KR20210045739A - Colorimetric sensor detecting the target, and preparation method of thereof - Google Patents

Abstract

The present invention relates to a colorimetric sensor detecting a target material and a manufacturing method thereof. The colorimetric sensor comprises: a substrate surface-modified with an amine functional group; tricosadiynoic acid (TCDA) liposomes immobilized to the amine functional group; plasmonic metal particles immobilized on the surface of the TCDA liposome; and an aptamer that specifically binds to the target material immobilized to the surface of the TCDA liposome.

Description

A color conversion sensor for detecting a target substance, and a manufacturing method thereof TECHNICAL FIELD [COLORIMETRIC SENSOR DETECTING THE TARGET, AND PREPARATION METHOD OF THEREOF}

The present invention relates to a color conversion sensor for detecting a target material, and a method for manufacturing the same, and more specifically, using the optical properties of 12-Tricosadiynoic acid (TCDA), a conjugated polymer. Thus, it relates to a color conversion sensor for detecting a target material, and a method of manufacturing the same.

In order to improve the optical and electrical properties of plasmonic nanoparticles, studies are being conducted to combine plasmonic nanoparticles and conjugated polymers. Among these conjugated polymers, polydiacetylene (hereinafter referred to as PDA) has been widely studied to utilize a colorimetric transition. This characteristic of PDA is due to structural modifications of both chains of the conjugated backbone by stimuli such as temperature, chemicals, pH, ligand receptor interaction and mechanical stress.

In the liquid phase, the color transition of PDA is induced by the repulsion in the liposome and the structural change of the spinal cord-coupled TCDA side chain of the liposome-binding agglutination modification within the TCDA liposome, leading to a strong optical transition of TCDA. Further, compared to the solid phase, higher concentrations of PDA liposomes may be available on the sensing component than at a fixed surface area. Therefore, PDA-based sensors mainly adopt liquid phase analysis, but the solid phase sensor is more practical than the liquid phase sensor, which requires finding a means to increase the optical transition of the PDA for solid phase access, thus reducing the optical resonance of the sensing platform. In order to improve, research is being conducted to introduce plasmonic nanoparticles such as gold nanoparticles (AuNP).

One of the studies is to investigate the signal enhancement of PDA by introducing AuNP into PDA as an additional mechanical stimulator for the purpose of signal enhancement. However, there is a problem in that an additional reaction step such as a sandwich method is required to improve a signal for a target material. In addition, as another example of the use of AuNP in PDA sensors, there is also a method of using diacetylene self-assembled on AuNP, but since diacetylene exists on the surface of AuNP, diacetylene-modified AuNP is applied to a robust platform. There is a problem that is very difficult to do.

An object of the present invention is to provide color conversion sensors for detecting a target material having an improved degree of color change, including polydiacetylene-based polymers and plasmonic metal particles.

Another object of the present invention is to provide a method of manufacturing a color conversion sensor that detects the target material.

A color conversion sensor for detecting a target material for an object of the present invention includes a substrate surface-modified with an amine functional group; Tricosadiynoic acid (TCDA) liposomes immobilized on the amine functional group; Plasmonic metal particles immobilized on the surface of the 10,12-Tricosadiynoic acid liposome; And an aptamer that specifically binds to a target substance bound to the surface of the TCDA liposome.

The liposome is a shell formed of TCDA, and may have a hollow shape with an empty core.

In one embodiment, when the sensor is exposed to a sample containing the target material, the target material is subjected to a binding reaction with the aptamer and a mechanical stress is added to the TCDA liposome to convert the color of the sensor. This can happen.

In one embodiment, the plasmonic metal particles are spaced apart at regular intervals so as not to contact each other on the surface of the 10,12-Tricosadiynoic acid liposome, and the surface of the TCDA liposome may be partially exposed. have.

In one embodiment, the plasmonic metal particles may be surface-modified with an amine functional group.

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

In one embodiment, the aptamer may include an amine or thiol functional group.

In one embodiment, the target material may be thrombin, DNA, enzyme, virus, and cell membrane protein in which a receptor is present.

In one embodiment, the target material is thrombin, and the aptamer may be a DNA oligonucleotide.

