KR101824303B1 - Paper sheet type diagnosis sensor for prostate cancer, method for preparing the same, and diagnosis method using the same - Google Patents

Abstract

The present invention provides a paper type prostate cancer diagnostic sensor utilizing a carbon-based nano-structure and filter paper, a method for producing the same, and a method for diagnosing prostate cancer using the same, Can be diagnosed. In addition, it can be applied to various biomarkers to diagnose diseases and thus can be used for a wide range of medical diagnoses.

Description

TECHNICAL FIELD [0001] The present invention relates to a paper type prostate cancer diagnosis sensor, a method of manufacturing the same, and a diagnostic method of prostate cancer using the same. BACKGROUND ART < RTI ID = 0.0 >

The present invention relates to a prostate cancer diagnosis sensor including a paper-type sensing unit, a method for manufacturing the same, and a method for diagnosing prostate cancer using the same.

Prostate cancer is a form of cancer that is expressed in the prostate gland, the gland in the male reproductive tract. The cancer cells can be transferred from the prostate to other body parts, particularly bone and lymph nodes.

Diagnostic rates for prostate cancer vary widely across the world, with diagnoses in Southeast Asia being lower than in Europe and the United States. Prostate cancer is one of the most common types of cancer in men, and the incidence rates increase after age 50. Early prostate cancer usually has no symptoms, and most are found in Prostate Specific Antigen or rectal examinations, and the treatment is delayed, leading to death due to cancer metastasis to surrounding tissues.

Prostate specific antigen (PSA) is a serine protease that is expressed at a high level in the prostate epidermis with a molecular weight of 34,000 Dalton. Currently, many kits for measuring the amount of prostate specific antigen (PSA) There is less than 2 ng / mL of PSA in the blood of healthy men and the amount of prostate cancer is diagnosed when the cancer develops.

Therefore, when the amount of PSA in the blood increases, it indicates the occurrence of prostate diseases such as prostate cancer and enlargement of the prostate. It is generalized that the cut-off of PSA used for diagnosing prostate cancer is 3 to 4 ng / mL And the probability of prostate cancer is 67% when the PSA level is 10 ng / mL or more.

However, since the conventional blood-based ELISA type diagnostic kit requires special equipment and specialized personnel such as an enzyme-linked immunosorbent assay (ELISA) reader and an automation equipment, it is applied in a region where the medical environment is poor And there is a problem that it takes a long time to obtain the test result.

Therefore, it is required to develop a prostate cancer diagnostic sensor having high practicality and reliability for successful treatment through early diagnosis of prostate cancer.

Korean Patent No. 10-1495131 Korean Patent Publication No. 10-2010-0075452

It is an object of the present invention to provide a prostate cancer diagnostic sensor capable of diagnosing prostate cancer with high reliability, enabling rapid diagnosis at low cost, and enabling diagnosis at an early stage for easy treatment.

It is still another object of the present invention to provide a method of manufacturing the above-described prostate cancer diagnostic sensor.

It is still another object of the present invention to provide a method for effectively and reliably diagnosing prostate cancer in an early stage using the obtained prostate cancer diagnosis sensor.

Accordingly, the present inventors have found that a paper-type sensing unit comprising an antibody-carbon based nanostructure and a filter paper deposited by sifting filtering the antibody-carbon based nanostructure; And a detection unit capable of measuring a change in electrical resistance of the sensing unit, wherein the paper-type sensing unit is obtained by radially cutting in the direction of the edge from the center of the circle of the filter paper after the sludge filtering deposition, A diagnostic sensor is provided.

In one example, the antibody may be, but is not limited to, a prostate specific antigen (PSA).

In one example, the carbon-based nanostructure may be selected from single wall carbon nanotubes, double wall carbon nanotubes, multi wall carbon nanotubes, graphite, and graphene, but is not limited thereto.

In one example, the filter paper may be, but is not limited to, a cellulose ester filter paper having micropores of, for example, 0.3 to 0.5 um in size.

In one example, the paper-type sensing portion obtained by radially cutting the filter paper may be a rectangle of 5 x 2 mm ² , but is not limited thereto.

In one example, the PSA concentration can be measured at a sensitivity of 1 ng / mL in the range of 0 ng / mL to 500 ng / mL, but is not limited thereto.

In one example, the diagnostic sensor includes a substrate on which a paper-like sensing portion is to be placed; And electrodes formed on both ends of the sensing unit.

