KR20220108312A - Biosensor and method for measuring prostate specific antigen using the same - Google Patents

Abstract

The present invention relates to a biosensor, a method for manufacturing the same, a method for measuring prostate-specific antigen using the same, a kit for diagnosing prostate cancer, and a method for providing information for diagnosing diseases. According to the present invention, the biosensor comprises: a substrate; a titanium electrode positioned on the substrate; a platinum electrode positioned on the substrate; and a fixing part modified with an aminosilane-based compound on at least a portion of the surface of the substrate. The biosensor of the present invention can be reused three or more times with a simple method.

Description

Biosensor and method for measuring prostate-specific antigen using the same

The present invention measures a prostate-specific antigen (PSA) using a repeatable, label-free, interdigitated capacitor (IDC) biosensor and the biosensor. it's about how to

Prostate cancer (PCa) is the most prevalent cancer among the male population, and its proportion is increasing so rapidly that it is counted as the second leading cause of cancer mortality worldwide, including Korea. In particular, the risk of prostate cancer is so serious that it accounts for 6% of the total cancer mortality in men.

Currently, for prostate cancer screening, there are two major methods, digital rectal examination or prostate-specific antigen detection.

Prostate-specific antigens are potential biomarkers of prostate cancer and are used to monitor disease recurrence after diagnosis and treatment. It cannot be a cancer-specific marker because prostate-specific antigen levels can also be increased in other noncancerous diseases of the prostate. However, since there is no alternative biomarker for early detection of prostate cancer, the measurement of prostate-specific antigen is important.

A biosensor is a receptor that recognizes and binds enzymes, antibodies, and DNA, which are target substances with specific functions of an organism, is combined with a signal converter, and converts biochemical interactions and recognition reactions into electrical signals for analysis. It refers to an electrochemical sensor that can selectively detect a substance.

A biosensor is a device that is expected to be widely used for bio, chemical, and environmental applications depending on the type of target material because it can rapidly quantify the concentration of various physiologically active substances.

In order to detect and analyze a target material using an electrochemical sensor, it must have high sensitivity so that the signal can be significantly changed even in the microscopic characteristics of the target material. It must have unaffected physical stability. In addition, it is required to be able to use the existing measurement platform for easy use, and to be manufactured in a structure that is easy to mass-produce for economical efficiency and practicality.

There is a high demand for a high-sensitivity, real-time, and reliable biosensor for prostate-specific antigen measurement. Measurement of prostate-specific antigens is performed by various methods such as various immunoassays, fluorescence-based detection, enzyme-linked immunosorbent assay (ELISA), and surface plasmon resonance (SPR), but expensive and complex antibodies or labels are required. It has limitations such as low sensitivity, or requiring high professional knowledge.

Accordingly, the present inventors have invented a completely new biosensor that does not require a label (label free), is simple, has very high sensitivity, enables real-time detection with an ultra-fast response time (within 30 seconds), and is reusable.

In order to solve the above problems, the present invention is to provide an interdigitated interdigital capacitor (IDC) biosensor for diagnosing prostate cancer.

In another aspect, an object of the present invention is to provide a biosensor capable of measuring a prostate-specific antigen without labeling, rapid, highly sensitive and reusable, and a method for measuring prostate-specific antigen in semen using the same.

In another aspect, it is an object to provide a biosensor that can be reused three or more times in a simple way.

One aspect of the present invention, a substrate; a titanium electrode positioned on the substrate; a platinum electrode positioned on the substrate; and a fixing part modified with an aminosilane-based compound on at least a portion of the substrate surface.

The biosensor can be reused after being washed and activated in a simple way, and can retain its properties even when reused three or more times.

The substrate in the present invention is silicon (silicon, Si), silicon oxide (silicon oxide, SiO ₂ ), silicon nitride (silicon nitride, Si ₃ N ₄ ), sapphire (sapphire), diamond (diamond), silicon carbide (silicon carbide) , gallium nitride (GaN), germanium (Ge), indium gallium arsenide (InGaAs), gallium arsenide (GaAs), lead sulfide and/or their It may include any suitable material, such as a combination, and in the present embodiment, silicon oxide (SiO ₂ ) was used, but is not limited thereto.

Because of its high tissue specificity and high sensitivity, PSA is used as a screening test, diagnosis, cancer risk prediction, and recurrence marker for prostate cancer. On the other hand, the disadvantage of PSA is that it has no cancer specificity. Specifically, the level of PSA can be increased not only in prostate cancer but also in benign prostate diseases such as prostatitis and prostatic hyperplasia.

In general, the reference range for PSA is less than 4.0 ng/mL, a PSA level of 10.0 ng/mL or higher is associated with a high risk of prostate cancer, and a PSA level between 4.0 and 10.0 ng/mL is a gray zone. , prostatic hyperplasia, and prostatitis.

As mentioned above, although it is difficult to detect prostate cancer at an early stage with only the PSA level, it is possible to detect prostate cancer at an early stage by combining PSA density (density), velocity (velocity), and %fPSA (free/total PSA ratio). have.