A color conversion sensor for detecting a target material for another object of the present invention includes a substrate coated with plasmonic metal particles; TCDA liposomes immobilized on the plasmonic metal particles; And an aptamer that specifically binds to the target substance bound to the TCDA liposome.

The liposome is a shell formed of TCDA, and may have a hollow shape with an empty core.

In one embodiment, when the sensor is exposed to a sample containing the target material, the target material is subjected to a binding reaction with the aptamer and a mechanical stress is added to the TCDA liposome to convert the color of the sensor. This can happen.

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

In one embodiment, the aptamer may include an amine or thiol functional group.

In one embodiment, the target material may be thrombin, DNA, enzyme, virus, and cell membrane protein in which a receptor is present.

In one embodiment, the target material is thrombin, and the aptamer may be a DNA oligonucleotide in which a receptor is present.

According to the color conversion sensor for detecting a target material of the present invention, and a method of manufacturing the same, it is easy to use and can check the sensing result of the target material with the naked eye through the discoloration of the sensor without separate analysis equipment. In addition, the degree of discoloration can be improved by applying mechanical stress to the polymer by the use of plasmonic metal particles, and the degree of discoloration can be further improved by synergistic action through the plasmonic effect of the plasmonic metal particles. It can have improved performance.

1 is a view for explaining a color conversion sensor according to an embodiment of the present invention.
2 is a diagram for evaluating characteristics of a color conversion sensor according to an embodiment of the present invention. a is a Uv-vis spectrum for evaluating the change of plasmonic metal nanoparticles before/after UV polymerization of TCDA. b is a Uv-vis spectrum for evaluating the properties according to the amount of plasmonic metal nanoparticles immobilized on the TCDA surface. c is a SEM image diagram for evaluating the structural characteristics according to the amount of plasmonic metal nanoparticles fixed on the TCDA surface.
3 is a Uv-vis spectrum for determining red-shift of a color conversion sensor according to an embodiment of the present invention.
FIG. 4 is a diagram for evaluating the detection effect of a target substance according to the amount of plasmonic metal nanoparticles immobilized on a TCDA surface in a color conversion sensor according to an embodiment of the present invention.
5 is a diagram for evaluating the properties of the plasmonic nanoparticles of Examples 1 and 2 of the present invention.
6 is a diagram for evaluating CR characteristics according to thrombin concentration of the color conversion sensors of Examples 1 to 4 of the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the present invention, various modifications may be made and various forms may be applied, and specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific form disclosed, it should be understood to include all changes, equivalents, or substitutes included in the spirit and scope of the present invention. In describing each drawing, similar reference numerals have been used for similar elements.

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.

Metal nanoparticle-bound polymer composites attracted great attention because of their ability to enhance the surrounding plasmon field. On the other hand, Tricosadiynoic acid, a conjugated polymer, has a unique optical property of discoloration from blue to red caused by distortion of the conjugated main chain by various stimuli. In this specification, as a new method of increasing the degree of color change of a TCDA-based sensor, target materials each prepared by two methods using plasmonic nanoparticles immobilized directly on the surface of TCDA and TCDA immobilized on a single layer of plasmonic nanoparticles are used. It provides color conversion sensors to detect. The color conversion sensor for detecting a target substance of the present invention has an effect of showing a colorimetric ratio 4 times higher in the presence of thrombin as compared to a TCDA-based color change sensor without plasmonic nanoparticles.

The color conversion sensor for detecting a target material provided by the present invention provides two types of color conversion sensors. First, the first type of color conversion sensor (aptamer-plasmonic metal particle-TCDA) is a sensor including plasmonic metal particles fixed directly on the TCDA surface, and will be described with reference to FIG. 1.

Referring to Figure 1, the color conversion sensor of the present invention is a substrate surface-modified with an amine functional group; Tricosadiynoic acid (TCDA) liposomes immobilized on the amine functional group; Plasmonic metal particles immobilized on the surface of the TCDA liposome; And an aptamer that specifically binds to a target substance bound to the surface of the TCDA liposome.

The substrate surface-modified with an amine functional group may be surface-modified with an amine functional group using 3-aminopropyl triethoxysilane (APTES). However, the present invention is not limited thereto, and any one of materials capable of surface modification of the substrate with an amine functional group may be used. In this case, the substrate may be a solid substrate. For example, the substrate may be a glass substrate.