Another aspect of the present invention provides a method of manufacturing a paper-type prostate cancer diagnostic sensor, comprising the steps of:

A carboxylation step of attaching a carboxyl group to the carbon-based nanostructure;

Binding the carbonylated carbon-based nanostructure to a prostate cancer-specific antibody to obtain an antibody-bound carbon-based nanostructure;

Depositing the resulting antibody-bound carbon-based nanostructure on a filter paper by syringe filtering; And

Cutting the silyfiltered filter paper radially from the center of the circle toward the edge to obtain a paper-type sensing unit;

In one example, the carboxylation can be performed by refluxing the carbon-based nanostructure at 20O < 0 > C for 3 hours at 105 < 0 > C with 3.0 M nitric acid.

In one example, the step of obtaining the antibody-bound carbon-based nanostructure comprises: an antibody accepting step in which the antibody is added to a carbon-based nanostructure mixture and reacted by stirring at room temperature for 8 hours overnight; And

And a step of incubating the carbon-based nanostructure reacted with the antibody with a blocking agent at room temperature to 40 ° C for 1 to 3 hours to obtain an antibody-bound carbon-based nanostructure.

The concentration of MWCNT is 6.5 ug / mL and the purified volume of Ab-MWCNT solution per filter paper is 200 mL.

Another aspect of the present invention relates to a diagnostic method of prostate cancer using the paper-type prostate cancer diagnosis sensor.

In one example, the antigen-antibody reaction is performed by culturing the paper-type sensing part in a wet state at 30 to 40 ° C for 1 to 3 hours with a biological sample;

Sensing the electrical resistance by assembling the paper type sensing unit with a detection unit capable of measuring electrical resistance change after drying; And

And comparing the measured electrical resistance with a basic resistance to obtain a relative electrical resistance (R / R ₀ ) value.

In one example, prostate cancer can be diagnosed at an early stage if the concentration of PSA antigen converted based on the relative electrical resistance (R / R ₀ ) value is greater than 4 ng / mL.

The present invention provides a paper-type biosensor based on a carbon-based nanostructure and a filter paper. According to this, for initial screening of prostate cancer, the PSA level changed the electrical resistance of the sensor by more than 150%. The maximum detection range is 500 ng / mL and the detection limit is 1.18 ng / mL. Paper-based biosensors are 23 times cheaper and 12 times faster than ELISA-based detection methods. Moreover, the maximum detection value is about 50 times higher. The detection range and sensitivity are sufficient to diagnose early stage prostate cancer. It is expected to be a very useful diagnostic tool because biomarkers that specifically react with various target biomolecules as well as PSA can be applied to CNTs.

FIG. 1 is a reaction scheme 1 showing the process of obtaining an antibody-MWCNT in which MWCNT is activated with a PSA antibody according to an embodiment of the present invention.
FIG. 2 is a schematic diagram illustrating a process of manufacturing a paper-type sensing unit and a PSA detection process according to an embodiment of the present invention.
FIG. 3 is a reaction formula 3 illustrating a process of diagnosing prostate cancer using a paper-type prostate cancer diagnosis sensor according to an embodiment of the present invention.
Figure 4 shows the IR spectra of MWCNT, carboxylated MWCT dipped in nitric acid for 3 days, and Ab-MWCNT.
5 is a SEM and AFM image of MWCNT-based sensor units; a and b are the SEM and AFM images of the carboxylated MWCNT, c and d are the SEM and AFM images of the Ab-MWCNT, and e and d are the SEM and AFM images of the PSA-Ab-MWCNT.
Figure 6 shows IV curves for MWCNT, carboxyl-activated MWCNT, Ab-MWCNT, and PSA-Ab-MWCNT at current ranges of 1.40 mA (a) and 1.00 uA (b).
Figure 7 shows the change in electrical resistance due to MWCNT surface modification. Data points represent the carboxylated MWCNT, Ab-MWCNT, and PSA-Ab-MWCNT (PSA; 1000 ng / mL) from the left.
Figure 8 is a graph showing the relative resistance of a biosensor at (a) a wide range (0-1000 ng / mL) and (b) a narrow and low range (0-10 ng / mL) PSA concentration.
9 is a graph showing the results of measurement of resistance uniformity according to the cutting method of paper-type sensor, (a) resistance uniformity of the sensor cut in the longitudinal radial direction of the present invention and (b) resistance uniformity of the sensor cut in the transverse radial direction .

Unless defined otherwise, all technical terms used in the present invention have the following definitions and are consistent with the meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to herein are incorporated herein by reference.

The term "about" is used herein to refer to a reference quantity, a level, a value, a number, a frequency, a percentage, a dimension, a size, a quantity, a weight or a length of 30, 25, 20, 25, 10, 9, 8, 7, Level, value, number, frequency, percentage, dimension, size, quantity, weight or length of a variable, such as 4, 3, 2 or 1%.