Specifically, the PSA density is calculated by using the volume of the prostate measured by transrectal ultrasound, which observes the prostate by taking ultrasound through the anus-rectum, to calculate the PSA per unit volume. In patients who have a negative digital rectal examination and a PSA level of 4 to 10 ng/mL and an increased PSA density, the risk of prostate cancer is judged to be high. In addition, the rate of PSA is to calculate the rate of increase in PSA over time, and it is determined that the higher the rate of increase, the higher the probability of prostate cancer. %fPSA is a calculation of the percentage of free PSA in total PSA, and since complex PSA increases in prostate cancer, it is determined that the risk of prostate cancer is high when the ratio of free PSA is low.

Specifically, in the biosensor of the present invention, an antibody of a prostate-specific antigen (PSA) may be immobilized in the fixing part, and the PSA-antigen and PSA present in the target sample By measuring the capacitance that changes in the binding process of , it is possible to detect the level of prostate-specific antigen (PSA) in the target sample to determine whether or not prostate cancer.

In an embodiment, each of the titanium electrode and the platinum electrode may have an interdigital interdigital structure in which at least one branch is alternately arranged with each other at intervals of 50 μm to 200 μm. The interdigital structure of the interdigital structure may mean that one electrode and the other electrode are alternately arranged as if the branches are interdigitated fingers.

In one embodiment, the width of each of the branches may be 50 ~ 200 μm.

In one embodiment, each of the titanium electrode and the platinum electrode may be composed of 8 to 16 branches.

In one embodiment, the thickness of the titanium electrode and the platinum electrode may be 50 ~ 200 nm, respectively.

In one embodiment, the fixing part is an aminosilane-based compound, for example, 3-aminomethylmethoxysilane, 3-aminoethylmethoxysilane, 3-aminopropyl to the hydroxyl group (-OH) formed on the substrate. Methoxysilane, 3-aminomethylethoxysilane, 3-aminoethylethoxysilane, 3-aminopropylethoxysilane and the like may be reacted and modified.

In an embodiment, the fixing part may be further modified with an aldehyde-based compound.

The fixing part may be one in which a compound is combined on a substrate to form a kind of chemical layer.

In one embodiment, an antibody (antibody) of a specific antigen (Prostate-Specific Antigen; PSA) may be immobilized in the immobilization portion.

In an embodiment, the biosensor may detect the level of a prostate-specific antigen (PSA) in a target sample. The level of the prostate-specific antigen may be measured through a change in capacitance of the biosensor. For example, the log value of the prostate-specific antigen concentration may be linearly proportional to the capacitance value, but is not limited thereto.

Another aspect of the present invention provides a kit for diagnosing prostate cancer, including the above-described biosensor.

In one embodiment, the prostate cancer diagnostic kit may have a solution, a lyophilized powder, a frozen solution, or a strip form, and each form may be formulated in a conventional manner in the art. For example, a detection kit in the form of a solution may be formulated by separately or mixing a protein or a primer in a buffer such as sodium-phosphate, potassium-phosphate, tris-hydrochloric acid, and other various types of buffers, and, if necessary, It can also be frozen or freeze-dried.

The detection kit may use an immunolateral flow strip method. A lateral flow assay is a method for detecting a protein or a nucleic acid based on a chromatography method. This lateral flow analysis method is widely used in various fields such as pregnancy diagnosis, cancer diagnosis, the presence of other specific proteins or genes, or detection of microorganisms. The lateral flow analysis method is based on a specific reaction between two substances, such as an antigen-antibody reaction, and has the advantages of high sensitivity and specificity, and the ability to confirm results in a short time, enabling diagnosis and rapid preventive measures. .

In the detection kit of the present invention, various reagents required for experiments (eg, PCR) and result confirmation required for detecting prostate cancer-related biomarkers using the biosensor of the present invention, for example, PCR composition, restriction enzyme, agarose , a buffer solution required for hybridization and electrophoresis may be additionally included. Specifically, the PCR composition comprises an appropriate amount of a DNA polymerase (eg, Thermusaquaticus (Taq), Thermusthermophilus (Tth), Thermusfiliformis, Thermisflavus, Thermococcusliteralis or thermostable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), dNTPs, PCR buffer and water (H ₂ O). The PCR buffer solution may include Tris-HCl (Tris-HCl), MgCl ₂ , KCl, and the like.

Another aspect of the present invention, (a) obtaining a subject sample from the subject; (b) contacting the sample with the biosensor described above; (c) detecting a capacitive change as the target material present in the target sample is bound to a receptor in the biosensor; and (d) detecting the degree of capacitive change to determine the level of the target material in the target sample; provides an information providing method for diagnosing a disease, including.

In one embodiment, the target sample of step (a) may be one or more selected from the group consisting of tissue, cells, whole blood, plasma, serum, blood, saliva, sputum, lymph, cerebrospinal fluid, intercellular fluid, semen and urine. have.