The TCDA liposome may be a 10,12-Tricosadiynoic acid liposome, and is a polydiacetylene-based polymer. The TCDA is a polymer having optical properties in which color changes due to twisting of a conjugated backbone. In addition, since the terminal may have a carboxylic acid, a crosslinking reaction with an amine group may be performed. In addition, the liposome in the present invention is a shell formed of TCDA, and may have a hollow shape with an empty core. In one embodiment, the size of the TCDA of the present invention may be characterized to be about 150 nm to 400 nm through dynamic light scattering (DLS).

The TCDA liposome immobilized on the amine functional group is 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide, hereinafter referred to as EDC)/N- It may be fixed through a coupling reaction of hydroxysuccinimide (N-hydroxysuccinimide, hereinafter referred to as NHS). Specifically, carboxylic acid and EDC react at the end of the TCDA to prepare an unstable intermediate, and the intermediate reacts with NHS to form an amine reaction ester, so that the amine functional group on the substrate and the TCDA are The carboxylic acid can be bonded to the terminal. At this time, the EDC/NHS coupling reaction may affect the pH environmental conditions. In the present invention, the pH environment was adjusted using triethylamine, but the present invention is not limited thereto.

Plasmonic metal particles are immobilized on the surface of the TCDA liposome. The plasmonic metal particle may be a particle surface-modified with an amine functional group. In addition, the plasmonic metal particles may be any one selected from aluminum, copper, silver, platinum, and gold particles. Preferably, the plasmonic metal particles may be gold particles. The gold particles (hereinafter referred to as AuNPs) may be particles that strongly resonate with light in a visible light region. In addition, the sensitivity of the color conversion sensor of the present invention may be controlled according to the size of the plasmonic metal particles.

The fixation of the plasmonic metal particles may be performed using an EDC/NHS coupling reaction. In one embodiment, the plasmonic metal particles immobilized on the surface of the TCDA liposome may be fixed by crosslinking the amine functional group and the carboxylic acid of the TCDA liposome surface-modified on the plasmonic metal particle.

In this case, the plasmonic metal particles are spaced apart at regular intervals so as not to contact each other on the surface of the TCDA liposome, and the surface of the TCDA liposome is partially exposed. When there are too many plasmonic metal particles immobilized on the surface of TCDA liposomes, that is, when they are fixed in contact with each other on the surface of TCDA liposomes, and the exposed area of the TCDA liposome surface is very small, the plasmonic metal particles are the main chain of TCDA. There is a problem in that the sensitivity of the color conversion sensor is inhibited by inhibiting distortion.

In addition, on the surface of the TCDA liposome, an aptamer that specifically binds to a target substance together with plasmonic metal particles is immobilized. The aptamer may include an amine or thiol functional group. The fixation of the aptamer may be performed using an EDC/NHS coupling reaction. In one embodiment, an amine of an aptamer containing an amine functional group and a carboxylic acid present in TCDA may be crosslinked and fixed through an EDC/NHS coupling reaction. In one embodiment, the aptamer may be a DNA oligonucleotide.

The target material may be thrombin, DNA, enzyme, virus, cell membrane protein, etc. in which a receptor is present. Preferably, it may be thrombin.

In one embodiment, the target material is thrombin, and the aptamer may be a DNA oligonucleotide.

In another embodiment, the target material is thrombin, and the aptamer may be a DNA oligonucleotide in which a receptor is present.

The second type of color conversion sensor (aptamer-TCDA-plasmonic metal particle) of the present invention is a sensor including a TCDA fixed to a single layer of plasmonic metal particles. The color conversion sensor may include a substrate coated with plasmonic metal particles; TCDA liposomes immobilized on the plasmonic metal particles; And an aptamer that specifically binds to the target substance bound to the TCDA liposome.

In the substrate coated with the plasmonic metal particles, the substrate may be a solid substrate, for example, a glass substrate. However, it is not necessarily limited thereto. The plasmonic metal particles may be any one selected from aluminum, copper, silver, platinum, and gold particles. Preferably, the plasmonic metal particles may be gold particles. In this case, the sensitivity of the color conversion sensor may be controlled according to the size of the gold particles present on the substrate.