Throughout this specification, the words "comprises" and "comprising ", unless the context requires otherwise, include the steps or components, or groups of steps or elements, Steps, or groups of elements are not excluded.

In the present invention, "prostate cancer" is described, for example, in Prostate. In: Edge SB, Byrd DR, Compton CC, et al., Eds .: AJCC Cancer Staging Manual. 7th ed. New York, NY: Springer, 2010, pp 457-68] (hereby incorporated by reference). In addition, the abnormal prostate condition can be classified into one or more of benign prostatic hyperplasia (BPH), androgen sensitive prostate cancer, androgen non sensitive or resistant prostate cancer, invasive prostate cancer, non-invasive prostate cancer, metastatic prostate cancer and non-metastatic prostate cancer have.

The term "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to ascertain whether or not prostate cancer has developed.

The term "antibody" is used in its broadest sense and includes, without limitation, a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., bispecific antibody), and an antibody fragment that exhibit the desired antigen- (Including but not limited to) a variety of antibody constructs. Diagnostic antibodies may be those which are provided as raw materials, such as purified monoclonal anti-PSA, rabbit anti-PSA, goat anti-mouse IgG, which are raw materials of conventional commercially available antibodies that can be used in the production of strips. In a preferred example, the antibody may be a prostate specific antigen (PSA).

Paper type prostate cancer diagnosis sensor

Another prostate cancer diagnosis sensor according to an embodiment of the present invention includes a paper type sensing unit and a detection unit. The paper-type sensing unit includes an antibody-carbon-based nanostructure having an antibody attached thereto and a filter paper deposited by sieving filtering the antibody-carbon-based nanostructure.

The present invention provides a paper-based sensor utilizing a carbon-based nanostructure and a filter paper. When the carbon-based nanostructures are formed into a mat shape, these mutual contact barriers determine electrical conductivity. After the antibody is attached to the outer wall of the structure It is a principle to determine the concentration of biomolecules such as antigens by measuring the change of electrical conductivity by changing the contact barriers between carbon-based nanostructures according to the content of antigens to be detected.

Syringe filtering is used to convert the carbon-based nanostructures into layered mat forms. Syringe filtering is performed by placing a solution containing carbon nanotubes in a syringe, using a pressure to apply the solvent to the filter And the nanotubes are filtered through the filter so as to be made into a mat shape. Using this syringe filtering, an antibody-carbon based nanostructure can be deposited, which is a very useful method because it can form a uniform layer with a uniform thickness, shorten the deposition time, and reduce the possibility of contamination. The thickness of the deposited antibody-carbon based nanostructure layer may be about 3 to 5 um.

In the present invention, a layer composed of the antibody-carbon based nanostructure is deposited on the filter paper by the syringe filtering, and then the filter paper is radially cut in the edge direction from the center of the circle. This is because the uniformity of the antibody-carbon based nanostructure layer is the most excellent in the radial cut portion, so that it is very effective to keep the basic resistance constant throughout the sensing portion. The shape of the radial cut portion is a rectangular shape having a long length from the center of the circle to the edge direction, and in the embodiment of the present invention, a rectangular shape of 5 x 2 mm ² is used, but the present invention is not limited thereto.

The carbon-based nanostructure refers to a nano-sized structure composed of a carbon component, and may include a reduced acid component included in the manufacturing process. In an embodiment of the present invention, a multi-walled carbon nanotube is used, but the present invention is not limited thereto. For example, single-walled carbon nanotubes, double-walled carbon nanotubes, multi-walled carbon nanotubes, graphite, graphene, Plate or the like can be applied. Hereinafter, the carbon-based nanostructure may be abbreviated as 'carbon structure' as the case may be.

It is preferable that the filter paper has a uniform micro-pore structure so that the antibody-carbon structure layer can be easily formed. In one example, micro-pores having a size of 0.3 to 0.5 μm are used. And a filter paper made of a cellulose ester material.

The maximum detection range of the paper type prostate cancer diagnosis sensor according to the present invention is 500 ng / mL and the detection limit is 1.18 ng / mL. The present inventors have experimentally confirmed that the contact potential between carbon structures increases due to the antibody-antigen binding reaction, and the change in relative resistance is almost linearly dependent on the concentration of the antibody. For example, in the case of PSA antibody, the concentration can be measured with a sensitivity of 1 ng / mL in the range of 0 ng / mL to 500 ng / mL. Generally, if the PSA level is higher than 4 ng / mL, it can be judged to be an early stage of prostate cancer. Therefore, diagnosis of the prostate cancer can be performed using the diagnostic sensor of the present invention.

