KR20210032763A - Colorimetric bio sensor, and method for detecting target materials using thereof - Google Patents

Abstract

The present invention provides a color conversion biosensor, and a target material detection method using the same, wherein the color conversion biosensor comprises: a substrate surface-modified with a metal affinity functional group; first plasmonic metal particles fixed to the metal affinity functional group; and a first aptamer specifically binding to a target material bound to the first plasmonic metal particles. When the biosensor is exposed to a sample containing the target material, by coupling reaction of the target material with the first aptamer, the plasmonic metal particles agglomerate to occur color conversion of the biosensor.

Description

Color conversion biosensor, and target substance detection method using the same {COLORIMETRIC BIO SENSOR, AND METHOD FOR DETECTING TARGET MATERIALS USING THEREOF}

The present invention relates to a color conversion biosensor, and a target substance detection method using the same, and more specifically, to a color conversion biosensor using the local surface plasmon resonance change, and a target substance detection method using the same.

Thrombin, a coagulation protein, acts as a serine protease in blood coagulation and is involved in many physiological and pathological processes such as inflammation, blood coagulation, thrombus, metastasis, cardiovascular function and tumor growth. Thrombin levels in the blood are essential biomarkers for tumors and other diseases associated with coagulation abnormalities. Therefore, a simple, rapid and sensitive detection method for detecting thrombin at the time of treatment is required.

Aptamer is a single-stranded oligonucleotide and can bind to an analyte with high specificity and affinity. Aptamers are considered effective receptors and are widely used in medical diagnostics and environmental and biological analyses because they are small, easy to handle and relatively stable. Since thrombin has two binding sites capable of reacting with a biomolecule, a 2 thrombin-binding aptamer (TBA) can be synthesized. One is a 15-mer oligonucleotide (5'-GGT TGT TGT TGGG-3), which interacts with the fibrinogen-recognition exosite of thrombin, while the other is a 29-mer oligonucleotide (5' AGC AGC TGG TG TGA-3') interacts with heparin-binding exosites.

Recently, gold nanoparticles (AuNPs) have attracted great attention due to their high extinction coefficient, chemical stability, and easy surface modification. To take advantage of the unique optical properties of AuNPs, a biosensor based on the aggregation of AuNPs has been developed for disease detection and clinical treatment. Aggregation-based AuNP detection has various advantages because it is a label-free and cost-effective method that does not require complex analytical equipment. In addition, the biosensor can be used as a simple analysis device capable of detecting with the naked eye using a localized surface plasmon resonance (LSPR) signal of AuNP. However, in general, LSPR-based biosensors deployed in solution have several disadvantages. Typically, color interference from colored target samples, non-specific binding occurs, ionic strength of the solution, PH and temperature, etc. I can. Therefore, in order to prevent non-specific aggregation and improve stability, various methods of attaching nanoparticles to a solid support, such as modifying a glass substrate with Thiol, amino groups, or polymers, have been developed. have.

An object of the present invention is to provide a color conversion biosensor capable of detecting a target material with the naked eye and having a higher detection sensitivity than a conventional sensor.

Another object of the present invention is to provide a method for detecting a target material using the color conversion biosensor capable of simple and rapid detection by using a local surface plasmon resonance change.

A color conversion biosensor for one object of the present invention includes a substrate surface-modified with a metal affinity functional group; A first plasmonic metal particle immobilized on the metal affinity functional group; And a first aptamer that specifically binds to a target material bound to the first plasmonic metal particle.

The fixation is that the plasmonic metal particles fixed to the substrate are completely attached to the substrate and do not fall on the substrate, which is not in an immovable state, but when a small physical force is applied from the outside, the x-axis and y-axis of the substrate It means enough to be able to move in the direction.

In one embodiment, when the sensor is exposed to a sample containing the target material, the plasmonic metal particles aggregate due to a binding reaction between the target material and the first aptamer, resulting in color conversion of the sensor. .

In one embodiment, the metal affinity functional group may be any one selected from an amine group, a thiol group, a vinyl group, a cyano group, and a carboxyl group.

