KR102043321B1 - Nano-biosensor with interdigitated electrode for enhanced sensing TNF-alpha by deposition of nanoparticle - Google Patents

Abstract

The present invention relates to an electrode and a nanobiosensor using the same by depositing gold nanoparticles on a comb-shaped electrode and binding a TNF-alpha antibody to increase the measurement sensitivity for TNF-alpha.
According to the present invention as described above, using an IDE deposited gold nanoparticles, there is an effect of providing a platform that binds to the receptor molecule better and persists biological activity.
Specifically, an interdigitate electrode-based immunosensor with deposited gold nanoparticles, including TNF-alpha antibody, which has increased measurement sensitivity to TNF-alpha has been developed, showing improved sensitivity compared to general IDE. Prevent incorrect attachment. It was confirmed that the capacitance (capacitance) value was influenced depending on the trace TNF-alpha concentration of 1 pg / ml to 10 ng / ml, and the concentration of 0.83 pg / ml can be measured, so that the concentration of TNF-alpha in human plasma was maintained as it is. It can be measured.
Biosensors with improved sensitivity do not require expensive fluorescent dyes, reagents, or equipment, which have been consumed in the existing methods, and can be applied to various TNF-alpha concentrations in the diagnosis of diseases of patients.

Description

Nano-biosensor with interdigitated electrode for enhanced sensing TNF-alpha by deposition of nanoparticle}

The present invention relates to an electrode and a nanobiosensor using the same by depositing gold nanoparticles on a comb-shaped electrode and binding TNF-alpha antibody to increase the measurement sensitivity for TNF-alpha.

Electrochemical biosensors, due to their sensitivity, selectivity, simplicity and low cost, are attracting attention in the fields of therapeutic diagnostics, food analysis and environmental monitoring and are becoming a major analytical tool. For this reason, new strategic sensor platforms using electrochemical biosensors with high sensitivity and selectivity are steadily evolving.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine that appears to play a pivotal role in immune and inflammatory responses, including allergic rhinitis and conjunctivitis. TNF-alpha is a soluble homotrimer of the 17 kD protein subunit (Smith, 1987). TNF-alpha is derived from monocytes and macrophages, along with other cell types. Modulation of TNF-alpha has been proposed as a therapeutic strategy for allergic conjunctivitis and other symptoms associated with TNF-alpha activation.

Precise detection of TNF-alpha can be applied to pharmacological response testing for clinical diagnostics and drug development.

Most studies of electrochemical biosensors have focused on the mixing of metal nanoparticles in the design of sensitive electrochemical sensors. In order to obtain nanoparticle inherent properties that do not appear in bulk structures, such as catalytic activity, optical, electrical and magnetic forces, much effort has recently been focused on nanoparticle deposition using electricity.

The inventors have made a new nano biosensor composed of Au nanoparticle (NP) hybrid structure deposited on an indium tin oxide (ITO) microdisk national array (MDEA) chip, and applied it as a TNF-alpha nanobiosensor. It was confirmed that the present invention was completed.

An object of the present invention is to prepare a comb-shaped electrode (AuNP = IDE) in which gold nanoparticles are deposited by material properties and nanostructures, and to improve measurement sensitivity than conventional IDEs, thereby making economical and stable trace amounts of TNF-alpha materials. It is an object of the present invention to provide a nanobiosensor that can be detected. In addition, to provide a method for manufacturing such a nanobiosensor.

In order to achieve the above object, the present invention provides an interdigitate electrode, the nanoparticles are deposited on the surface, including the bound antibody.

Preferably, the nanoparticles are characterized in that the gold (Au) nanoparticles.

Preferably, the bound antibody is characterized in that the TNF-alpha antibody.

Preferably, the bond is characterized in that the covalent bond.

Preferably, the interdigitate electrode is characterized in that a plurality of comb-like fingers are arranged spaced apart from each other.

In another aspect, the present invention provides an immune sensor for measuring TNF-alpha comprising any one of the above electrodes.

