KR102352999B1 - Novel fluorescent material, nanobead comprising the same and diagnostic kit using the same - Google Patents

Abstract

The present invention relates to a novel fluorescent material, nanobeads containing the same, and a diagnostic kit using the same.
The novel fluorescent compound of the present invention includes azide groups at both ends of the compound, and has excellent fluorescence sensitivity.

Description

Novel fluorescent material, nanobead comprising same, and diagnostic kit using same

The present invention relates to a novel fluorescent material.

Specifically, the present invention relates to a fluorescent material that can be used in a diagnostic device such as a point-of-care diagnostic device.

The in vitro diagnostic device market is a market that is expected to grow continuously due to various factors such as changes in health and medical trends, the expansion of target countries, an aging population, and new infectious diseases. Main areas include immunochemical diagnosis, self-diagnosis, point-of-care, and molecular diagnosis. Among them, the point-of-care diagnostic device is mainly used for the purpose of wanting quick results or an environment that is not equipped with facilities for an emergency site or diagnosis. In particular, the technology is being developed so that there are no errors even if it is performed by general inspectors, not skilled personnel, including inspection subjects that require quick inspection results, and the trend is expanding to other subjects.

Types of on-site diagnostic devices include heart disease biomarkers, drug addiction tests, and test devices for diagnosing sexually transmitted diseases, such as pregnancy diagnosis, cholesterol levels, and blood gas concentrations. Most of these devices are expensive foreign products, and domestic products are used only for urine tests, some rapid tests, and immune tests.

In the case of gold nanoparticles used as a representative labeling material in existing point-of-care diagnostic devices, they have disadvantages of high price and low sensitivity. To overcome this, various fluorescent materials such as organic dyes, quantum dots, and carbon dots have been applied as materials to replace gold nanoparticles. However, in the case of these materials, since the Stokes shift, which is the difference between the positions of the maximum values of the absorption and emission spectra of the electron transition, is small, it is difficult to distinguish the detection signal from the background noise, making it difficult to diagnose.

Accordingly, the inventors of the present invention completed the present invention while conducting intensive research in order to overcome the above problems occurring in the existing diagnostic kits.

An object of the present invention is to provide a novel fluorescent material having a large excitation-emission shift value.

Another object of the present invention is to provide a fluorescent material capable of excellently improving fluorescence sensitivity and exhibiting a high-sensitivity detection signal even in a low concentration sample, and a diagnostic material using the same.

To this end, the present invention according to one aspect provides a compound represented by the formula (1).

[Formula 1]

N ₃ -[A]-N ₃

The present invention according to another aspect provides a nano-bead comprising the compound.

The present invention according to another aspect provides a diagnostic kit including the nano-beads.

The present invention according to another aspect provides a method for providing data for medical diagnosis, comprising measuring the fluorescence intensity of the nano-beads.

Since the compound of the present invention has a large excitation-emission shift, the use of the compound of the present invention can improve the difficulty of detection caused by background noise caused by various substances dissolved in blood.

In addition, by inserting a large amount of the compound into the nanobead, and then immobilizing an antibody that selectively binds to an antigen, signal amplification can be generated. As a result, it is possible to detect a sample with a small concentration, thereby exhibiting superior diagnostic sensitivity compared to a conventional fluorescent material-based diagnostic device.

Accordingly, it is possible to show the effect of contributing to on-site diagnosis of various diseases and prevention of the spread of diseases with rapidity and accuracy, economical efficiency and convenience of use.

The following drawings attached to this specification illustrate preferred embodiments of the present invention, and serve to further understand the technical spirit of the present invention together with the above-described content of the invention, so the present invention is limited to the matters described in those drawings It should not be construed as being limited.
1 is a schematic diagram of the configuration of a simple test (Lateral Flow Assay, LFA) diagnostic kit according to an embodiment of the present invention.
2 is a schematic diagram of a synthesis route of a fluorescent compound according to an embodiment of the present invention.
3 is an NMR graph of a fluorescent compound synthesized according to an embodiment of the present invention.
4 is a schematic diagram of a method for manufacturing a diagnostic kit according to an embodiment of the present invention.
5 is an image as a result of confirming fluorescence on the LFA membrane according to an embodiment of the present invention.
6 is an image of the result of confirming the fluorescence of LFA using the nanobead containing the fluorescent compound according to an embodiment of the present invention according to the concentration of the fluorescent compound contained therein.
7 is a schematic diagram of a device that can be used to provide data for medical diagnosis using nanobeads according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention according to one aspect provides a compound represented by the formula (1).

[Formula 1]

N ₃ -[A]-N ₃

The compound may include an azide group (-N ₃ ) at both ends, and the two azide groups act as a fluorophore for generating a fluorescence signal.