In the diagnostic sensor, the detection unit may include a substrate on which the paper type sensing unit is disposed; And electrodes formed on both ends of the sensing unit, and electric wires may be connected to the electrodes to allow a current to flow. These detectors can detect the change in resistance due to the change of the contact potential between the carbon structures, as well as the charge transfer model between the biomolecule and the antibody-carbon structure affecting the conductivity.

The substrate may be made of glass, silicon, or a mixture thereof. In addition, the electrode may be selected from the group consisting of gold, platinum, chromium, copper, aluminum, nickel, palladium, and titanium.

The paper-type prostate cancer diagnosis sensor according to the present invention can be used to diagnose prostate cancer incidence in a subject having an increased risk of developing prostate cancer. Subjects with increased risk of developing prostate cancer should be monitored for additional signs or symptoms of prostate cancer. The diagnostic methods provided in the present invention for identifying subjects with increased risk of developing prostate cancer are not limited to other known risk factors or signals of prostate cancer such as, for example, urinary stream loss, urgency, hesitation, nighttime urine incontinence bladder emptying) and age.

Manufacturing method of paper type prostate cancer diagnosis sensor

A method of manufacturing a paper-type prostate cancer diagnosis sensor according to an embodiment of the present invention will be described with reference to Schemes 1 and 2 of FIGS. 1 and 2. FIG. A carboxylation step (110) of attaching a carboxyl group to the carbon-based nanostructure; Binding the antibody to the carboxylated carbon-based nanostructure to obtain an antibody-bound carbon-based nanostructure (120); And depositing (130) the antibody-bound carbon-based nanostructure on a filter paper by syringe filtering; (140) cutting the silyfiltered filter paper radially from the center of the circle toward the edge to obtain a paper-type sensing unit (140).

The carboxylation (110) can be performed by refluxing the carbon-based nanostructure at 105 DEG C for 3 hours to 3 days in a nitric acid solution.

The step 120 of obtaining the antibody-bound carbon-based nanostructure may include an antibody accepting step 121 in which the antibody is added to the carbon-based nanostructure mixture and reacted at room temperature for 6 to 12 hours to react; And a step (122) of adding a blocking agent to the antibody-containing carbon-based nanostructure and incubating the mixture at room temperature to 40 ° C for 1 to 3 hours to obtain an antibody-bound carbon-based nanostructure.

That is, the non-specific antigen binding can be prevented in advance by first reacting the antibody with the carbon structure, and then adding a non-reactive blocking agent such as BSA to attach the antibody to the non-antibody binding site in the carbon structure layer.

Next, the antibody-bound carbon-based nanostructure deposition step 130 forms a nonspecific site-blocked antibody-carbon structure layer on the circular filter paper by the syringe filtering method. Syringe filtering is well known in the art, and known methods are equally applicable to the present invention.

Finally, the step 140 of cutting the filter paper is cut in a rectangular shape having a long length toward the edge from the center side of the circular filter paper in a radial manner, and the cutting method is not particularly limited.

Prostate cancer diagnosis using paper type prostate cancer diagnosis sensor

A method for diagnosing prostate cancer according to an embodiment of the present invention will be described with reference to the reaction formula 3 in FIG.

(210) performing an antigen-antibody reaction by incubating the paper-type sensing part in a wet state at 30 to 40 ° C for 1 to 3 hours with a sample;

(220) drying the paper-type sensing unit at room temperature and assembling the paper-type sensing unit with a detector capable of measuring a change in electrical resistance to measure electrical resistance; And

And comparing the measured electrical resistance to a basic electrical resistance to obtain a relative electrical resistance (R / R ₀ ) value 230 (not shown).

First, a specimen is put into a paper-type sensing unit where an antibody-carbon structure layer is formed, and an antigen-antibody reaction 210 is induced. At this time, the sensing part should not be in a dry state, and preferably, the reaction proceeds while being kept in a wet state at 30 to 40 ° C for 1 to 3 hours.

Next, when the antigen-antibody reaction is completed, the paper-type sensing unit is dried at room temperature for about 30 minutes to about 2 hours (221). The dried sensing part is placed on a substrate (slide glass), electrodes are connected to each other, assembled with the detection part, and the electrical resistance is measured (222).