In addition, the target material of step (c) may be a biomarker, specifically a screening biomarker, a prognostic biomarker, a stratification biomarker, an efficacy biomarker, It may be one or more selected from the group consisting of a toxicity biomarker, a predictive biomarker, and a pharmacodynamic biomarker, and most specifically, a prostate-specific antigen (PSA) may be, but is not limited thereto.

In one embodiment, the disease is diabetes, rheumatoid arthritis, leukemia, lymphoma, hematopoietic malignancy, cervical cancer, sarcoma, testicular cancer, malignant melanoma Malignant melanoma, endocrine tumor, bone cancer, prostate cancer, uterus cancer, breast cancer, bladder cancer, brain cancer , liver cancer, stomach cancer, pancreas cancer, skin cancer, lung cancer, larynx cancer, head and neck cancer, esophageal cancer ), colorectal cancer, and ovarian cancer can be diagnosed by detecting a biomarker specifically expressed in one or more diseases selected from the group consisting of, most specifically, prostate cancer. However, the present invention is not limited to the above diseases and is applicable to any disease in which a specific biomarker exists according to the onset or progression of the disease. In one embodiment, the disease may be prostate cancer.

Those skilled in the art will appreciate that, with reasonable experimentation, the same or similar results may be obtained depending on the particular process and material used, even if the order of various steps in these methods is changed. That is, the order and process in which the various steps described herein are performed should not be considered in a limiting sense, and those skilled in the art will appropriately modify these methods within the spirit and scope of the present disclosure. so it can be applied. In various embodiments, one or more of the illustrated steps may be omitted. A person skilled in the art will recognize that factors such as, for example, the particular substance used, the quality of the substance, available reagents, available equipment, etc., can be omitted while still obtaining the desired result. can be decided based on

Another aspect of the present invention comprises the steps of treating a prostate cancer patient sample with a prostate cancer inhibitor or a prostate cancer therapeutic agent; And it provides a method for monitoring a prostate cancer inhibitor or a prostate cancer therapeutic agent, comprising the step of measuring the PSA level before and after the treatment of the prostate cancer inhibitor or the prostate cancer therapeutic agent in the sample.

In the present invention, the prostate cancer inhibitor or prostate cancer therapeutic agent may be in the form of a small molecule, compound, antibody, immunotherapy, virus, etc. Any agent that can be treated or improved can be applied without limitation.

In the present invention, by measuring a change in the PSA level of a sample treated with a prostate cancer inhibitor or a prostate cancer therapeutic agent, the cancer inhibiting ability and cancer treatment effect of the prostate cancer inhibitor or prostate cancer therapeutic agent can be monitored. For example, when the PSA level in the sample is significantly within the range of a normal sample according to the treatment of the prostate cancer inhibitor or the prostate cancer therapeutic agent, the cancer inhibiting ability and the cancer therapeutic effect of the prostate cancer inhibitor or the prostate cancer therapeutic agent are excellent. can judge

Another aspect of the present invention comprises the steps of treating a prostate cancer treatment candidate to a prostate cancer patient sample; and measuring the level of PSA before and after processing the candidate substance in the sample.

In the present invention, by measuring the change in the PSA level of a sample treated with a candidate for treating prostate cancer, it is possible to determine the prostate cancer inhibitory ability and the therapeutic effect of the candidate for prostate cancer. For example, when the level of PSA in the sample is significantly within the range of a normal sample according to the treatment of the candidate material, the candidate material may be selected as a therapeutic agent for prostate cancer disease.

In another aspect of the present invention, (i) a glass wafer having a thickness of 300 to 1,200 μm is organically cleaned using acetone, isopropyl alcohol (IPA) and deionized water (DI), and then HCl for 1 to 100 seconds: H ₂ O is washed with an acid mixture in a ratio of 1:5-15; (ii) coating a negative photo-resist (PR) layer of 2.5 μm or more on the wafer with a spin coater; (iii) homogenizing the thickness of the photoresist layer; (iv) depositing titanium (Ti) and platinum (Pt) to a thickness of 50 to 200 nm through electron beam deposition; and (v) using acetone, isopropyl alcohol (IPA) and deionized water to remove the photoresist layer within 60 to 150 seconds.

In one embodiment, the (iv) electron beam deposition step is performed under a pressure of 1.0 x 10 ^-6 ~ 10.0 x 10 ^-6 mTorr, the electron energy is fixed at 5 ~ 15 kV, the minimum deposition of 0.5A / s It may be done under speed conditions, but is not limited thereto.

In one embodiment, after step (v), (vi) washing the surface of the biosensor with a Piranha solution at 60-100° C. for 15-25 minutes; (vii) immersing in 1-5% by weight 3-aminopropyltriethoxysilane (APTES) solution at room temperature for 2-4 hours; And (viii) 25 to 50% by weight of glutaraldehyde (Glutaraldehyde, C ₅ H ₈ O ₂ ) It may further include the step of fixing for 2 to 4 hours.

In one embodiment, the used biosensor may be reused by performing steps (vi) to (viii). The biosensor can be processed and reused in a simple way, and its performance as a sensor can be maintained even when reused multiple times.