The plasmonic metal particles immobilize TCDA liposomes on the surface. The fixation may be fixed using an EDC/NHS coupling reaction.

The TCDA liposome immobilizes an aptamer that specifically binds to a target substance on the surface. The aptamer may be an aptamer containing an amine or thiol functional group. The immobilization may be immobilized due to crosslinking of the amine of the aptamer containing the amine functional group and the carboxylic acid of the TCDA. Preferably, it may be crosslinked using an EDC/NHS coupling reaction. In one embodiment, the aptamer may be a DNA oligonucleotide.

The target material may be thrombin, DNA, enzyme, virus, cell membrane protein, etc. in which a receptor is present. Preferably, it may be thrombin.

In one embodiment, the target material is thrombin, and the aptamer may be a DNA oligonucleotide in which a receptor is present.

Two types of color conversion sensors provided by the present invention, when the sensor is exposed to a sample containing the target material, mechanical stress on the TCDA liposome due to the binding reaction of the target material with the aptamer. ) May be added, and the color conversion of the sensor may occur due to the distortion of the main chain of the TCDA to which the stress is applied.

In addition, the present invention can provide a method of manufacturing a color conversion sensor for detecting a target material. The method of manufacturing a color conversion sensor for detecting a target material of the present invention comprises the steps of: surface modification of a substrate with an amine functional group; Immobilizing a TCDA liposome on the amine functional group; Binding plasmonic metal particles to the surface of the TCDA liposome; And binding an aptamer that specifically binds to a target substance on the surface of the TCDA liposome.

The combining step is 1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide (1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide) and N-hydroxysuccinimide (N-hydroxysuccinimide) It can be combined by carrying out a coupling reaction of.

Before performing the step of binding the plasmonic metal particles to the surface of the TCDA liposome, the step of surface modification of the plasmonic metal particles with an amine functional group; may further include.

Prior to performing the step of binding the aptamer that specifically binds to the target substance on the surface of the TCDA liposome, surface modification of the aptamer with an amine functional group; may further include.

Example 1: Color conversion sensor for detecting a target substance (TBA-AuNP-TCDA)

① Preparation of surface-modified AuNP with amine group

First, 22.8 μl of 1.42 mM HAuCl ₄ , 400 μl of 213 mM cysteamine hydrochloride, and 40 mL of deionized water were mixed, and stirred at room temperature and dark room for 20 minutes to prepare a mixed solution. 10 mM NaBH₄10 µl solution was rapidly added to the prepared mixed solution in a dark place with vigorous stirring for 60 minutes to prepare a solution containing AuNP surface-modified with an amine group. As the amount of AuNP increased, the color changed from yellow to red grapes. The solution containing the surface-modified AuNP with the amine group was stored at 4°C.

② Preparation of TCDA liposome

In the flask, the monomer of TCDA was dissolved in chloroform, and the solvent was evaporated through a rotary evaporation system. Then, 18.2 MΩ·cm of deionized water was added to the flask to prepare a 1 mM TCDA solution. Sonication was performed at 70° C. for 15 minutes until the TCDA solution was clearly dissolved. The sonicated solution was filtered through a 0.8 μm cellulose acetate syringe filter to remove liposomes having a size of 0.8 μm or less, and then cooled at 4° C. overnight to prepare TCDA liposomes.

③ Fixing AuNP and TCDA on the substrate

_{First, after plasma treatment of O 2} on a glass substrate at 50 sccm and 100 w/min power for 1 minute, 3-aminopropyltriethoxysilane (APTES) and ethanol It was immersed in the mixed solution for 1 hour to prepare a glass substrate surface-modified with an amine group.

Then, a 10 mM mixed solution of the same volume of EDC and NHS solution was added to the prepared TCDA liposome solution in 0.1 volume, incubated at 4°C for 30 minutes, and then surface-modified with amine groups at room temperature for 3 hours. It was made to react with the glass substrate. After the reaction, the substrate was washed with deionized water and then dried with pure N2 gas. Then, a 1 mM mixed solution of the same volume of EDC and NHS solution was incubated on a TCDA-bonded glass substrate for 30 minutes with a solution containing AuNP surface-modified with 0.5 volume of amine group. After incubation, it was gently washed with deionized water and dried with pure N₂ gas. In addition, in order to control the amount of fixed AuNP, 1 mM triethylamine (hereinafter referred to as TEA) was used to adjust the pH.