Finally, the relative electrical resistance (R / R ₀ ) value is obtained by comparing the measured electrical resistance with the basic resistance value of the antibody-carbon structure layer not bound to the antigen. In one example, prostate cancer can be diagnosed at an early stage if the concentration of PSA antigen converted based on the relative electrical resistance (R / R ₀ ) value is greater than 4 ng / mL.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

Experimental material

Nitric acid (64.0 ~ 65.0%) was purchased from DUKSAN science. PBS (pH = 7.2) buffer solution was purchased from Biosesang Inc. 1 M 2- (N-morpholino) ethanesulfonic acid (MES, pH = 5.0) and 0.05% Tween 20 + 1% BSA (bovine serum albumin) in PBS were purchased from Tech & Innovation. (EDC), N-hydroxysuccinimide sodium salt (NHSS), monoclonal anti-KLK3 antibody (species And PSA (prostate specific antigen from human semen) were purchased from Sigma-Aldrich. Microporous filter paper (cellulose ester blend, diameter 25 mm, pore size 0.45 um) was obtained from Toyo Roshi Kaisha, Ltd. Lt; / RTI > MWCNT (multi-wall carbon nanotubes, diameter 20 nm, length 5 μm) was purchased from Carbon Nano-material Technology Co. MWCNT was produced by CVD using iron and molybdenum catalysts.

Abbreviation Description

- MWCNT; Multi-Walled Carbon Nanotubes

- carboxylated MWCNT; MWCNT < / RTI > through the carboxylation step (110)

- Ab-MWCNT; After the antibody binding step (120), the MWCNT

< Example  1>

1-1. Carboxylation step of carbon-based nanostructures

Reaction 1 shown in FIG. 1 shows the activation of MWCNT and PSA antibody. The detection system is performed by incubating at 37 DEG C for 1.5 hours. Hereinafter, the present invention will be described in more detail with reference to Scheme 1. The first step is the carboxylation step (110) of the carbon-based nanostructure.

First, MWCNT (300 mg) was refluxed in 20 mL of 3.0 M nitric acid at 105 ° C for 3 hours to form a carboxyl group on the MWCNT. The functionalized MWCNT solution is centrifuged at least 10 times with distilled water so that the pH approaches 7. The carboxylated MWCNT was dried in a 100 &lt; 0 &gt; C oven for 24 hours. The carboxyl groups on the MWCNT surface were confirmed by Fourier transform infrared spectrometer (FT-IR, JASCO FT / IR-4100).

1-2. Steps to Obtain Antibody-Carbon-Based Nanostructures

The carboxylated MWCNT is capable of accepting the PSA antibody in a next step 120. The carboxylated MWCNT (1.3 mg) is dispersed with 0.4 mmol EDC and 0.1 mmol NHSS in 4 mL of 0.1 M MES buffer solution. The dispersion was sonicated for 1 minute with an ultrasonic shaker (Jeio Tech, UC-02) and mixed with a vortex mixer (Scientific Industries, Inc) for 15 minutes at room temperature. The obtained mixed solution is centrifuged (Hanil Science Industrial Co., Ltd., HA-1000-3) at 3000 rpm for 10 minutes, and the supernatant is discarded. Wash with PBS at least 5 times to remove excess EDC and NHSS from the precipitate. Add 0.5 mL (0.01 mg / mL) of PSA antibody to the mixture and stir at room temperature overnight to attach to the MWCNT. This process is the PSA antibody accepting reaction 121 in the second step of the reaction scheme 1.

The next step is to produce an MWCNT-based biosensor, which inhibits unwanted reactions between the PSA antibody-attached MWCNT and the target PSA (step 122). Add a mixture of 0.05% Tween 20 in 1% BSA (100 uL) to the mixed solution (2 mL) to which the PSA antibody has been added to prevent the attachment of PSA on the MWCNT surface. BSA completely encapsulates the MWCNT surface to prevent indiscriminate (non-specific) PSA attachment. Tween 20 clears the binding site of the PSA antibody. The mixture was incubated at room temperature for 1.5 hours, centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. Remove any free PSA antibody and BSA by washing the supernatant with PBS buffer more than 5 times and centrifuging. The step (122) of reacting the MWCNT with the PSA antibody and the blocking agent (BSA) to obtain the antibody-bound MWCNT. The obtained sample is referred to as Ab-MWCNT, which means the MWCNT to which the PSA antibody is attached.

1-3. Filter paper deposition step

The Ab-MWCNT is deposited on the microporous filter paper by syringe filtering. This step is the first step of Scheme 2 (see Fig. 2). The Ab-MWCNT deposited on the microporous filter paper should not be dry until the sensing is complete for the activity of the antibody.

The Ab-MWCNT deposited on the filter paper is cut into a size of 5 x 2 mm ² in the circumferential direction at the center of the filter paper to obtain a paper-type sensing portion. This radial cutting method is very important to maintain the resistance uniformity of all the sensing parts.