The biosensor according to the present invention has the effect of quantifying the concentration of PSA with ultra-high sensitivity using a small amount of sample.

In addition, since there is no need for a label, the concentration of PSA can be quantified in a short time by a method using capacitance rather than analyzing a signal of a marker, and it is economically excellent because it can be reused.

1 is (a) a manufacturing process, (b) structure, (c) an exemplary schematic diagram of a PSA biomolecule secreted from the prostate of the capacitance-based biosensor according to the present invention, (d) PSA biosensor by the biosensor Scanning images showing the mechanism of marker detection, (e) biochip configuration, (f) morphological behavior of the biochip in the presence of anti-PSA, and (g) the boundary between metal fingers in the presence of PSA.
Figure 2 shows the subsequent process of the IDC biosensor, and shows an overview of the aspect of surface modification, Aβ antibody immobilization, and binding of PSA on the surface.
3 is an exemplary schematic diagram proposed for the mechanism of action of the biochip by the antibody-antigen interaction.
4 is (A) capacitance-concentration curve; (B) Current density, equivalent circuit ADS simulation by capacitive coupling effect; (C) Shows the mechanism of action in which the Ti/Pt-based IDC equivalent circuit and the binding of PSA bioparticles interfere with the electric field between the metal electrodes (metal fingers) to show the capacitance-concentration curve.
5 is a view showing AFM values of a bare IDC chip, a bare IDC chip-APTES, a bare IDC chip-APTES-Ab, and a bare IDC chip-APTES-Ab-Ag.
6 is (a) FT-IR spectrum of bare IDC chip, APTES functionalized IDC chip, antibody-coupled IDC chip and PSA-induced IDC chip, (b) IDC chip surface-modified with anti-PSA (inset: Bare SEM image of IDC chip), (c) SEM image of PSA-induced IDC glass substrate.
7 shows a graph between the log value of the PSA concentration and the capacitance to confirm the linearity.
8 is a result of confirming the self-lifespan stability of the capacitive biosensor chip in the presence of a glutaraldehyde solution and an anti-PSA antibody for 15 days.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention belongs It is provided to fully inform the possessor of the scope of the invention, and the present invention is only defined by the scope of the claims.

Accordingly, in some embodiments, well-known process steps, well-known structures, and well-known techniques have not been specifically described in order to avoid obscuring the present invention.

The terminology used herein is for the purpose of describing the embodiments and is not intended to limit the present invention. In this specification, the singular also includes the plural, unless specifically stated otherwise in the phrase. As used herein, includes and/or comprising refers to the presence or addition of one or more other components, steps, operations and/or elements other than the recited elements, steps, operations and/or elements. It is used in the sense of not being excluded. And, “and/or” includes each and every combination of one or more of the recited items.

Spatially relative terms "below", "beneath", "lower", "above", "upper", etc. It can be used to easily describe the correlation between an element or components and other elements or components. The spatially relative terms should be understood as terms including different orientations of the device during use or operation in addition to the orientation shown in the drawings.

In addition, the embodiments described herein will be described with reference to cross-sectional and/or schematic diagrams that are ideal illustrative views of the present invention. Accordingly, the shape of the illustrative drawing may be modified due to manufacturing technology and/or tolerance. Accordingly, the embodiments of the present invention are not limited to the specific form shown, but also include changes in the form generated according to the manufacturing process. In addition, in each of the drawings shown in the present invention, each component may be enlarged or reduced to some extent in consideration of convenience of description. Like reference numerals refer to like elements throughout.

The biosensor according to the embodiments of the present invention analyzes biomolecules included in a biosample, so that abruptly such as gene expression profiling, genotyping, and SNP (Single Nucleotide Polymorphism). It is used to detect mutations and polymorphisms, analyze proteins and peptides, screen for potential drugs, and develop and manufacture new drugs. The biosensor employs probes or receptors suitable for the target of the biosample to be analyzed. Examples of the probe that can be employed in the biosensor include a DNA probe, an enzyme or an antibody/antigen, a protein probe such as bacteriorhodopsin, a microorganism probe, a neuron probe, and the like. A biosensor manufactured in the form of a chip is also referred to as a biochip. For example, it may also be referred to as a DNA chip, a protein chip, a cell chip, a neuron chip, etc. depending on the type of probe employed.

The biosensor according to some embodiments of the present invention may include an oligomeric probe as a probe. The oligomeric probe implies that the number of monomers of the probe employed is at the level of oligomers. Here, the oligomer may be used to refer to a polymer having a molecular weight of about 1000 or less among polymers composed of two or more covalently bonded monomers. Specifically, it may contain about 2-500 monomers, preferably 5-30 monomers. However, the meaning of the oligomer probe is not limited to the above numerical values.

The monomer constituting the oligomer probe can be modified depending on the type of the biosample to be analyzed, and may be, for example, a nucleoside, a nucleotide, an amino acid, or a peptide.

