detection results are as follows: Figure 4 As shown, the oxidation peak current increases linearly with the scan rate. The oxidation peak (Ipa) and reduction peak (Ipc) currents are related to the square root of the scan rate (v). 1 / 2 Linear fitting, such as Figure 2 As shown in C, a good linear relationship (R) 2 =0.99) indicates that the redox reaction on the electrode surface is a diffusion-controlled process. For example... Figure 2 As shown in Figure D, the microscopic electroactive areas of the bare CNT-PDMS electrode, AgNWs-coating / AuNPs electrode, and free-standing AgNWs / AuNPs electrode can be calculated using the Randles-Sevcik equation, and are 0.0043 mm², respectively. 2 33.68mm 2 and 60.29mm 2 The trend of microscopic effective surface area and Figure 2 The change in peak current in A is consistent.

[0069] 2. Electrochemical characterization of the fabrication process of free-standing-AgNWs / AuNPs electrochemical biosensors

[0070] To demonstrate the gradual success of electrode manufacturing, Figure 5 A, Figure 5 In section B, the CV and electrochemical impedance spectroscopy (EIS) of the modified electrode were tested. For example... Figure 5 As shown in Figure A, compared to the bare CNT-PDMS electrode (curve I), the oxidation peak current of the free-standing AgNWs / AuNPs electrode increases to 500 μA (curve II), which is due to the excellent conductivity of AgNWs and AuNPs. However, connecting MUA-MPA / EDC-NHS to the AuNPs reduces the oxidation peak current to 300 μA (curve III). The specific connection process is shown below.

[0071]

[0072] Ester group combinations of free-standing AgNWs / AuNPs / MUA-MPA / EDC-NHS (curve IV). Correspondingly, [Fe(CN)6] 3- / 4- The oxidation peak current dropped to 200 μA. This may be due to the negative charge of the amino group in the anti-IL-6 capture antibody hindering the reaction of [Fe(CN)6]. 3- / 4- The diffusion was observed. Finally, the elimination of N-Au bonds (between AuNPs and IL-6) with 1% bovine serum albumin (BSA) had a significant effect on electron transfer ability (curve V).

[0073] like Figure 5 As shown in Figure B, the Rct values ​​in the EIS exhibit opposite trends, indicating the successful fabrication of the electrochemical biosensor. The enhanced steric hindrance and electrostatic repulsion of the anti-IL-6 capture antibody and IL-6 specifically hindered the capture of [Fe(CN)6]. 3- / 4- Diffusion to the electrode surface leads to a decrease in the peak current associated with IL-6 concentration.

[0074] 3. Measurement of IL-6 using a biosensor based on free-standing AgNWs / AuNPs

[0075] DPV technology was used in a solution containing 5 mM [Fe(CN)6] 3- / 4- Different concentrations of IL-6 were measured in 10 mM PBS.

[0076] from Figure 6 A shows that, in the range of 0.001 to 100 ng / ml -1 Within this range, the peak current of DPV decreased from 66 μA to 20 μA with increasing IL-6 concentration. This is because the combination of IL-6 and the anti-IL-6-capturing antibody blocks [Fe(CN)6]. 3- / 4- Electron transport. For example... Figure 6 As shown in Figure B, the peak current of the DPV exhibits a good linear relationship with the logarithm of different IL-6 concentrations, and the linear regression equation is I = 38.53 - 8.44lg c(R). 2 =0.988), and the low detection limit of 29.8fg (S / N=3) was calculated by equation (2), indicating that the biosensor is superior to or comparable to other electrochemical biosensor strategies for IL-6 detection.

[0077]

[0078] Where X b1 S is the average response of the biosensor to a blank measurement. b1 is the standard deviation of the blank measurement, k is a numerical factor chosen according to the required confidence level, a is the intercept of the calibration curve, and b is the slope of the calibration curve. Generally, a value of 3k is used in the equation. For example... Figure 6 As shown in Figure C, the CV response shows the same trend, indicating that the electrochemical biosensor can be successfully used to detect IL-6.

[0079] To explore whether biosensors can specifically recognize IL-6 protein, the levels of IL-6 protein were measured using 1 ng / ml... -1 Peak current of sensors incubated with CEA, CTNI, IgG, and IL-6, such as Figure 6 As shown in Figure D. The results indicate that the peak current in the presence of the target IL-6 is much lower than that of other proteins, indicating that the sensor has excellent selectivity for the target IL-6. Furthermore, the reproducibility of the biosensor in detecting IL-6 was also investigated. Five biosensors developed from free-standing AgNWs / AuNPs electrodes (i.e., five biosensors prepared using the same fabrication method of this invention, through the same fabrication steps, to test the consistency of the detection performance of the biosensors of this invention) were used to measure the same IL-6 concentration (1 ng / ml). -1 DPV response, such as Figure 6 As shown in Figure E, the RSD is 3.77% (<5%), indicating that the biosensor prepared in this invention has good reproducibility. Figure 6 As shown in Figure F, the biosensor retained 93.5% of its current response after 25 days, meeting the experimental requirements.