In one embodiment, the metal affinity functional group is 3-aminopropyltriethoxysilane (APTES), 3-aminopropyltrimethoxysilane (3-Aminopropyl)trimethoxysilane, APTMS), and 3-mercaptopropyl. It may be any one functional group selected from methyldimethoxysilane (3-Mercaptopropyltrimethoxysilane).

In one embodiment, the plasmonic metal particle may be any one selected from aluminum, copper, silver, platinum, and gold particles.

In one embodiment, the target material may be any one of DNA, enzyme, virus, thrombin, and cell membrane protein among materials having two binding sites capable of specifically binding to an antibody or an aptamer.

In one embodiment, the target material is thrombin, and the first aptamer may be a 15-mer oligonucleotide (single-stranded oligonucleotide) or a 29-mer oligonucleotide (single-stranded oligonucleotide).

In one embodiment, the second plasmonic metal particles immobilized on the metal affinity functional group; And a second aptamer that specifically binds to a target material bound to the second plasmonic particle, wherein the first plasmonic metal particle and the second plasmonic metal particle are the same or different particles, and the The first aptamer and the second aptamer may be different materials.

In one embodiment, the target material is thrombin, the first aptamer is a 15-mer oligonucleotide, and the first aptamer is a 29-mer oligonucleotide. I can.

A method for detecting a target material using a color conversion biosensor for another object of the present invention includes the steps of exposing a sample to the color conversion biosensor; And confirming a change in local surface plasmon resonance of the sensor.

In an embodiment, in the step of confirming the change in the local surface plasmon resonance, when color conversion of the sensor occurs, it may be determined that the target material has been detected.

In one embodiment, the sample may be serum, and the target material may be thrombin.

A method for detecting a target substance using a color conversion biosensor for another object of the present invention includes the steps of exposing a sample to a color conversion biosensor; After the step, further exposing the second plasmonic metal particles to which the second aptamer is bound to the sensor; And confirming a change in local surface plasmon resonance of the sensor.

In one embodiment, the first plasmonic metal particle and the second plasmonic metal particle may be the same or different particles, and the first aptamer and the second aptamer may be different materials.

In an embodiment, in the step of confirming the change in the local surface plasmon resonance, when color conversion of the sensor occurs, it may be determined that the target material has been detected.

In one embodiment, the sample may be serum, and the target material may be thrombin.

According to the color conversion biosensor of the present invention, and the target substance detection method using the same, the change in the spectral signal and the absorption peak are increased, so that the color change can be visually detected even at a low target substance concentration. It shows improved sensitivity and detection efficiency.

In addition, it has excellent selectivity with target biomolecules by using the aptamer as the basis, and exhibits high clinical applicability.

1 is a diagram illustrating a color conversion biosensor of the present invention and a method of detecting a target substance using the same.
2 is a diagram showing a UV-vis spectrum measured when a target substance is detected using a biosensor manufactured according to Example 1 of the present invention. Figure 2 (a) is a view showing the UV-vis spectrum of the TBA1 bonded AuNP on the APTES-coated substrate, (b) is a view showing the UV-vis spectrum of the TBA2 bonded AuNP in the solution.
3 is a view for explaining the result of Experimental Example 2 showing the color conversion biosensor analysis according to the thrombin concentration of the present invention. Figure 3 (a) is a view showing the UV-vis spectrum measured in the first step of contacting the first sensor and thrombin of the present invention, (b) is the second sensor after performing the first step It is a diagram showing the Uv-vis spectrum measured in the second step, (c) is a diagram showing the color conversion biosensor after performing the first and second steps according to the thrombin concentration, and (d) is It is a diagram showing the measured peak shift after performing the first step and the second step.
4 is a view for explaining the result of Experimental Example 3 for evaluating the target substance binding specificity of the color conversion biosensor of the present invention. Figure 4 (A) is a diagram showing detection in various interference analytes using the color conversion biosensor of the present invention, (B) is a diagram showing a function of thrombin concentration and peak shift at 520 nm, upper left The graph inserted in is a diagram showing a linear relationship between the LSPR peak shift and the added thrombin concentration.