④ TCDA functionalization

In the above, 497.5 µl of EDC (1 mM), 497.5 µl of NHS, and 5 µl of aminated aptamer (0.05 mM) were added to the glass substrate on which AuNP-TCDA was fixed, and then incubated for 3 hours to bind amide. As a result, the aptamer was bound to the TCDA. Then, the substrate was washed three times with deionized water and dried with an inert nitrogen gas to prepare a color conversion sensor for detecting a target material according to Example 1 of the present invention.

Example 2: Color conversion sensor for detecting a target substance (TBA-AuNP-TCDA)

A color conversion sensor according to Example 2 of the present invention was manufactured through the same process as Example 1 of the present invention, except that 300 μl of 213 mM cysteamine hydrochloride was used.

Example 3: Color conversion sensor for detecting a target substance (TBA-TCDA-AuNP)

① Preparation of AuNP (13 nm AuNP)

_{After adding 2 ml of 50 mM HAuCl 4} to 98 ml of distilled water, the temperature is increased, and then 10 ml of 38.8 mM sodium citrate is added, followed by stirring at high speed for 40 minutes. A red 13 nm AuNP was prepared.

② Preparation of TCDA liposome

In Example 1 of the present invention, TCDA liposomes were prepared by performing the same process as the manufacturing process of TCDA liposomes.

③ Fixing AuNP and TCDA on the substrate

_{First, after plasma treatment of O 2} on a glass substrate at 50 sccm and 100 w/min power for 1 minute, 3-aminopropyltriethoxysilane (APTES) and It was immersed in a mixed solution of ethanol for 1 hour to prepare a glass substrate surface-modified with an amine group.

Then, in order to attach the AuNP by reacting the 13 nm AuNP prepared above with a glass substrate surface-modified with an amine group, and to attach a TCDA liposome to the AuNP attached to the substrate, TCDA 1 mM, EDC 1 mM and 1 mM NHS was mixed and stirred for 1 hour to prepare a mixed solution. The substrate to which the AuNP was attached was immersed in the mixed solution for 1 hour to fix AuNP and TCDA on the substrate.

④ Functionalization of TCDA

The substrate on which the AuNP and TCDA prepared above were immobilized was _{immersed in a solution of 1 mM EDC, 1 mM NHS, and 0.05 mM aptamer modified with NH 2} for 4 hours, thereby binding the aptamer to the TCDA. . Then, the substrate was washed three times with deionized water and dried with an inert nitrogen gas to prepare a color conversion sensor for detecting a target material according to Example 3 of the present invention.

A color conversion sensor according to Example 3 of the present invention was manufactured.

Example 4: Color conversion sensor for detecting a target substance (TBA-TCDA-AuNP)

① Preparation of AuNP (30 nm AuNP)

1.42 mM HAuCl ₄ 22.8 µl, 213 mM Cysteamine hydrochloride 400 µl and 40 ml of deionized water were mixed to prepare a first mixed solution, and then in a room-temperature atmosphere in an environment where light was blocked. After stirring for 20 minutes, 10 mM NaBH ₄ 10 ul solution was additionally added to the stirred second mixed solution and stirred for 60 minutes to prepare a second mixed solution, and then stored refrigerated at 4° C. to obtain 30 nm AuNP. Was prepared.

② Preparation of TCDA liposome

In Example 1 of the present invention, TCDA liposomes were prepared by performing the same process as the manufacturing process of TCDA liposomes.

③ Fixing AuNP and TCDA on the substrate

Except that 30 nm AuNP was used, the same process as the manufacturing process of the TCDA liposome was performed in Example 3 of the present invention, and the AuNP and TCDA were fixed on the substrate.

④ Functionalization of TCDA

In Example 3 of the present invention, the same process as that of Au-TCDA functionalization was performed to perform TCDA functionalization, and a color conversion sensor according to Example 4 of the present invention was manufactured.