[Example 2] Diagnosis of prostate cancer using a paper type diagnostic sensor

2-1. Antigen-antibody reaction step

In the paper type sensing unit PSA-Ab-MWCNT manufactured in Example 1-3, BSA-blocked PSA-Ab-MWCNT was deposited on the micro pore filter paper.

Add 100 uL of PBS buffer solution containing 0 to 1000 ng / mL of PSA concentration to the paper type sensor. To maintain the biological activity of all proteins, paper-based sensors should not be dried. That is, it must remain wet until the site-specific reaction between PSA antibody and PSA is complete. The site-specific reaction was completed by incubating the sensing part at 37 DEG C for 1.5 hours.

2-2. Electrical resistance measurement step

After the culture, the paper-type sensing unit is continuously dried in the air at room temperature for 30 minutes or more. Next, assemble in the form of a biosensor and measure the electrical resistance (see equation 3). The resistance of the dried paper-type sensing portion can be measured using a digital multimeter (Hewlett-Packard Co. multimeter 3478A or Agilant 34401) under atmospheric conditions. A silver paste electrode is used as a detecting portion to maintain electrical contact between the sensing portion and the copper wire. The basic morphological information of the material is observed with an AFM (Park scientific instruments) and a scanning electron microscope (SEM, HITACHI S-2400).

2-3. Step of obtaining relative electric resistance

The paper sensor, in contact with a PBS buffer solution (100 uL) without PSA (PSA concentration 0 ng / mL), was incubated at 37 ° C for 1.5 h and the sensor resistance measured for a specific concentration of PSA was measured for 30 min Compare to the basic electrical resistance (background) measured after drying. The measured sensor resistance is standardized considering the physical shape of the sensor. The thickness of the sensor is not considered. Because the background resistance and all resistance values were compared, and all the sensors were made of one filter paper. The most important factor to keep the basic resistance of the sensor constant is the uniformity of the MWCNT layer deposited on the fine pore filter paper.

The uniformity of the MWCNT layer can be obtained by adjusting the concentration of MWCNT to 6.5 ug / mL. The volume of Ab-MWCNT solution used per filter paper is 200 mL. The thickness of the deposited MWCNT was ~4.5 um. In addition, as shown in Reaction Formula 2 in Fig. 2, the radial cutting method of the sensing portion which cuts from the center to the circle is very important for maintaining a constant resistance.

This method of measuring the PSA concentration has an advantage over the widely used ELISA such as portability, time saving and cost reduction.

< Experimental Example  1> Verification of antibody-carbon structure binding

Figure 4 shows the IR spectra of MWCNT, nitric acid-saturated carboxylated MWCT for 3 days, and Ab-MWCNT. The peak position corresponding to the carbon-carbon double bond of MWCNT was found at 1630 cm ^-1 . And 1715 cm ^-1 corresponds to the C = O bond of the carboxyl group, which is generated by treatment of MWCNT with nitric acid. The carboxyl group may be located on the surface of the MWCNT and react with the amine group of the L-lysine unit in the antibody. This is a simple substitution reaction to form an amide group. Before the reaction takes place, the carboxylated MWCNT is treated with EDC / NHSS to induce a mutual exchange reaction of the hydroxyl group of the carboxyl group and the antibody. The IR spectrum of Ab-MWCNT is shown by the blue line in FIG. 1, and a complex peak corresponding to the amide bond appears at around 1550 cm ^-1 .

< Experimental Example  2> Verification of antibody-carbon structure binding

5 is SEM and AFM images of MWCNT-based elements. Samples were deposited on filter paper and dried at ambient conditions for 2 hours or more for image capture. Figures 2 (a) and 2 (b) are SEM and AFM images of carboxylated MWCNT, respectively. In both images, the diameter of MWCNT was about 20 nm. This corresponds to the diameter of the receptor MWCNT. No damage was observed on the surface of the carboxylated MWCNT. All MWCNTs were distinguishable.

Figures 5c and d are SEM and AFM images of Ab-MWCNT. The diameter of the antibody-attached MWCNT was similar to that of the carboxylated MWCNT. Any irregularly attached object was observed on the tube surface. This means that the attachment of the antibody may not be coated over the entire MWCNT surface. A lump, such as a cotton ball, on the SEM image is presumed to be an attached antibody.

5E and 5F are SEM and AFM images of PSA-Ab-MWCNT. Most SEM images of MWCNT are covered with proteins (PSA antibody and PSA). Sommon-like areas are spread over the entire surface of the deposited film. The shape of the sommon-like part is not clearly observed in the AFM image due to the softness of the protein. The densities of selectively observed deposited films were in the order of carboxylated MWCNT Ab.MWCNT> PSA-Ab-MWCNT. From the AFM images of the film, the RMS (RMS) roughness was in the same order as the naked eye observation. MWCNT, Ab-MWCNT, and PSA-Ab-MWCNT were 38, 32, and 61 nm, respectively.