The terms used in the present application are only used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present application, terms such as "comprise" or "have" are intended to designate the presence of features, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features or steps. It is to be understood that it does not preclude the possibility of addition or presence of, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein including technical or scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present application. Does not.

The present invention is a solid-phase color conversion biosensor using the aggregation of plasmonic metal particles fixed on a substrate coated with a metal affinity functional group for detection of a target material, and a target material detection method using the same Provides.

Referring to Figure 1, the color conversion biosensor of the present invention, and a method for detecting a target material using the same will be described.

First, hereinafter, the color conversion biosensor of the present invention will be described.

The color conversion biosensor of the present invention includes a substrate having a metal affinity functional group surface-modified; A first plasmonic metal particle immobilized on the metal affinity functional group; And a first aptamer that specifically binds to a target material bound to the first plasmonic metal particle.

First, the substrate having the metal affinity functional group surface-modified may have a form in which a plurality of metal affinity functional groups are attached over the entire surface of the substrate. The substrate may be a solid substrate, and may be prepared by treating the substrate with a solution containing a metal affinity functional group.

The metal affinity functional group may be any one selected from an amine group, a thiol group, a vinyl group, a cyano group, and a carboxyl group. Preferably, it may be an amine group. In addition, the metal affinity functional group is 3-aminopropyltriethoxysilane (APTES), 3-aminopropyltrimethoxysilane (3-Aminopropyl)trimethoxysilane, APTMS), and 3-mercaptopropylmethyldimethoxy It may be any one functional group selected from silane (3-Mercaptopropyltrimethoxysilane). Preferably, it may be 3-aminopropyltriethoxysilane (APTES). However, the present invention is not necessarily limited thereto.

The first plasmonic metal particles surface-modified with the metal affinity functional group may be prepared by immersing the substrate coated with the metal affinity functional group in a solution containing the first plasmonic metal particles.

The plasmonic metal particles may be passivated plasmonic metal particles to prevent aggregation and non-specific binding from other molecules. In one embodiment, the passivation of the plasmonic metal particles may be performed using polyvinylpyrrolidone (PVP). The plasmonic metal particles may be any one selected from aluminum, copper, silver, platinum and gold particles, and preferably gold particles. However, the present invention is not necessarily limited thereto.

The plasmonic metal particles may be stabilized through electrostatic interaction with a metal affinity functional group surface-modified on the substrate, and thus may be fixed on the substrate. Here, fixation means that the plasmonic metal particles fixed to the substrate are completely attached to the substrate and do not fall on the substrate, which is not in an immovable state, but when a small physical force is applied from the outside, the x-axis of the substrate, y It means the degree to be able to move in the axial direction.

The first aptamer that specifically binds to the target material bound to the first plasmonic metal particles may be prepared by immersing the substrate on which the plasmonic metal particles are immobilized in a solution containing the first aptamer.

The target material may be a biomolecule present in a living body. In detail, the target material may be any one of DNA, enzyme, virus, thrombin, and cell membrane protein among substances having two binding sites capable of specifically binding to an antibody or an aptamer. Preferably, the target material may be thrombin.

When the target material is thrombin, the first aptamer may be a 15-mer oligonucleotide (single-stranded oligonucleotide) or a 29-mer oligonucleotide (single-stranded oligonucleotide).

Additionally, the color conversion biosensor of the present invention comprises: a second plasmonic metal particle fixed to the metal affinity functional group; And a second aptamer that specifically binds to the target material bound to the second plasmonic particle. In this case, the first plasmonic metal particle and the second plasmonic metal particle may be the same or different particles, and the first aptamer and the second aptamer may be different materials.

In one embodiment, when the target material is thrombin, the first aptamer may be a 15-mer oligonucleotide, and the second aptamer may be a 29-mer oligonucleotide.