In another aspect, the present invention, (a) depositing nanoparticles on the surface of the comb-shaped electrode by electrodeposition (Electrodeposition); And (b) covalently fixing the antibody to the comb-shaped electrode which has been subjected to the step (a). It provides a method for producing an immune sensor for measuring TNF-alpha, comprising: a.

Preferably, the nanoparticles of step (a) are gold (Au) nanoparticles, the antibody of step (b) is characterized in that the TNF-alpha antibody.

In another aspect, the present invention, (1) obtaining a calibration curve for the measurement of the immune sensor for measuring TNF-alpha; (2) measuring a capacitance by placing a sample in an electrode of an immune sensor for measuring TNF-alpha and connecting the electrode; And (3) obtaining the TNF-alpha content of the sample from the capacitance value obtained in step (2) using a calibration curve. to provide.

According to the present invention as described above, by using a comb-shaped IDE on which gold nanoparticles are deposited, there is an effect of providing a platform that binds to receptor molecules better and persists biological activity.

Specifically, an interdigitate electrode-based immunosensor with gold nanoparticles deposited, including TNF-alpha antibody, which has increased measurement sensitivity to TNF-alpha has been developed, showing improved sensitivity compared to general IDE. Prevent incorrect attachment. It was confirmed that the capacitance (capacitance) value was influenced depending on the trace TNF-alpha concentration of 1 pg / ml to 10 ng / ml, and the concentration of 0.83 pg / ml can be measured, so that the concentration of TNF-alpha in human plasma was maintained as it is. It can be measured.

Biosensors with improved sensitivity do not require expensive fluorescent dyes, reagents, or equipment, which have been consumed in the existing methods, and can be applied to various TNF-alpha concentrations in the diagnosis of diseases of patients.

값을 비교하고 Kd값을 구한 것. 그래프 안의 그래프는 농도가 0-500 pg/ml일 때의 농도범위만 확대한 것이다.1 is a photograph of the appearance of the finally produced IDE. (a) is a photograph of an IDE with eight reaction chambers. (b) is an enlarged photograph of the reaction chamber. You can check the shape of the comb-shaped electrode. (c) Electron micrograph of a comb-type IDE surface. There is no additional deposition. (d) is an electron microscope photograph of the surface of a comb electrode on which gold nanoparticles were deposited.
FIG. 2 is a graph of AuNP deposited on one IDE arm (WE) and two IDE arms (WE + CE), and analyzing the impedance and capacitance (capacitance) of IDE in 1 × PBS solution. (a) is an impedance graph. (b) is a capacitance (capacitance) graph.
Figure 3 is a graph of the results of the measurement of the change in impedance amount and capacitance (capacitance) according to the change in concentration. (a) is the Bode curve of impedance amount | Z |, (b) is the reactive capacitance curve, which is the data obtained by the TNF-alpha sensor to detect TNF-alpha in 1X PBS solution, (c) and (d) Curves of impedance amounts | Z | and reactive capacitance values obtained at different TNF-alpha concentrations ranging from 10 to 10 ⁴ pg / ml, (e) and (f) varying from 10 to 10 ⁴ pg / ml. Curve of impedance amount | Z | and reactive capacitance values obtained at HS-TNF-alpha concentration
FIG. 4 is a graph of the resulting capacitance (capacitance) modulus variation of the sensor with varying TNF-alpha concentrations ranging from 10 to 10 ⁴ pg / ml. (a) is the curve for several TNF-alpha concentrations, (b) is the curve for several HS-TNF-alpha concentrations, and (c) is the Bare IDE and AuNP when the TNF-alpha concentration is 1-3 pg / ml. A comparison of the capacitance measurement sensitivity of the IDE. (d) shows the concentration of TNF-alpha in humans with and without plasma.

Comparing values and obtaining Kd values. The graph in the graph only shows the concentration range when the concentration is 0-500 pg / ml.

Hereinafter, the present invention will be described in detail.

The present invention provides an interdigitate electrode, in which nanoparticles are deposited on a surface and comprising bound antibodies.

Preferably, the nanoparticles are characterized in that the gold (Au) nanoparticles.

Preferably, the bound antibody is characterized in that the TNF-alpha antibody.

Preferably, the bond is characterized in that the covalent bond.