&Lt; Experimental Example 3 & gt; Measurement of electrical resistance change of sensor part

Figure 6 shows the I-V curves for MWCNT, carboxyl-activated MWCNT, Ab-MWCNT, and PSA-Ab-MWCNT at current ranges of 1.40 mA (a) and 1.00 uA (b). Table 1 also shows the relative resistance values of MWCNT, carboxylated MWCNT, Ab-MWCNT, and PSA-Ab-MWCNT calculated in FIG.

Initial amount of CNTs (mg) I-V curve slope Resistance (Ω) MWCNTs 1.16 0.117 117 Carboxylated MWCNTs 1.16 0.585 585 Ab-MWCNTs 1.30 0.916 916 PSA-Ab-MWCNTs 1.30 1.36 1360

Referring to FIG. 6 and Table 1, all MWCNTs follow Ohm's law at high current levels (from -1.4 mA to +1.4 mA) as well as in the low current range (-1.0 uA to +1.0 uA) . The higher the amount of protein attached to the MWCNT, the higher the slope of the I-V curve.

The electrical resistance of each MWCNT is shown in Table 1 and can be estimated from the linear slope shown in FIG. The electrical resistance was increased by 500% by carboxylation of MWCNT. Decrease in carbon-sp ² character, which is the origin of conductivity during the carboxylation process in MWCNT, can cause this resistance increase. When the PSA antibody was attached to the carboxyl group, the electrical resistance of the Ab-MWCNT was increased by about 160% more than that of the carboxylated MWCNT. The increased resistance is believed to be due to an increase in the contact potential barrier between the MWCNTs by the additional attachment of the protein (PSA-antibody). For the same reason, the resistance of PSA-Ab-MWCNT (PSA, 1000 ng / mL) was about 150% higher than that of Ab-MWCNT. This stepwise increase of CNT is clearly shown in Fig.

Figure 7 shows the change in electrical resistance due to MWCNT surface modification. Data points represent MWCNT, carboxylated MWCNT, Ab-MWCNT and PSA-Ab-MWCNT (PSA; 1000 ng / mL) from the left.

When biomolecules attached to CNTs take electrons from CNTs with normal p-type conductivity at normal temperature and pressure, the conductivity will increase (off current increase) and the threshold voltage (V _TH ) of the field transistor Due to the increase of the p-character, the voltage shifts to a negative voltage. If biomolecules provide electrons to CNTs, the opposite phenomenon will be observed. In addition to the charge transfer model, the increase in the contact energy barrier between conductive CNT strands due to the adsorption of biomolecules on the CNT surface causes a decrease in conductivity without changing the V _TH of the FET.

Therefore, the value of the target biomolecule that can be attached to the CNT can be determined by the resistance change of the biomolecule-CNT and the V _TH movement of the FET. However, in the CNT-FET formed between the Au microelectrodes on SiO ₂ / Si wipers, the movement of V _TH due to biomolecule addition was not observed, and only the decrease of the off-current of the FET due to biomolecule exposure I could observe. This means that there is no electron transfer from the CNT to the biomolecule, which means that the charge carrier concentration is constant and the contact energy between the CNTs due to biomolecule adhesion is increased. In recent years, it has been confirmed that an FET assembled using metal-attached CNTs has excellent hydrocarbon detection capability for this reason.

(R / R _0, where R is the resistance of a sensor that senses a specific concentration of PSA, and R ₀ is the sensing value of a buffer solution that does not contain PSA). Is the resistance value of one sensor) is shown in FIG. FIG. 8 (a) shows that the change in relative resistance is almost linearly dependent on the concentration of PSA in the range of 0 to 500 ng / mL. This means that if the concentration of PSA is less than 500 ng / mL, the concentration of PSA can be estimated by measuring the electrical resistance of the sensor. However, if the concentration of PSA is> 500 ng / mL, the resistance of the sensing part appears to be saturated.

Figure 8 (b) shows the change in relative resistance in the low PSA concentration range (0-10 ng / mL). In this range, the relative resistance was linearly dependent on the PSA concentration. As shown in the sensor graph, a very low level of PSA found in the early stages of prostate cancer can be detected. If PSA levels are higher than 4 ng / mL, prostate cancer may be at an early stage.