When the color conversion biosensor is exposed to a sample containing the target material, the plasmonic metal particles aggregate through a binding reaction between the target material and the first aptamer, thereby causing color conversion of the sensor.

Hereinafter, the present invention describes a method of detecting a target material using the color conversion biosensor. There are two methods of detecting a target substance using the color conversion biosensor provided in the present invention.

First, the first target substance detection method is to use single sensing. The first detection method, a method of detecting a target substance using a color conversion biosensor, includes the steps of exposing a sample to a color conversion biosensor; And confirming a change in local surface plasmon resonance of the sensor.

The sample is a sample to be tested whether it contains a target material to be detected. Preferably, the sample may be a sample obtained from a living body, and more preferably, the sample may be a serum.

The step of exposing the sample to the color conversion biosensor may be performed by dropping the sample onto the sensor in the form of a sample drop or touching the sample by placing the sensor on the sample. However, the present invention is not limited thereto.

In the step of confirming the change in local surface plasmon resonance of the sensor, when the color conversion of the color conversion biosensor occurs, it may be determined that the target material has been detected. The color conversion of the color conversion biosensor occurs through a phenomenon in which the target material binds to the aptamer of the color conversion biosensor, and the plasmonic nanoparticles bound to the aptamer aggregate on the substrate. Therefore, when the color conversion of the color conversion biosensor occurs, since the target material is bound to the aptamer that specifically binds to the target material to be detected, it can be determined that the sample contains the target material.

In this case, as a method of checking the color conversion, the color change can be checked with the naked eye or the color conversion can be checked using a device.

In one embodiment, when detecting thrombin in serum, when thrombin is present in the serum, the color conversion biosensor may change from red (red) to purple. In this case, the aptamer of the color conversion biosensor used may be any one selected from a 29-mer oligonucleotide (single-stranded oligonucleotide) or a 29-mer oligonucleotide (single-stranded oligonucleotide).

The second target substance detection method is to use double sensing.

The second detection method provided by the present invention, a method for detecting a target substance using a color conversion biosensor, includes: exposing a sample to a color conversion biosensor; After the step, further exposing the second plasmonic metal particles to which the second aptamer is bound to the sensor; And confirming a change in local surface plasmon resonance of the sensor.

In the step of further exposing the second plasmonic metal particles to which the second aptamer is bound, the target material bound to the first aptamer and the second aptamer are combined to fix the second plasmonic metal particles on the substrate, The first plasmonic metal particles and the second plasmonic metal particles are fixed on the substrate of the color conversion sensor, so that sensing can be performed more sensitively.

In this case, the first plasmonic metal particle and the second plasmonic metal particle may be the same or different particles, and the first aptamer and the second aptamer may be different materials.

Hereinafter, an embodiment of the present invention using the second target substance detection method of the present invention will be described.

When detecting thrombin in serum, the first aptamer used in the color conversion biosensor is a 15-mer oligonucleotide, and the first aptamer is a 29-mer oligonucleotide. ), and the metal affinity functional group may be an amine group.

First, a sample is exposed to the color conversion biosensor for serum to be detected. Preferably, the color conversion biosensor may be dropped as a serum drop, or the color conversion biosensor may be brought into contact with the serum. At this time, when thrombin is present in the serum, thrombin binds to the first aptamer, and thus, the first plasmonic metal particles aggregate on the substrate.

Thereafter, a step of further exposing the second plasmonic metal particles to which the second aptamer is bound to the color conversion biosensor is performed. In this case, it may be performed by injecting the color conversion biosensor into a substrate in a solution containing the second plasmonic metal particles to which the second aptamer is bound. At this time, when thrombin is present in the serum, thrombin and the second aptamer bound to the first aptamer and the second aptamer are bound, through which plasmonic metal particles bound to the second aptamer are fixed on the substrate.

Then, a step of confirming a change in local surface plasmon resonance of the sensor is performed. The confirmation can be confirmed with the naked eye or by using a device. In this case, when thrombin is present in the serum, an increase in absorption peak and red-shift can be confirmed.