Preferably, the interdigitate electrode is characterized in that a plurality of comb-like fingers are arranged spaced apart from each other.

In another aspect, the present invention provides an immune sensor for measuring TNF-alpha comprising any one of the above electrodes.

In another aspect, the present invention, (a) depositing nanoparticles on the surface of the comb-shaped electrode by electrodeposition (Electrodeposition); And (b) covalently fixing the antibody to the comb-shaped electrode which has been subjected to the step (a). It provides a method for producing an immune sensor for measuring TNF-alpha, comprising: a.

Preferably, the nanoparticles of step (a) are gold (Au) nanoparticles, the antibody of step (b) is characterized in that the TNF-alpha antibody.

In another aspect, the present invention, (1) obtaining a calibration curve for the measurement of the immune sensor for measuring TNF-alpha; (2) measuring a capacitance by placing a sample in an electrode of an immune sensor for measuring TNF-alpha and connecting the electrode; And (3) obtaining the TNF-alpha content of the sample from the capacitance value obtained in step (2) using a calibration curve. to provide.

Hereinafter, the present invention will be described in more detail through experimental examples. These experimental examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not interpreted to be limited by these experimental examples.

Experimental Example 1. AuNP / IDE Array Fabrication

3-mercaptopropionic acid (MPA), N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA) and poly (Dimethylsiloxane) (PDMS) was purchased from Sigma-Aldrich. Recombinant TNF-alpha was purchased from Enzo Life Sciences, Inc. TNF-alpha monoclonal antibody was purchased from Santa Cruz Biotechnology. Human serum was collected with the consent of Yonsei Severance Hospital (Seoul, South Korea). Phosphate buffered saline (1X PBS, 137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, and 1.4mM KH2PO4, pH 7.4) and 2- (N-morpholino) ethane sulfonic acid (MES; 0.1M, pH 6.0) are Tech Obtained from and Innovation. Deionized water (distilled water) (18.2 MΩcm) was obtained from the Milli-Q system and used throughout the experiment. All other chemicals were used in analytical grade.

Au / NP IDE arrays were prepared on slide glass (75 × 25 × 1 mm ³ ) substrates. The glass substrate was cleaned by sonication in 70% methanol and the lift-off photoresist (DNR L300-30) was spin-coated onto the substrate and patterned to have an inverse pattern of electrodes, transmission lines and terminal pads. Ti (25 nm) and Au (50 nm) were deposited sequentially on the patterned substrate by sputtering and the sacrificial layer was removed to form an IDE. Subsequently, an insulating substrate (SU-8 2002) was patterned over the IDE substrate to expose the IDE sensing area and the terminal pad. The width and spacing of the IDE comb electrodes are 50 μm. In order to form AuNPs on the IDE and preserve the reaction solvent used, a PDMS chamber was placed on the prepared IDE (see FIGS. 1A and 1B). Au (HAuCl 4 .3H 2 O; 0.5 mM) solution in distilled water was added to the IDE. A deposition voltage of -0.9 V relative to Ag / AgCl was applied to the IDE for 20 seconds at room temperature. After deposition, the resulting AuNP / IDE array was washed with distilled water and dried under a nitrogen gas (N2) environment. Finally on the glass slide, an AuNP / IDE array was formed.

Scanning electron microscopy (SEM) measurements were performed at 20 kV operating voltage (COXEM) to confirm AuNP deposition in the IDE (see FIGS. 1C and 1D). The electrical properties of the manufactured sensors were examined at room temperature using an electrical impedance spectroscopy (EIS) analyzer (IVIUM CompactStat). Measurements were performed at an input potential of 0.1 V in PBS solution, scanning with a frequency range of 1-10 ⁵ Hz. Prior to electrical impedance measurement, oxygen was removed from the PBS electrolyte solution by bubbling with a pure nitrogen gas stream. The measured impedance spectra were characterized by nonlinear curve fitting in the designed equivalent circuit model. In order to better understand the electrical characteristics of AuNP / IDE, the electric field and current distribution of the device were simulated by COMSOL multiphysics. The dimensions of the modeled electrode structure are similar to those of the manufactured IDE, but AuNP on the IDE is simplified as a uniform hemisphere with a radius of 50 nm.