Experimental Example 1: Evaluation of properties

In order to evaluate the characteristics of the TBA-AuNP-TCDA color conversion sensor manufactured according to Example 1 of the present invention, first, the unit before combining the TBA in the TBA-AuNP-TCDA color conversion sensor manufactured according to Example 1 (“ AuNP-TCDA”) was obtained using a UV-visible spectrophotometer (V-770, JASCO), and the results are shown in FIG. 2A.

Referring to FIG. 2A, it can be seen that the AuNP-TCDA (black curve) before UV polymerization has a peak at 525 nm. AuNP-TCDA (red curve) after UV polymerization can confirm peaks at 525 nm, 580 nm and 640 nm, whereas TCDA without AuNP (blue dotted line) shows peaks only at 580 and 640 nm.

Through this, it can be seen that the AuNPs immobilized on the TCDA surface did not change the AuNP during the TCDA polymerization process in the UV polymerization of TCDA.

In addition, the UV-vis spectrum of TBA-AuNP-TCDA after TBA was introduced into AuNP-TCDA was obtained using an ultraviolet visible light spectrophotometer (V-770, JASCO), and the results are shown in FIG.

Referring to FIG. 3, TBA-AuNP-TCDA after introducing TBA into AuNP-TCDA shows a slight red-shift from 525 nm to 528 nm, indicating successful modification of TBA in TCDA.

Experimental Example 2: Evaluation of properties according to the amount of AuNP fixed on the surface of TCDA-1

In order to evaluate the characteristics according to the amount of AuNPs immobilized on the TCDA surface with respect to the sensitivity of the color conversion sensor for detecting the target substance of the present invention, various concentrations of AuNPs for TCDA were tested. The AuNP fixation on the TCDA surface was performed through the EDC/NHS reaction, and taking into account that the EDC/NHS reaction is active at pH 8.5-9.5, the EDC/NHS reaction environment pH was controlled by changing the amount of TEA. The addition of TEA induced the reaction environment from a basic pH to an acid pH environment. The three representative concentrations of the AuNP solution 6.6 μM, 20 μM and 26.5 μM are the degree of AuNP fixed to TCDA according to TEA (“Low-conc”, “Medium-conc” and “High concentration ( High-conc)” (named AuNP). For each of the color conversion sensors manufactured according to the amount of AuNP fixed on the TCDA surface, structural characteristics were evaluated through Uv-vis spectrum and SEM image. The structural characteristic evaluation was performed using an ultraviolet visible light spectrophotometer (V-770, JASCO) and a field emission scanning electron microscope (JSM-750F, JEOL, Glass coverslip (18mm x 18mm)), and the results are shown in FIGS. It is shown in 2c.

Referring to FIG. 2B, it can be seen that the absorption band intensity at 525 nm increases as the pHrk of the EDC/NHS reaction environment increases. That is, it can be seen that the amount of AuNP fixed on the TCDA surface increased according to the adjustment of the TEA concentration.

In addition, referring to FIG. 2C, it can be seen that the number of AuNPs attached to the TCDA surface is smaller in the Low-TCDA AuNP compared to the Midium-conc AuNP. In the case of High-conc AuNP, compared with Low-TCDA AuNP and Midium-conc AuNP, only AuNP can be confirmed, and it can be confirmed that it is difficult to confirm the structure of TCDA liposome. The structure of the liposome of High-conc AuNP can be expected to shrink and distort due to the pH change of the reaction environment and the attachment of the liposome on the solid substrate.

Experimental Example 3: Evaluation of properties according to the amount of AuNP fixed on the surface of TCDA-2

For each of the color conversion sensors manufactured according to the amount of AuNPs immobilized on the TCDA surface, in order to evaluate the sensitivity of each color conversion sensor depending on the amount of AuNPs immobilized on the TCDA surface, color transition conduction was quantified and performed. Thrombin was used as the target material, and after thrombin was added, the colorimetric response (CR, %) of each sensor was at 543 nm and 640 nm in _{the absence (PB 0} ) and presence (PB _{1) of thrombin, respectively.} It was calculated through the following equation 1 by determining the change in the absorbance measurement ratio of. PB ₀ is the blue wavelength region, and PB ₁ is the degree of change of _{PB 0} after thrombin addition.