These results can appear as the following phenomena.

When thrombin is present in the serum, in the step of further exposing the second plasmonic metal particles to which the second aptamer is bound, the structure in the form of a sandwich (first plasmonic metal particles-first aptamer-thrombin-second pressure Tamer-second plasmonic metal particle) is formed, and the plasmonic metal particle is colored through electrostatic attraction between the plasmonic metal particle and the thiol group of the aptamer, which is relatively stronger than the binding force of the amine group and the plasmonic metal particle. It becomes free on the surface of the substrate of the conversion biosensor, causing aggregation and movement of the plasmonic metal particles.

As the distances between the plasmonic metal particles become closer to each other, and as aggregation occurs, the local surface plasmon coupling phenomenon increases, so that the change in the absorption spectrum peak is not only observed by ultraviolet visible light spectroscopy (UV-visible spectrometer). It shows a significant color change that can be detected visually.

Hereinafter, a color conversion biosensor of the present invention and a method of detecting a target material using the same will be described in more detail through specific examples and experimental examples. However, the embodiments of the present invention are only some embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

Thrombin was selected as a target material in one embodiment and experimental examples of the present invention.

Primary thrombin-binding aptamer TBA1 (5'-thiolated-TTT TTT TTT TTT TTT GGT TGG TGT GGT TGG-3') and secondary thrombin-binding aptamer TBA2 (5 '-thiolated-TTT TTA GTC CGT GGT AGG GCA GGT TGG GGT GAC T-3') was synthesized by Bioneer (Korea). Human serum, human IgG, osteopontin (OPN), bovine serum albumin (BSA), trisodium citrate, tetrachloroauric(III) acid) (HAuCl4 ), APTES, acetate buffer (pH 5.12), Tris (2-carboxyethyl) phosphine hydrochloride (Tris(2-carboxyethyl)phosphine hydrochloride) (TCEP), polyvinylpyrrolidone (PVP) , Ethanol, and methanol were purchased from Sigma-Aldrich (Korea).

Example 1: Color conversion biosensor

① Manufacturing of surface-modified substrate

Under 100 W and 50 cm ³ /min conditions, functionalized glass coverslips (18 mm ㅧ 18 mm, thickness 0.12 mm) were treated with 0.1% APTES solution for 1 hour, followed by washing with ethanol and nitrogen. (N ₂ ) It was dried using gas, and solidified in an oven at 120°C for 2 hours to obtain a substrate surface-modified with APTES.

② Preparation of gold nanoparticles (AuNPs)

AuNPs were synthesized according to Turkevich's method. All used glass articles to make gold nanoparticles were washed with an aqua regia solution for 30 minutes prior to preparation of the nanoparticles. Briefly, 100 mL of 1 mM HAuCl ₄ solution was boiled with vigorous stirring at 100° C. to prepare a gold precursor solution, and 38.8 mM sodium citrate was rapidly injected into the gold precursor solution. The color of the mixed solution changed from transparent to purple and then red. Then, the mixture was further stirred for an additional 40 minutes and then cooled to room temperature to synthesize AuNP. The synthesized AuNP solution was passivated with PVP to prevent aggregation and non-specific binding from other molecules.

③ Preparation of TBA1 and TBA2 solutions

TBA1 solution was prepared by dissolving TBA1 (0.1 μL, 0.5 mM), TCEP (0.1 μL, 10 mM) and acetate buffer (pH 5.12, 0.1 μL, 500 mM) in distilled water to obtain a 3 mL mixed solution, and then conjugation. It was stored for 1 hour before to prepare a TBA1 solution.

TBA2 solution was prepared by dissolving TBA2 (0.1 μL, 0.5 mM), TCEP (0.1 μL, 10 mM), and acetate buffer (pH 5.12, 0.1 μL, 500 mM) in distilled water to obtain a 3-mL solution. Before conjugation, TBA2 solution was prepared by holding for 1 hour in order to divide into disulfide groups of aptamers.