Nucleosides and nucleotides include known purine and pyrimidine bases as well as methylated purines or pyrimidines, acylated purines or pyrimidines, and the like. In addition, nucleosides and nucleotides include conventional ribose and deoxyribose sugars, as well as modified sugars in which one or more hydroxyl groups are substituted with halogen atoms or aliphatic groups or to which functional groups such as ethers and amines are attached.

Amino acids may be L-, D-, and nonchiral amino acids of amino acids found in nature, as well as modified amino acids, or amino acid analogs.

A peptide refers to a compound produced by an amide bond between the carboxyl group of an amino acid and the amino group of another amino acid.

Unless otherwise specified, probes exemplarily assumed in the following examples are DNA probes, and oligomeric probes in which monomers of about 5-30 nucleotides are covalently bonded. However, the present invention is not limited thereto, and it goes without saying that the above-described various probes may be applied.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the present invention is not limited by the following examples.

Example 1: Sample and Equipment Preparation

All stock solutions were prepared using deionized water supplied from the water distributor system Milli-Q of Simplicity, Millipore, USA. Anti-Prostate-specific Antigen antibody (anti-PSA antibody, product number SAB4501531) produced in rabbit and prostate-specific antigen obtained from human semen (product number P3338-25UG), hydrogen peroxide (H ₂ O) ₂ ), sulfuric acid (H ₂ SO ₄ ), 3-aminopropyltriethoxysilane (APTES) 99% and a 70% solution of glutaraldehyde in H ₂ O were purchased from Sigma-Aldrich. 0.1 x PBS buffer solution (ph 7.4), ethyl alcohol was also used. An impedance analyzer or LCR meter was used to characterize the sandwich-type bioelectrode and calculate the PSA concentration. Capacitance measurements were performed at open circuit potentials in the frequency range of 1 kHz to 100 kHz.

To analyze the surface properties of the biocomposite, an atomic force microscope (AFM) was used in tapping mode (AFM (Model XE-Model XE-100, Park Systems, Korea)).

The thickness and the modified surface were checked with a high-resolution low vaccuum scanning electron microscope (SEM, FEI (Nova Nano SEM 200)). The prepared sample grids were checked with FT-IR iS 10 of the Korea Institute of Science and Technology (KIST), and the capacitance of the prepared bioelectrode was measured using an LCR meter (Gohm, U1730C Series). Measurements are obtained by a 0 V DC bias voltage of 1 kHz frequency across the IDC biosensor.

Example 2: Simulation and Fabrication of IDC Chips

The IDC biosensor according to an embodiment of the present invention is inverted on EagleXG glass wafer so that 24 fingers become a capacitor assembly having a width of 100 μm and an interdigital spacing of 100 μm using simulation software (Advanced Design System; ADS). made by photolithography.

In the first step, for the titanium (Ti)/platinum (Pt) electron beam deposition process, 700 μm thick EagleXG glass substrates were subjected to isopropyl alcohol (IPA) cleaning and organic cleaning using an ultrasonic acetone bath, followed by HCl and H for 60 seconds. To perform an acidic wet washing step in which ₂ O is mixed in a ratio of 1:10. The cleaning process is an important process to make an effective photoresist coating for titanium (Ti)/platinum (Pt) metal deposition. They can be easily dislodged together (Fig. 1(a); Step 1).

As a second step, a negative photoresist was coated on a glass wafer to a thickness of 3 μm using a spin coater, and then an edge bead removal process was performed so that the thickness of the coated resist was uniform to clarify the boundaries of the metal layer. Then, acid cleaning of the surface is performed (Fig. 1(a); step 2).

In the third step, the electron beam deposition process, titanium (Ti) is first used for adhesion on a glass substrate, and platinum (Pt) is deposited on titanium (Ti) because it cannot be directly deposited on the glass surface. Titanium (Ti)/platinum (Pt) was deposited by two e-beam processes, and the thicknesses of titanium (Ti) and platinum (Pt) were 0.15 μm and 0.15 μm, respectively. This deposition process is carried out under a pressure of 5.0 x 10 ^-6 mTorr, and the electron energy is fixed at 10 kV. Also, set the minimum deposition rate of 0.5 Å/s to obtain the correct layer thickness (Fig. 1(a); step 3).

Then, the photoresist is removed by a lift-off machine within 90 seconds using acetone/IPA/DI water (Fig. 1(a); step 4). In the simulation, the frequency of 3.75 pF was set to 1 MHz and the measurement result was set to 3.8 pF as an embodiment. Advance Design System (ADS Keysight ⓒ) software is used for IDC (Inter-digital Capacitor) biochip structure with a size of 4.5 x 9 mm ² (Fig. 1(b)). The prepared IDCs were shown in Figure 1(b) with W _e = 100 μm, W _c = 100 μm, G _e = 100 μm, G _c = 100 μm, and L = 3.8 mm.

The biosensor according to the present invention is measured with an LCR meter (handheld, 1kHz, 2kH, 200pF, 0.2Gohm, U1730C series) connected to a PC for data collection, and the measured value is 0 V DC bias voltage of 1 kHz frequency. obtained by

Example 3: Sample preparation of PSA bioelectrodes

As a first step, for the piranha etching process, the IDC surface of the substrate is put into a piranha solution composed of 1:1 H ₂ O ₂ and H ₂ SO ₄ at 80° C. to remove organic contaminants for 15 to 20 minutes, A real machine was formed (Fig. 2(a)).