<Equation 1>

Referring to Equation 1, as described above, the higher the ratio of CR, the more effective color conversion from blue to red. The results of the sensitivity evaluation are shown in FIG. 4.

Referring to FIG. 4, in (a), when thrombin is added to a color conversion sensor including TCDA to which TBA is not bound, it can be seen that the absorbance is increased. This increase in absorbance can be expected to be highly likely due to the non-specific binding of thrombin occurring under the negative charge of the alkyl tail group of TCDA liposomes. On the other hand, in (b), it can be seen that the color conversion sensor including TCDA to which TBA is bound has a slight response to thrombin. It can be seen that the relatively insignificant response of the color conversion sensor to such contact of the target substance (thrombin) is because the color conversion sensor is based on a solid phase approach. This approach typically indicates a lack of interliposomal repulsion and a low concentration of TCDA compared to the liquid approach.

On the other hand, in (c), in the case of the color conversion sensor (Low-conc AuNP) in which low concentrations of AuNP and TBA are combined on the TCDA surface, it can be seen that the absorbance response of the color conversion sensor to the addition of the target substance (thrombin) is slightly improved. have. In comparison, in the case of the color conversion sensor (Medium-conc AuNP) in which an intermediate concentration of AuNP and TBA was combined on the TCDA surface by adjusting the pH of the reaction solution in (d), a color transition change greater than that of Low-conc AuNP appeared. Can be confirmed. However, in (e), in the case of a color conversion sensor (High-conc AuNP) in which a high concentration of AuNP and TBA are combined on the TCDA surface by adjusting the pH of the reaction solution, it can be seen that the absorbance decreases.

Through this, it can be seen that the color conversion sensor in which too much AuNP is fixed to the TCDA worsens the detection response. This can be expected that when an excessive amount of AuNPs are fixed on the TCDA surface and AuNPs are attached to each other or exist in close proximity to each other, since the repulsion of the TCDA backbone is suppressed, the sensitivity is remarkably reduced. Therefore, it can be seen that it is important to adhere at an appropriate amount of AuNP concentration that does not interfere with the distortion of the backbone of TCDA. In addition, it can be seen that the mechanical stress on TCDA of AuNP and the plasmonic effect of AuNP affect the sensitivity of the color conversion sensor of the present invention.

In (f), it can be seen that the Middle-conc AuNP has a CR 2.5 times higher than that of a color conversion sensor including TCDA to which TBA without AuNP is bound. If the amount of AuNP fixed to TCDA does not interfere with the distortion of the TCDA backbone, it can be confirmed that CR is most effective in Middle-conc AuNP.

Experimental Example 4: Influence of AuNP size and adjacent location on TCDA

First, through each of the color conversion sensors manufactured according to Examples 1 and 2 of the present invention, the influence of the size of AuNP on TCDA on the target material was evaluated. The evaluation was performed under different concentrations (10, 510, 100 and 1000 ng/ml) of the target substance thrombin, and ultraviolet visible light spectrophotometer (V-770, JASCO), field emission scanning electron microscope (JSM- 750F, JEOL, Glass coverslip (18mm x 18mm)) and colorimetric response (CR, %) were used, and the results are shown in FIGS. 5 and 6.

First, referring to FIG. 5, (a) 27.8 nm-AuNP in the solution in Example 1 was found to have 525 nm, and (c) 8.1 nm-AuNP in the solution in Example 2 had 535 nm. Able to know. In addition, (b) it can be seen that the average diameter of 27.8 nm-AuNP in the solution in Example 1 is 27.8±5.0, and (d) the average diameter of 8.1 nm-AuNP in the solution in Example 2 is 48.1±5.0. I can confirm that.

Referring to FIG. 6, in the case of the color conversion sensor (Medium-conc AuNP (27.8 nm)) manufactured according to Example 1, than the color conversion sensor (Medium-conc AuNP (48.1 nm)) manufactured according to Example 2). It can be seen that the value of CR is small. This is similar to the case of the color conversion sensor including the high concentration AuNP bound to the TCDA identified above, it was found that the large size AuNP tends to interfere with the morphological distortion of the TCDA backbone in the target blister binding.