④ Manufacturing of color conversion biosensor

The substrate surface-modified with APTES prepared above was immersed in a solution containing AuNP overnight to prepare an AuNP-immobilized substrate. The AuNP-immobilized substrate was immersed in a TBA1 solution (3 mL, 16.6 nM) for 16 hours to combine TBA1 and AuNP, then washed with distilled water and dried with nitrogen, color conversion bio according to an embodiment of the present invention. The sensor was prepared. The prepared color conversion biosensor was referred to as an AuNP-TBA1 sensor, and the appearance of the particles in which TBA1 was bound to AuNP was referred to as AuNP-TBA1 particles.

In addition, the 2mL solution of AuNP prepared above was mixed with the TBA2 solution to prepare a mixed solution, and AuNP and TBA2 were combined for 16 hours. Subsequently, the mixed solution was centrifuged twice for 15 minutes at 17,000 rpm at 23 to 25° C. to remove excess unconjugated TBA2 to prepare AuNP-TBA2 particles having TBA2 bound on AuNP.

Experimental Example 1: Target substance detection analysis using a color conversion biosensor

First, thrombin (500 μL, 1-10 μg/mL) was injected into the AuNP-TBA1 sensor prepared according to Example 1, and incubated for 30 minutes to perform the first step, and then the first step was performed. In order to increase the sensitivity to the sensor, a second step of injecting a solution containing 500 μL of AuNP-TBA2 particles was performed to perform double bond detection. All experiments were performed by repeating 3 times. In each of the above steps, the LSPR signal was detected by a UV-vis spectrometer at a wavelength of 400-900 nm, and images were obtained using a scanning electron microscope (SEM), and the results are shown in FIGS. 1 and 2.

First, referring to FIG. 1, it can be confirmed that gold nanoparticles are aggregated through a scanning electron microscope (SEM) image, and the color of the sensor is changed to purple through the aggregation (the upper right image of each of the scanning electron microscope images of FIG. 1 )can confirm. In addition, it can be confirmed that an increase in the amount of absorbance and a red shift of the absorbance spectrum were induced through the UV spectrum.

Through this, it can be seen that AuNPs aggregate through the first step of contacting the AuNP-TBA1 sensor with thrombin. In addition, through the second step of contacting the AuNP-TBA2 particles, the thrombin molecule acts as a linker between the AuNP-TBA1 particles and the AuNP-TBA2 particles to induce cross-linking between the molecules, resulting in AUNP-TBA1-thrombin-TBA2. It can be seen that the -AUNP sandwich complex can be formed and an increase in the absorption peak and red shift can be further induced.

Referring to FIG. 2, when comparing the absorption peak of AuNPs in (a) and the absorption peak after TBA1 is bonded to AuNP, it can be seen that the absorption peak after TBA1 is bonded to AuNP is shifted red. This was set as a change in the refractive index of the AuNP surface due to the binding of TBA1, and for this reason, it can be expected that the absorption peak is shifted red.

In (b), compared with the absorption peak of AuNPs, it can be seen that the absorption peak of PVP passivation for AuNPs has a red shift, and then, it can be confirmed that an additional red shift is induced by binding of TBA2.

Experimental Example 2: Target material analysis

For the color conversion biosensor prepared according to Example 1 as described above, detection was performed using various concentrations of thrombin (500 μL, 1-10 μg/mL), and the detection limit was a peak deviated from the blank peak position. It was calculated as the average value of the peak shift. The results are shown in FIG. 3.

Referring to FIG. 3, it can be seen that the characteristic LSPR at about 520 nm is red shifted as the absorbance increases due to the induced AuNP aggregation proportional to the thrombin concentration.

In Figure 3 (a) in the first step (STEP 1) of contacting the AuNP-TBA1 sensor with thrombin, as the concentration of thrombin increases, the absorbance increases, and it can be seen that the red shift of the absorption spectrum of AuNP LSPR is induced. . These results show that the TBA1-thrombin-TBA1 connection of the AuNP-TBA1 sensor is formed and the aggregation of AuNP is induced.