Experimental Example 2 Preparation of AuNP / IDE-based TNF-alpha Immune Sensor

Confirmation of effective binding with TNF-alpha antigen was performed according to standard procedures. The prepared AuNP / IDE was treated by soaking in ethanolic 10 mM solution of 3-MPA for 6 hours at room temperature. Then, 400 mM EDC solution and 100 mM NHS solution were gently mixed on the IDE while gently mixed, and reacted with 3-MPA for 1 hour on the surface of the fixed IDE. Then 10 μl of antibody solution (100 μg / ml) was dropped onto the activated IDE surface. Bovine serum albumin (BSA, 1 ng / ml) dissolved in PBS (10 mM, pH 7.4) was used to block nonspecific adsorption on the electrode surface. Various amounts of TNF-alpha were mixed in PBS or human serum solution to obtain final concentrations of 1, 10, 100, 500, 1,000 and 10,000 pg / ml. 10 μl aliquots of each sample were then applied to the prepared sensors and incubated at 4 ° C. for 60 minutes.

Experiment result

(1) Electrochemical Characteristics of AuNP / IDE-Impedance and Capacitance (Capacitance)

Bare where nothing was deposited, AuNP deposited on one arm of IDE (WE) electrode, and AuNP deposited on both arms of IDE (WE + CE) electrode of 1X PBS, measured impedance magnitude (| Z | ) And the corresponding capacitance (capacitance) graph are shown in FIG. 2. The impedance characteristics of the fabricated electrode can be modeled as an equivalent circuit containing a series resistance to solution resistance (R _S ) and a constant phase element for electrode interface impedance (CPE), which is 1 / T (jω) ^CPE-P (Where CPE-T and CPE-P are adjustable values, j is an imaginary unit, and ω is each frequency). From the results of the data fitting analysis below (Table 1), the deposition of AuNP induces a decrease in 1 / CPE-T and CPE-P reflecting the increase in the roughness and area of the electrode surface, and the CPE in bare IDE. The -P value is close to 1, indicating that the electrical interfacial impedance is mostly due to capacitive reactance. The degree of decrease in electrode interface impedance due to AuNP deposition depends on the degree of AuNP deposition.

Table 1

The electric field and current density distributions were simulated in both Bare IDE and AuNP / IDE (see Figure 2). In the case of the Bare IDE, the highest electric field value is observed at the edge of the electrode, and a relatively low electric field slope is observed in the other regions of the electrode (see c in FIG. 2). This result indicates that the measurement sensitivity is maximum at the edge rather than the center of the electrode as compared with the rest of the electrode. However, in the case of AuNP / IDE, a strong electric field is observed at both the edge of the electrode and around the region where AuNP is deposited (see d in FIG. 2). Due to the concentrated electric field strength and the current density well distributed in the AuNPs on the electrodes, the high sensitivity region of the sensor is expanded.

For direct detection of TNF-alpha, the formation of SAM and functionalization of AuNP / IDE surface is assessed by EIS. Linkers and proteins exhibit complex electrical properties with charge and structure, but due to sequential formation on the electrodes, the impedance magnitude (| Z |) increases and conversely, the reactive capacitance (capacitance) decreases (see FIG. 3). After surface functionalization and protein immobilization, a decrease in total capacitance was observed. This is presumed to be derived from the combination of each immobilized layer on the electrodes forming a series of dielectric layers. The formation of SAM on the AuNP / IDE surface is thought to produce series capacitance (capacitance) C _SAM along with the impedance of the electrode interface. In the same way, the immobilization of the TNF-alpha antibody (CaTNF-alpha) and the binding of TNF-alpha form additional series-connected capacitors (CaTNF-alpha or CTNF-alpha). Therefore, the total capacitance (capacitance) of the sensor after immobilizing SAM, aTNF-alpha, and TNF-alpha can be expressed by the following equation.

Overall, the decrease in capacitance (| Z |) is associated with the binding affinity and antigen-antibody response of SAM molecules. Through impedance analysis, it is possible to check the surface state of the sensor sequentially coated with SAM molecules and proteins.