After the surface was washed with deionized water and ethanol, a 2 wt % solution of 3-aminopropyl triethoxysilane (APTES) was drop-casted and functionalized with an amine functional group (FIG. 2(b)).

Then, the antibody was designed in a cross-linking manner by applying a 25% glutaraldehyde (C ₅ H ₈ O ₂ ) solution for 3 hours at room temperature (FIG. 2(c)).

After the aldehyde group was formed on the IDC surface, the rabbit-produced anti-PSA antibody (10 μl mL ^-1 in PBS, pH 7.4) was bound overnight at room temperature (Fig. 2 (e)).

Formation of the chemical layer and immobilization of anti-PSA responded to the change in capacitance of the biochip due to the binding of the target analyte (Fig. 2(f)).

After preparation of anti-PSA, various concentrations of PSA cultured at room temperature were immobilized on the biological surface and the capacitance value was confirmed by LCR measurement.

The PSA-based biosensor showed positive capacitance after incubation with APTES and glutaraldehyde solution, and was treated with a reference capacitance in consideration of the increase in capacitance due to surface-bound APTES and glutaraldehyde solution. Finally, as a result of checking with the IDC biosensor and LCR meter, the capacitance variable was changed according to the addition of the target protein (PSA) having various concentrations of 0.1 to 10 μl mL ^-1 .

The change of the IDC biosensor was recorded by measuring the capacitance value at each step (FIG. 3).

(a) Bare-APTES IDC biosensor chip;

(b) an antibody-immobilized IDC biosensor chip;

(c) a PSA-induced biosensor chip;

The biosurface was activated due to a fast and non-destructive controlled biological system, providing a balanced and non-drift signal of a complete biosensor isolation system. To confirm the reusability of the capacitive biosensor, PSA was repeatedly detected under numerous cycles of regenerative equilibrium injection using various bioelectrodes. The reproducibility of the assay was evaluated by confirming the change in capacitance within a response time of 3 seconds, which is a very fast response time based on the conventional first-generation IDC biosensor, at various concentrations of 0.1 to 10 μl mL ^-1 .

The biosensor was able to detect a capacitive sensitivity of a level of pF to nF, which is a typical PSA concentration, within 3 seconds. The sandwich-based layer-by-layer arrangement reduced the analysis time and enabled real-time sensing.

The IDC biosensor of the present invention was designed and manufactured in consideration of the specific antigen and antibody characteristics of the prostate-specific antigen (PSA) biomaterial.

Specifically, the main concept of the biosensor is based on controlling charge distribution, dielectric constant, and conductivity.

When the analyte material is placed in the IDC biosensor, it reacts with the dielectric to change the charge distribution and the conductivity of the antibody and antigen together, and the change in these sensing parameters corresponds to the capacitive reactance of the IDC.

Therefore, the sensing response of the IDC biosensor can be obtained by observing the capacitive reactance through the IDC voltage.

The PSA antibody immobilized on the IDC surface was functionalized with 2 wt% APTES solution, and the substrate was 0.1 μl mL ^-1 , 0.2 μl mL ^-1 , 1.0 μl mL -1 by AFM, SEM, FT ^- IR and ultrafast capacitive measurement, respectively. It was assayed at various standard PSA concentrations of ¹ , 5.0 μl mL ⁻¹ , and 10 μl mL ⁻¹ . Antigen-antibody binding is helpful for quantification as it exhibits a change in capacitance depending on the amount of antigen captured by the anti-PSA on the glass substrate at various PSA concentrations.

Capacitive-based sensing technology is known to have high sensitivity and short response time compared to other methods. However, the biosensor has significantly improved sensitivity and selectivity compared to the prior art by providing Ti/Pt having a biological composition on a glass substrate. This unique design leads to more accurate results, and the biological mechanism of action of PSA particles affecting the capacitance of the sensing system can be represented by the equivalent circuit representation model of FIG. 4 .

Referring to FIG. 4 , this circuit shows the number of digits of the electrode capacitance (Cdl) of the Ti/Pt metal layer capacitance, the physical capacitance of the electrode (Cg), the resistance of the surrounding bioparticle solution (Rs), and the impedance (Z) of the bound nanoparticles. is composed of a combination of PSA can bind to specific biological substances with extremely high selectivity, making it a very useful biomarker for the detection of prostate cancer. They are generally configured to change through a capacitance versus concentration curve in combination with the substrate (FIG. 4).

Example 4: Surface properties of capacitive-based biochips

The characteristics of the surface modification of the IDC chip were analyzed through capacitance measurement, AFM, SEM, and FT-IR analysis. According to the capacitance measurement, APTES surface modification generally forms voids on the surface, causing a change in the capacitance value, and it was found that the capacitance value increased according to the adsorption of the anti-PSA. Characteristics changed according to the PSA concentration and they could be detected, and Ti/Pt IDC showed a unique tendency in layer-by-layer surface modification compared to other structures, showing better performance. Ti/Pt is more user-friendly, and allows for cheaper microfabrication of metal electrode fingers on glass substrates.