3B) In the second step (STEP 2) of contacting the AuNP-TBA2 particles after the first step, it can be seen that the red shift and intensity of the absorption spectrum increase, which is the substrate of the color conversion biosensor. This is greatly improved by increasing the number of AuNPs bound to and increasing the degree of AuNP aggregation.

In (c) of FIG. 3, it is possible to check the color change of the biosensor according to the concentration, and it can be seen that the color of the biosensor is changed and detected even at a low thrombin concentration.

In (d) of FIG. 3, it can be seen that the peak shift of the AuNP-TBA1 sensor and the AuNP-TBA2 particle together (STEP 2) is significantly improved than when the AuNP-TBA1 sensor is used (STEP 1). At 10 μg/mL thrombin concentration, it can be seen that the peak shift is about 4 times larger than when the AuNP-TBA1 sensor and AuNP-TBA2 particles are used together (STEP 2) and the detection using the AuNP-TBA1 sensor (STEP 1). have. In addition, when the AuNP-TBA1 sensor is used (STEP 1), the detection limit is 3.94 μg/mL, whereas when the AuNP-TBA1 sensor and AuNP-TBA2 particles are used together (STEP 2), the detection limit is 1.33 μg/mL. It can be seen that it is about 3 times lower than that of STEP 1.

Through this, it can be seen that the detection sensitivity and lower detection limit are improved when the dual sensor is used than when the single sensor is used.

Experimental Example 3: Analysis of target substance binding specificity of color conversion biosensor

In order to analyze the binding specificity of the color conversion biosensor of the present invention to thrombin, thrombin and various interference analytes (IgG, OPN, BSA and complex mixtures thereof) using the color conversion biosensor prepared according to Example 1 Was injected at a concentration of 10 μg/mL to confirm the effect of interference by other biomolecules, and all experiments were repeated three times to obtain an average value. The results are shown in FIG. 4.

Referring to FIG. 4, it can be seen that (A) has high selectivity to thrombin, and in (B), as the concentration of thrombin added increases, the peak shift increases. A change in the LSPR signal of the biosensor was observed in the presence of an interfering substance, but this is within a statistically acceptable range (p <0.05), so even in the presence of an interfering substance, the biosensor manufactured according to an embodiment of the present invention is It can be seen that it has a binding specificity.

Example 4: Evaluation of the clinical applicability of the biosensor of the color conversion biosensor

In order to evaluate the clinical applicability of the color conversion biosensor prepared according to an embodiment of the present invention, various concentrations of thrombin (1, 3, 5, 7 and 10 μg/mL) were added to 100-fold diluted human serum samples. A target substance solution was prepared by adding to the diluted human serum. The target material solution was injected into the color conversion biosensor of the present invention, and the recovery rate (%) was calculated using Equation 1 below. The results are shown in Table 1 below.

[Equation 1]

Serum sample
(Serum sample) Added
(μg/mL) Measured (μg/mL) Recovery (%) One One 1.08 107.76 2 3 2.08 67.02 3 5 5.22 104.35 4 7 7.84 112.02 5 10 9.59 95.91

Referring to Table 1, the five tested serum samples showed an average recovery rate of 94.4%. The complexity of human serum samples can have a major impact on thrombin and, consequently, the sensitivity of the biosensor. From the results shown in Table 1, since the color conversion biosensor of the present invention has high sensitivity even in a complex biological sample, it can be expected that clinical application of the biosensor is promising.

Through the experimental examples of the present invention, when the color conversion biosensor of the present invention is used, it can be confirmed that the spectral signal and peak shift are greatly improved to have high sensitivity, and it can be easily detected by checking the color change detectable with the naked eye. Can be seen. In addition, when thrombin is detected using the color conversion biosensor of the present invention, it can be detected visually through color conversion even at a low thrombin concentration of 3 μg/mL. When the target material is thrombin, it can be seen that the detection limit of thrombin using the color conversion biosensor of the present invention is 1.33 μg/mL. These results can be seen that when the detection method using the dual sensor of the present invention is used, it is three times lower than that of the detection method using a single sensor, thus showing improved sensitivity and detection efficiency. In addition, the analysis using the color conversion biosensor of the present invention showed excellent specificity and clinical applicability of the target material.