  When the blank sample (0 ng / mL of PSA) was detected from 11 sensors prepared on one filter paper, the detection limit was estimated to be 1.18 ng / mL as a result of 2 standard deviations from the signal average value.

< Experimental Example 4> Measurement of resistance uniformity according to the cutting method of paper-type sensor

In the paper-type sensor of the present invention, it was confirmed that the resistance uniformity according to the cutting method was varied. Thus, as shown in FIGS. 9 (a) and 9 (b) .

As a result of measuring the resistance uniformity of the longitudinally-radial cut paper type sensor of Fig. 9 (a), the standard deviation was found to be 0.00332 from 11 sensors made by vertical radial cutting and the range of R / R ₀ value was 0.99 to 1.00 And the resistance uniformity was constant. This results in no problem in detecting 1.18 ng / mL, which is the detection limit of the present invention sensor. In the paper type sensor of the present invention, two sensors of background detection indicator and detection indicator are used to exclude errors that may occur depending on humidity, temperature, and surrounding environment in the PSA detection process, and the difference in electric resistance between the background and the indicator R / R ₀ value to calculate the PSA concentration. Therefore, in order to compare the electrical resistances of the background and the indicator, the two detectors should have the same initial electrical resistance.

On the other hand, as a result of measuring the resistance uniformity of the transverse cutting paper sensor of Fig. 9 (b), the standard deviation of electrical resistance obtained from eleven sensors made by transverse cutting was 0.0438, which was 4 ng / mL Since the PSA is larger than the detection signal, it is inadequate to manufacture the sensor. Also, it was confirmed that the value of R / R ₀ was 0.91 as the lowest value and 1.06 as the maximum value, and the value was not constant, which was not suitable for the PSA detection method of the paper type sensor of the present invention. If the resistance uniformity is not ensured, the reproducibility of the experiment and the accuracy of the PSA concentration measurement are greatly reduced.

As a result, the paper-type sensor cut in the longitudinal radial direction according to the present invention exhibits a superior effect to the paper-type sensor cut in the transverse radial direction, and it is confirmed to be suitable as a sensor for diagnosis (see FIG. 9).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (13)

A paper-type sensing unit including an antibody-carbon-based nanostructure having an antibody attached thereto and a filter paper deposited by sieving filtering the antibody-carbon-based nanostructure; And
And a detection unit capable of measuring a change in electrical resistance of the sensing unit,
Wherein the paper-type sensing unit is obtained by radially cutting the paper-type sensing unit into a rectangle in the edge direction from the center of the circle of the filter paper after the sill filter filtering deposition. The method according to claim 1,
Wherein the antibody is a prostate-specific antigen (PSA). The method according to claim 1,
Wherein the carbon-based nanostructure is selected from single wall carbon nanotubes, double wall carbon nanotubes, multiwall carbon nanotubes, graphite, and graphene. The method according to claim 1,
Wherein the filter paper is a cellulose ester filter paper having fine pores having a size of 0.3 to 0.5 mu m. The method according to claim 1,
Wherein the paper type sensing part obtained by radially cutting the filter paper is a rectangle of 5 x 2 mm ² . The method according to claim 1,
Wherein the maximum detection range of the sensor is 500 ng / mL and the detection limit is 1.18 ng / mL. 3. The method of claim 2,
Wherein the PSA concentration can be measured at a sensitivity of 1 ng / mL in the range of 0 ng / mL to 500 ng / mL. The method according to claim 1,
Wherein the detection unit comprises: a substrate on which the paper-type sensing unit is disposed; And two electrodes formed on both ends of the sensing unit. A carboxylation step of attaching a carboxyl group to the carbon-based nanostructure;
Coupling the antibody to the carboxylated carbon-based nanostructure to obtain an antibody-bound carbon-based nanostructure; And
Depositing the antibody-bound carbon-based nanostructure on a filter paper by syringe filtering;
A step of radially cutting the syllable-filtered filter paper into a rectangle from the center of the circle toward the edge to obtain a paper-type sensing unit;
Wherein the method comprises the steps of: 10. The method of claim 9,
Wherein the carboxylation is carried out by refluxing the carbon-based nanostructure at 105 DEG C for 3 to 3 days in a nitric acid solution. 10. The method of claim 9, wherein obtaining the antibody-bound carbon-based nanostructure comprises:
Adding an antibody to the carbon-based nanostructure solution, and stirring the mixture at room temperature for 6 to 12 hours to react; And
(B) adding a blocking agent to the antibody-containing carbon-based nanostructure and incubating the mixture at room temperature to 40 ° C for 1 to 3 hours to obtain an antibody-bound carbon-based nanostructure;
Wherein the method comprises the steps of: delete delete

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