One) Atomic force microscopy studies (AFM) analysis

Atomic force microscopy (AFM) was used in tapping mode (AFM) to characterize the surfaces of the Bare IDC chip, Bare IDC chip-APTES, Bare IDC chip-APTES-Ab and Bare IDC chip-APTES-Ab-Ag as shown in Figure 5. (Model XE-Model XE-100, Park Systems, Korea)). The vibration frequency (320.30 Hz) and vibration amplitude (1 V) were used as experimental parameters. Imaging studies were done at 2 mm/s scanning speed and 256 x 256 pixel resolution.

2) Fourier transform infrared spectroscopy (FT-IR) analysis

As shown in Fig. 6(a), it was confirmed that a C-H aromatic ring having a weak intensity peak and a C-H alkene out of plane bending were formed. Sharp intermolecular H bonds were detected in the spectrum, and an aliphatic C-H stretching band was identified at 849.82 cm −1 .

Bare biochip (Sample 1) can confirm the weak intensity peak of C-H aromatic ring and C-H alkene out of plane bending.

In the APTES surface-modified biochip (Sample 2), it can be confirmed that APTES is fixed to the biochip from the R-NO ₂ nitro group and the ethoxy group due to the presence of CO.

The biofunctionalized biochip (Sample 3) can confirm the sharp peak of O-H and the nitro group resulting from the surface antibody.

In the PSA-induced biochip (Sample 4), R-NO ₂ nitro group, CO carboxyl ether and CH aromatic peaks can be confirmed.

3) SEM image

As shown in FIGS. 6(b) and 6(c), the surface morphology of the anti-PSA and PSA-coupled substrate on the IDC chip was examined with an ultra-high-resolution low-vacuum scanning electron microscope (SEM, FEI (Nova Nano SEM 200)). observed. The biochip was washed with ethanol and water for 10 minutes each. Prior to SEM observation, the electrodes were sputter coated with Au/Pd.

Example 5: PSA detection from human semen

In order to evaluate the reliability of the IDC biosensor of the present invention for clinical use, a detection experiment for PSA extracted from human semen was conducted.

The semen sample contains a 26 kDa single chain glycoprotein containing 237 amino acids encoded by the kallikrein gene mapped to human chromosome 19.

The change in capacitance of all samples showing the detection limit of 0.1 μl/mL to 10 μl/mL within 3 seconds is measured and shown in FIG. 7 , so the biosensor of the present invention has a short response time and high sensitivity confirmed the fact.

The detection of PSA at the μl/mL (0.1-10.0) level was successful, and the linear calibration curve between the log of PSA concentration and the capacitive response was good with a correlation coefficient (R ² ) of 0.89 in the range of 0.1-10.0 μl/mL. It showed linearity (FIG. 7).

Example 6: Confirmation of selectivity of IDC biosensors

To apply the functionalized biological complex, PSA-anti-PSA-APTES-Pt/Ti-free platform, to PSA measurement, it is a glutaraldehyde solution (C ₅ H ₈ O ₂ ) for various potential interfering compounds coexisting in biological samples. Selectivity was tested. Selectivity tests were performed by measuring 0.1 μl/ml and 10.0 μl/ml PSA standard solutions in the absence and presence of protein biomolecules. The change in capacitance was proportional to the amount of PSA binding, and very specific and sensitive sensing was possible. The selectivity of the biosensor chip was tested for different concentrations of PSA, and as a result, it was confirmed that the biosensor had high selectivity for PSA detection. In addition, the sensor chip self-life stability in the presence and absence of anti-PSA, an aqueous antibody solution before and after the test (C ₅ H ₈ O ₂ ) was evaluated for 15 days ( FIG. 8 ).

Example 7. Confirmation of Reusability and Reliability of IDC Biosensor

The reusability of the IDC biosensor of the present invention was evaluated by monitoring the capacitance value while varying the concentration of the standard PSA. This indicates that the biosensor chip recovered its original properties after washing and drying with a piranha solution at 80°C. It was confirmed that the IDC biosensor chip can be reused three or more times, thereby reducing product cost in the long run. In the example of the present invention, the same IDC biosensor was continuously used in the experiment, and it was confirmed that there was no difference in the results by showing excellent stability even after each analysis was repeated three times, so that the IDC biosensor of the present invention exhibited excellent reusability and stability. confirmed to have.