Although the above has been described with reference to preferred embodiments of the present invention, those skilled in the art will be able to variously modify and change the present invention without departing from the spirit and scope of the present invention described in the following claims. You will understand that you can.

Claims (16)

A substrate surface-modified with a metal affinity functional group;
A first plasmonic metal particle immobilized on the metal affinity functional group; And
Containing a first aptamer that specifically binds to a target material bound to the first plasmonic metal particle,
Color conversion biosensor.
The method of claim 1,
When the sensor is exposed to a sample containing the target material, the plasmonic metal particles aggregate as a result of a binding reaction of the target material with the first aptamer to cause color conversion of the sensor,
Color conversion biosensor.
The method of claim 1,
The metal affinity functional group is characterized in that any one selected from an amine group, a thiol group, a vinyl group, a cyano group and a carboxyl group,
Color conversion biosensor.
The method of claim 1,
The metal affinity functional group is 3-aminopropyltriethoxysilane (APTES), 3-aminopropyltrimethoxysilane (3-Aminopropyl)trimethoxysilane, APTMS), and 3-mercaptopropylmethyldimethoxysilane ( 3-Mercaptopropyltrimethoxysilane), which is any one functional group selected from,
Color conversion biosensor.
The method of claim 1,
The plasmonic metal particle is characterized in that any one selected from aluminum, copper, silver, platinum and gold particles,
Color conversion biosensor.
The method of claim 1,
The target material is characterized in that any one of DNA, enzyme, virus, thrombin, and cell membrane protein among substances having two binding sites capable of specifically binding to antibodies or aptamers,
Color conversion biosensor.
The method of claim 1,
The target material is thrombin,
The first aptamer is a 15-mer oligonucleotide (single-stranded oligonucleotide) or a 29-mer oligonucleotide (single-stranded oligonucleotide),
Color conversion biosensor.
The method of claim 1,
Second plasmonic metal particles immobilized on the metal affinity functional group; And
Further comprising a second aptamer that specifically binds to the target material bound to the second plasmonic particles,
The first plasmonic metal particle and the second plasmonic metal particle are the same or different particles,
The first aptamer and the second aptamer are characterized in that the material is different from each other,
Color conversion biosensor.
The method of claim 8,
The target material is thrombin,
The first aptamer is a 15-mer oligonucleotide (single-stranded oligonucleotide),
The first aptamer is a 29-mer oligonucleotide (single-stranded oligonucleotide),
Color conversion biosensor.
Exposing the sample to the color conversion biosensor according to any one of claims 1 to 9; And
Including; confirming the local surface plasmon resonance change of the sensor,
Target substance detection method using color conversion biosensor.
The method of claim 10,
In the step of confirming the local surface plasmon resonance change,
When the color conversion of the sensor occurs, it is determined that the target material has been detected,
Target substance detection method using color conversion biosensor.
The method of claim 10,
The sample is serum,
The target material is characterized in that thrombin,
Target substance detection method using color conversion biosensor.
Exposing the sample to the color conversion biosensor according to any one of claims 1 to 7;
After the step, further exposing the second plasmonic metal particles to which the second aptamer is bound to the sensor; And
Including; confirming the local surface plasmon resonance change of the sensor,
Target substance detection method using color conversion biosensor.
The method of claim 13,
The first plasmonic metal particle and the second plasmonic metal particle are the same or different particles,
The first aptamer and the second aptamer are different materials from each other,
Target substance detection method using color conversion biosensor.
The method of claim 13,
In the step of confirming the local surface plasmon resonance change,
When the color conversion of the sensor occurs, it is determined that the target material has been detected,
Target substance detection method using color conversion biosensor.
The method of claim 13,
The sample is serum,
The target material is characterized in that thrombin,
Target substance detection method using color conversion biosensor.

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