In addition, through each of the color conversion sensors prepared according to Examples 3 and 4 of the present invention, the adjacent position of the AuNP to the TCDA with respect to the target material was evaluated. The evaluation was performed using a colorimetric response (CR, %), and the results are shown in FIG. 6.

6, in the case of the color conversion sensor (13nm-AuNP monolayer) manufactured according to Example 3, it was confirmed that the value of CR was higher than that of the color conversion sensor (30nm-AuNP monolayer) manufactured according to Example 4 I can. In addition, the 13nm-AuNP monolayer had a higher CR value than that of Midium-conc AuNP (27.8nm) and Midium-conc AuNP (48.1nm), showing the highest CR reaction to the target material.

Through this, it can be seen that the TBA-TCDA-AuNP color conversion sensor of the present invention enables higher plasmon enhancement than the TBA-AuNP-TCDA color conversion sensor. That is, it can be seen that the color conversion sensor in which AuNP is fixed on the substrate exhibited a higher plasmon effect than the color conversion sensor in which AuNP is fixed on the surface of the TCDA liposome. These results can be expected to be due to two reasons. First, the first reason is that AuNPs were uniformly distributed on the glass substrate, and the second reason is that the mass of AuNPs decorated on the surface of TCDA liposomes is the structure of TCDA. This is because it can hinder the degree of deformation. In conclusion, it can be seen that the size and position of the TCDA-based AuNP play an important role in the sensitivity of the TCDA-based color conversion sensor.

The color conversion sensor for detecting a target substance based on TCDA of the present invention can be applied to molecular diagnosis of a portable color conversion sensor. In addition, the present invention for improving the sensitivity to the color-forming polymer liposome can further promote the development and change of a biological sensing platform.

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 (14)

A substrate surface-modified with an amine functional group;
Tricosadiynoic acid (TCDA) liposomes immobilized on the amine functional group;
Plasmonic metal particles immobilized on the surface of the TCDA liposome; And
Containing; an aptamer that specifically binds to a target substance bound to the surface of the TCDA liposome
Color conversion sensor to detect target substances.
The method of claim 1.
When the sensor is exposed to a sample containing the target material, the target material applies mechanical stress to the TCDA liposome due to a binding reaction with the aptamer to cause color conversion of the sensor,
Color conversion sensor to detect target substances.
The method of claim 1,
The plasmonic metal particles are spaced apart at regular intervals so as not to contact each other on the surface of the TCDA liposome, and the surface of the TCDA liposome is partially exposed,
Color conversion sensor to detect target substances.
The method of claim 1,
The plasmonic metal particles are surface-modified with an amine functional group,
Color conversion sensor to detect target substances.
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 sensor to detect target substances.
The method of claim 1,
The aptamer comprises an amine or thiol functional group,
Color conversion sensor to detect target substances.
The method of claim 1.
The target material is characterized in that the receptor is present in thrombin, DNA, enzyme, virus and cell membrane protein,
A color conversion sensor that detects a target substance,
The method of claim 1.
The target substance is thrombin,
The aptamer is a DNA oligonucleotide,
A color conversion sensor that detects a target substance,
A substrate coated with plasmonic metal particles;
TCDA liposomes immobilized on the plasmonic metal particles; And
Containing; an aptamer that specifically binds to a target substance bound to the TCDA liposome
Color conversion sensor to detect target substances.
The method of claim 9.
When the sensor is exposed to a sample containing the target material, the target material applies mechanical stress to the TCDA liposome due to a binding reaction with the aptamer to cause color conversion of the sensor,
Color conversion sensor to detect target substances.
The method of claim 9,
The plasmonic metal particle is characterized in that any one selected from aluminum, copper, silver, platinum and gold particles,
Color conversion sensor to detect target substances.
The method of claim 9,
The aptamer comprises an amine or thiol functional group,
Color conversion sensor to detect target substances.
The method of claim 9.
The target material is characterized in that the receptor is present in thrombin, DNA, enzyme, virus and cell membrane protein,
A color conversion sensor that detects a target substance,
The method of claim 9.
The target substance is thrombin,
The aptamer is a DNA oligonucleotide in which a receptor is present,
A color conversion sensor that detects a target substance,

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