An embodiment of the present invention is to manufacture a label-free capacitive biosensor using a biofunctionalized IDC electrode (Ti / Pt) for the detection of selectivity and sensitivity of PSA within 3 seconds. The biosensor was configured to perform real-time PSA detection required to manage the point of care (POC) based on patient symptom progression requirements, such a PSA diagnosis required very small sample volumes and the sensor lasted for up to several weeks. Reproducibility and stability were shown. Moreover, upon such reuse, the biosensor can detect PSA as well as other useful biomarkers and perform early stage diagnosis in a personalized way. The biosensor can be applied to a target disease management program that urgently requires rapid, sensitive and selective detection of biomarkers.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Claims (20)

Board;
a titanium electrode positioned on the substrate;
a platinum electrode positioned on the substrate; and
A biosensor comprising a fixing part modified with an aminosilane-based compound on at least a portion of the substrate surface.
According to claim 1,
Each of the titanium electrode and the platinum electrode is an interdigital interdigital structure in which at least one branch is alternately disposed with an interval of 50 to 200 μm, the biosensor.
3. The method of claim 2,
The width of each branch is 50-200 μm, biosensor.
3. The method of claim 2,
The titanium electrode and the platinum electrode are each composed of 8 to 16 branches, a biosensor.
According to claim 1,
Each of the titanium electrode and the platinum electrode has a thickness of 50 to 200 nm, a biosensor.
According to claim 1,
The fixing part is further modified with an aldehyde-based compound, biosensor.
7. The method of claim 6,
A biosensor in which an antibody (antibody) of a specific antigen (PSA) is immobilized on the fixing part.
8. The method of claim 7,
The biosensor is to detect the level of prostate-specific antigen (PSA) in the target sample, the biosensor.
A kit for diagnosing prostate cancer, comprising the biosensor of any one of claims 1 to 8.
10. The method of claim 9,
The prostate cancer diagnosis kit is in any one of a solution, a lyophilized powder, a frozen solution, or a strip form, the prostate cancer diagnosis kit.
10. The method of claim 9,
The prostate cancer diagnostic kit further comprises at least one selected from the group consisting of PCR composition, restriction enzyme, agarose, DNA polymerase, dNTP, Tris-HCl (Tris-HCl), MgCl ₂ and KCl, prostate cancer diagnostic kit .
(a) obtaining a subject sample from the subject;
(b) contacting the sample with the biosensor of any one of claims 1 to 8;
(c) detecting a capacitive change as the target material present in the target sample is bound to a receptor in the biosensor; and
(d) detecting the degree of capacitive change to determine the level of the target material in the target sample; Information providing method for diagnosing a disease comprising a.
13. The method of claim 12,
The target sample of step (a) is at least one selected from the group consisting of tissue, cells, whole blood, plasma, serum, blood, saliva, sputum, lymph, cerebrospinal fluid, intercellular fluid, semen and urine, information for diagnosing a disease How to provide.
13. The method of claim 12,
The target material of step (c) is a prostate-specific antigen (PSA), an information providing method for diagnosing a disease.
13. The method of claim 12,
The disease is diabetes, rheumatoid arthritis, leukemia, lymphoma, hematopoietic malignancy, cervical cancer, sarcoma, testicular cancer, malignant melanoma, Endocrine tumor, bone cancer, prostate cancer, uterus cancer, breast cancer, bladder cancer, brain cancer, liver cancer , stomach cancer, pancreas cancer, skin cancer, lung cancer, larynx cancer, head and neck cancer, esophageal cancer, colorectal cancer Cancer) and at least one selected from the group consisting of ovarian cancer, an information providing method for diagnosing a disease.
16. The method of claim 15,
The disease is prostate cancer (prostate cancer), information providing method for the diagnosis of the disease.
(i) A 300-1200 μm-thick glass wafer was organically cleaned using acetone, isopropyl alcohol (IPA) and deionized water (DI), followed by HCl:H ₂ O 1:5-15 for 1-100 sec. washing with a mixed acid at a ratio of;
(ii) coating a negative photo-resist (PR) layer of 2.5 μm or more on the wafer with a spin coater;
(iii) homogenizing the thickness of the photoresist layer;
(iv) depositing titanium (Ti) and platinum (Pt) to a thickness of 50 to 200 nm through electron beam deposition; and
(v) using acetone, isopropyl alcohol (IPA), and deionized water to remove the photoresist layer within 60 to 150 seconds, comprising a lift-off (lift-off) step, the manufacturing method of the biosensor.
18. The method of claim 17,
The (iv) electron beam deposition step is performed under a pressure of 1.0 x 10 ^-6 ~ 10.0 x 10 ^-6 mTorr, the electron energy is fixed at 5 ~ 15 kV, and is made under the condition of a minimum deposition rate of 0.5 A / s. Method of manufacturing the sensor.
18. The method of claim 17,
After step (v),
(vi) washing the surface of the biosensor with a Piranha solution at 60-100° C. for 15-25 minutes;
(vii) immersing in 1-5% by weight 3-aminopropyltriethoxysilane (APTES) solution at room temperature for 2-4 hours; and
(viii) 25 to 50% by weight of glutaraldehyde (Glutaraldehyde, C ₅ H ₈ O ₂ ) The method of manufacturing a biosensor further comprising the step of fixing for 2 to 4 hours.
20. The method of claim 19,
A method of manufacturing a biosensor, which is reused by performing steps (vi) to (viii) with the used biosensor.

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