(2) Electrochemical Characteristics of AuNP / IDE-Determination of TNF-alpha Concentration

Impedance measurement to determine the detection characteristics of the sensor manufactured in the present invention, compared with TNF-alpha concentration (ConcTNF-alpha) in PBS solution and TNF-alpha concentration (ConcHS-TNF-alpha) in human serum It became. At this time, a TNF-alpha concentration solution of 10 ⁴ pg / ml (equivalent to 588.24 fM to 588.24 pM) at 10 pg / ml dissolved in 10 mM PBS solution is used. As can be seen in (c) of FIG. 3 and (e) of FIG. 3, the impedance modulus value (| Z |) was found to increase with antigen concentration. In addition, the dependence of reactive capacitance (capacitance) on antigen concentration is reduced, as seen in Figures 3d and 3f.

The capacitance (capacitance) modulus value was extracted from the measured data as described by the following equation.

Where C is the actual capacitance (capacitance) after TNF-alpha binds the antibody at a specific concentration, and C ₀ is the capacitance (capacitance) value before binding. Increasing TNF-alpha concentration on the surface of the immune sensor increases | ΔC | (see FIG. 4A).

The feasibility of the detection platform developed for TNF-alpha in human serum was investigated. To obtain more reliable results, working curves were made using human serum diluted 200-fold as the analytical curve. As the concentration of TNF-alpha in human serum increases, the | ΔC | It also increases in the same manner as (b) of FIG. 4. These results indicate that TNF-alpha can be applied to complex biological structures without problems.

In addition, the sensitivity of Bare IDE and AuNP / IDE was investigated with low TNF-alpha concentration (1-3 pg / ml). AuNP / IDE was compared with Bare IDE. It was confirmed that the spectrum had high sensitivity (see c of FIG. 4). These results are in good agreement with the simulation results that AuNP / IDE is more sensitive than bare IDE. The dependence of the sensor response on the analyte concentration, the TNF-alpha concentration or the HS-TNF-alpha concentration is shown in (d in FIG. 4). The change in capacitance (capacitance) measured at 10 Hz increased with TNF-alpha concentration in PBS or human serum samples. For analyte in serum, a limit of detection (LOD) of 0.83 pg / ml was calculated from 3 (Sb / m). Where Sb is the standard deviation of the signal measured for m, which is blank and the slope of the analysis curve. However, it is not sufficient to judge the LOD only from the signal of being blank and the standard deviation obtained from the fitting curve. More accurate assessments of LOD can be obtained from experimental data measured at concentrations below 1 pg / ml.

To assess binding affinity with TNF-alpha, the dissociation constant Kd of TNF-alpha was calculated using an approach based on the Langmuir adsorption model. The equilibrium of antibody (Ab) and antigen (Ag) binding can be expressed as shown below.

If the surface coverage of the Ab-Ag conjugate is θ and the surface coverage of the unbound antibody is 1-θ, the dissociation constant Kd is

From the Langmuir adsorption model we find | ΔC | The value is directly related to antibody-antigen binding as follows.

Where | ΔC | sat is the maximum response of the sensor and | (C _sat -C ₀ ) / C ₀ | In the above equations, the following equation, which is a linearized form of the Langmuir isotherm equation, can be derived.

Using the above equations, Kd values can be obtained while avoiding reactions from low antigen concentrations. The Kd values of TNF-alpha observed in PBS and human serum were 73.98 and 103.53 pg / ml, respectively.

Good reproducibility is an important factor for the application of immune sensors in clinical diagnosis. In the current study, the reproducibility of the immune sensor is tested by intra-assay and internal assay relative standard deviation (RSD). Through the detection of TNF-alpha at a concentration of 100 pg / ml, the internal analysis precision measurement of the immunosensor was investigated in five iterations. Inter-assay precision was assessed by measuring TNF-alpha 100 pg / ml concentration samples with 5 differently prepared BSA / aTNF-alpha / MPA / AuNP / IDE. Intra-assay and inter-assay RSD values were 2.62 and 4.58%, respectively. This result indicates that the immune sensor of the present invention has acceptable accuracy and reproducibility. To regenerate the immune sensor, the electrode could be achieved by immersing the electrode in 0.2 M glycine-HCl solution (pH 2.3) for 10 minutes to break antibody-antigen binding. Nevertheless, biochemical reagents can be easily cleaned on the sensor surface for reuse and each modified sensor is used for single use in the experiment.