[0080] 4. Detection of IL-6 secreted by Caco-2 cells in intestinal organ-on-a-chip

[0081] To dynamically measure IL-6 secretion on the intestinal organ-on-a-chip (GOC), in Figure 7A microfluidic device comprising Caco-2 cells and a sensing system was assembled in section A. To dynamically perfuse culture medium in the GOC, controlled valves and microfluidic channels were programmed to allow solution to be introduced at predetermined times. Caco-2 cells (1.5 × 10⁻⁶) were then introduced. 5 cells / cm 2 The oxidation peak current was injected into the top layer of the GOC porous membrane, and the change in the value was continuously measured every 24 hours for 10 days. Figure 7 B, Figure 8 There was no significant difference in the peak oxidative current among cells cultured for 1–4 days. Within 5–10 days, the peak oxidative current decreased significantly from 65 μA to 56.5 μA. Correspondingly, the IL-6 concentration trend curve (…) Figure 7 C) The linear regression equation in 4b is I = 38.53 - 8.44lg c(R). 2 =0.988). When a confluent epithelial monolayer has not formed, IL-6 secretion is less than 0.77 pg ml⁻¹ (≤4 days). However, in cultures of 5–10 days (after the formation of an integrated cellular barrier), the concentration of IL-6 increases from 0.77 pg ml⁻¹. -1 Increased to 7.8 pg ml -1 This represents a 10.13-fold increase. This indicates that biosensors based on free-standing AgNWs / AuNPs electrodes can be used to detect real-world samples.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a free-standing AgNWs / AuNPs electrochemical biosensor, characterized in that, CNTs were obtained by mixing CNTs with PDMS and then processing under vacuum. PDMS composite material, with CNTs PDMS composite material is mixed with a curing agent and filled into an electrode template and cured to obtain a bare electrode. The bare electrode and a silver nanowire (AgNWs) suspension are placed in an electric field and subjected to electrochemical treatment, causing the AgNWs to be orderly embedded into the bare electrode along the electric field direction to obtain a modified electrode. Gold nanoparticles are electrodeposited on the modified electrode, and carboxyl groups are modified on the gold nanoparticles through Au-S bonds to activate the carboxyl groups at the ends of the gold nanoparticles on the modified electrode. Then, a capture probe containing an amino group is linked to the activated carboxyl group to obtain the modified electrode. The capture probe can specifically bind to IL-6. The concentration of AgNWs suspension was 1.5-2.0 mg / ml. -1 ; During the power-on process, an AC voltage of 150–160 kV and 8–12 kHz is applied; the power-on process takes 25–35 minutes.

2. The method for preparing the free-standing-AgNWs / AuNPs electrochemical biosensor as described in claim 1, characterized in that, CNTs and PDMS were mixed using a dry mixing method; Or, CNTs The mass percentage of CNTs in the PDMS composite material is 5–10 wt.%. Alternatively, the absolute pressure of vacuum treatment is 50–100 Pa, and the time is 20–40 min.

3. The method for preparing the free-standing-AgNWs / AuNPs electrochemical biosensor as described in claim 2, characterized in that, The rotation speed is 100–200 rpm, and the dispersion time is 12–36 h.

4. The method for preparing the free-standing-AgNWs / AuNPs electrochemical biosensor as described in claim 1, characterized in that, The curing agent is CNTs 5-15% of the mass of PDMS composite material; Alternatively, in the energized process, the bare electrode serves as one electrode of the electric field.

5. The method for preparing the free-standing-AgNWs / AuNPs electrochemical biosensor as described in claim 1, characterized in that, Gold nanoparticles were deposited onto the surface of modified AgNWs electrodes using an electrodeposition method, with chloroauric acid included in the dielectric solution for electrodeposition.

6. The method for preparing the free-standing-AgNWs / AuNPs electrochemical biosensor as described in claim 1, characterized in that, Using MUA and MPA via Au S-bonds are used to modify gold nanoparticles with carboxyl groups at the top end, and then the modified electrode is activated using NHS and EDC.

7. The method for preparing the free-standing-AgNWs / AuNPs electrochemical biosensor as described in claim 1, characterized in that, Through a self-assembly process, an amide bond is formed between the amino group of the capture probe and the activated carboxyl group, thus attaching the capture probe to the surface of the modified electrode.

8. The method for preparing the free-standing-AgNWs / AuNPs electrochemical biosensor as described in claim 1, characterized in that, After connecting the capture probe, it is immersed in bovine serum albumin solution for incubation.

9. A free-standing AgNWs / AuNPs electrochemical biosensor, characterized in that, Obtained by the preparation method described in any one of claims 1 to 8.

10. The use of the free-standing-AgNWs / AuNPs electrochemical biosensor of claim 9 in the detection of IL-6 or in the manufacture of a system for detecting IL-6.

11. An IL-6 detection kit, characterized in that, This includes the free-standing AgNWs / AuNPs electrochemical biosensor and buffer solution as described in claim 9, wherein the buffer solution contains [Fe(CN)6]. 3 / 4 .

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