Immune sensor of the present invention, even if the sensor of the BSA / aTNF-alpha / MPA / AuNP / IDE structure in PBS (10 mM, pH 7.4) for 1 week at 4 ℃ without apparent denaturation, EIS response ability even after storage Maintained. This indicates that the structure of the sensor is biocompatible enough to maintain the bioactivity of the antibody. In addition, the covalent binding action between the MPA-modified AuNP and the primary amine group of the antibody prevents the desorption of the TNF-alpha antibody. The advantage of the TNF-alpha detection method based on the proposed capacitance (capacitance) value is that, compared to other conventional methods, it requires no labeling material, is highly applicable, prepares the minimum sample required, and directly supplies the electrical signal. Reading is less expensive.

Immunosensors based on sensitive capacitance (capacitance) measurements for TNF-alpha concentrations have been successfully manufactured by simply immobilizing one TNF-alpha on AuNP / IDE. AuNP formation in the IDE was confirmed by SEM and EIS, and AuNP / IDE provides a robust platform for enhancing binding of receptor molecules and maintaining bioactivity. The manufactured AuNP / IDE-based capacitive (capacitance) measuring sensor can be directly applied to human serum, and has higher sensitivity and resistance to biocontamination compared to exposed IDE. The immune sensor calculated a detection limit of 0.83 pg / ml and obtained a calibration curve for measurement in the concentration range of 1 to 10 ⁴ pg / ml. These results also suggest that AuNP / IDE may be suitable for building biocompatibility platforms for measuring TNF-alpha concentrations, which makes it easy to implement disposable bioimmune sensors. The capacitive (capacitance) type biosensor developed in the present invention is an economical tool because it does not require expensive fluorescent dyes, reagents and sophisticated instruments. It will be an easy and versatile tool to detect TNF-alpha concentration levels from subjects for diagnosis of disease.

As mentioned above, specific portions of the present disclosure have been described in detail, and it is apparent to those skilled in the art that such specific techniques are merely preferred embodiments, and thus the scope of the present disclosure is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

In the immune sensor manufacturing method,
a) depositing nanoparticles on the surface of the comb electrode by electrodeposition;
(b) immersing the comb-shaped electrode which passed through step (a) in ethanolic solution of 3-MPA;
(C) reacting 3-MPA on the surface of the comb electrode by continuously dropping the EDC solution and the NHS solution in a molar ratio of 4: 1 on the surface of the comb electrode in the ethanolic solution of 3-MPA; And
(d) preparing the electrode by dropping the antibody onto the comb-shaped electrode to which 3-MPA reacted;
The nanoparticles of step (a) are gold (Au) nanoparticles,
The antibody of step (d) is an antibody of TNF-alpha, using a concentration of 100 ㎍ / ㎖,
Blocking nonspecific adsorption using bovine serum albumin (BSA) dissolved in PBS (Phosphate Buffered Saline) solution in the step of dropping the antibody on the comb-shaped electrode reacted with 3-MPA in step (d),
The immune sensor is composed of two electrodes,
The immune sensor is a method of manufacturing an immune sensor for measuring TNF-alpha, characterized in that used to measure the capacitance (capacitance) of the analyte of interest.
delete delete delete delete An immune sensor for measuring TNF-alpha prepared by the method of claim 1.
delete delete (1) obtaining a calibration curve for the measurement of the immune sensor for measuring TNF-alpha;
(2) Insert a sample containing PBS (Phosphate Buffered Saline) solution or human serum into the electrode of the immune sensor for measuring TNF-alpha of item 6, and connect the electrodes to adjust the capacitance at a frequency of 10 ¹ to 10 ⁴ Hz. Measuring; And
(3) measuring the TNF-alpha content of the sample by applying the capacitance value obtained in step (2) to the calibration curve for measurement.

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