A is a structure for separating the two azide groups of Formula 1 so that they are not directly bonded, C ₁ to C ₁₀₀ alkyl, a substituted or unsubstituted C ₁ to C _{80 containing at least one hetero atom} heteroalkyl, substituted or unsubstituted C ₂ to C ₈₀ alkenyl, substituted or unsubstituted C ₂ to C ₈₀ alkynyl, substituted or unsubstituted C ₁ to C ₁₀₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ to C ₁₀₀ haloalkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted amide, carbamate, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, It may include a structure selected from substituted or unsubstituted aralkyl, ketone, aldehyde, ester, polyalkylene oxide and acyl chloride.

In one embodiment, A may include multiple resonance structures within the structure.

The fluorescence sensitivity of the compound may be more excellently improved by including a multiple resonance structure in A so that the electron excitation-emission shift value is large.

In one embodiment, the azide group (-N ₃ ) of Formula 1 may have a non-conjugated structure with A.

The electron movement space may be expanded by including a multi-resonance structure of A by including a structure conjugated with the azide group of Formula 1, but in the present invention, A is a non-covalent group with the azide group of Formula 1 Having a liquefied structure may be preferable in terms of improving fluorescence sensitivity by allowing the electron excitation-emission shift value to be large.

In one embodiment, A may be represented by the formula (2).

[Formula 2]

In Formula 2, the R ¹ and R ² are C ₁ to C _20, each the same or different and each independently represent a substituted or unsubstituted C ₁ to C ₂₀ alkyl, at least one hetero atom substituted heteroalkyl, Substituted or unsubstituted C ₂ to C ₂₀ alkenyl, substituted or unsubstituted C ₃ to C ₂₀ alkynyl, substituted or unsubstituted C ₄ to C ₂₀ cycloalkyl and C substituted with at least one hetero atom ₁ to C ₂₀ selected from heterocycloalkyl,

wherein X ¹ and X ² are the same or different from each other, and are each independently selected from hydrogen, -OR ³ , -NR ³ ₂ , -COOH, -COH, R ⁴ and a halogen group, wherein R ³ is independently of each other hydrogen , substituted or unsubstituted C ₁ to C ₂₀ alkyl, at least one hetero atom-substituted C ₁ to alkenyl of C ₂₀ heteroalkyl, substituted or unsubstituted C ₂ to C ₂₀ a and a substituted or unsubstituted C ₃ to selected from alkynyl of C _20, R ⁴ is independently a substituted or unsubstituted C ₁ to heteroalkyl, substituted or unsubstituted of C ₂₀ alkyl, at least one hetero atom-substituted C ₁ to C ₂₀ of each other substituted C ₂ to C ₂₀ alkenyl, substituted or unsubstituted C ₃ to C ₂₀ alkynyl, substituted or unsubstituted C ₄ to C _{20 cycloalkyl, and C 1} to C ₂₀ substituted with at least one hetero atom of heterocycloalkyl, wherein Y ¹ and Y ² are the same or different, and each independently -CO, -SO ₂ , O ₃ , and -N ₃ may be selected from.

The compound was newly designed under a push-pull system based on an intramolecular donor-π-conjugation-acceptor structure.

The compound may be nonionic and may consist of a single benzene platform. In particular, the compound is characterized in that it can exhibit excellent light stability and efficient fluorescence in the blue range (about 400 nm) in both the solution state and the solid state. In addition, the compound is characterized by a large Stokes mutation.

Specifically, the compound may exhibit desirable characteristics as a fluorescent molecule for use in bioimaging. For example, because the emissive properties of the compound in the solid state help prevent self quenching, and in general, the Stokes shift helps prevent reabsorption of emitted photons, so the compound can be used for fluorescence imaging can enable high contrast in

In one embodiment, the compound may be represented by Formula 3.

[Formula 3]

Specifically, the compound represented by Formula 3 may act as a very strong donor group having a high HOMO due to the 1,4-diamino benzene unit. In addition, although the mechanism of the present invention is not limited thereto, the compound may exhibit very strong fluorescence intensity due to a push-and-pull system between amino and sulfonyl groups supported by hydrogen bonds within and between molecules.

The present invention according to another aspect provides a nano bead (Nano bead) comprising the above-described compound.

As used herein, the term “nano-beads” refers to beads having a nano-sized particle size. Specifically, the nano-beads have an average particle diameter (D50) of 1 μm or less, and may refer to beads having a size of 150 to 200 nm.

In one embodiment, the nano-beads containing the fluorescent compound may be introduced into a living body and used to detect a detection target. In this case, in consideration of the ease of introduction into the living body of the nano-beads, the nano-beads may be, for example, 150 to 200 nm.

The nanobeads may be formed using a material such as a single molecule, a metal, or a polymer. For example, the nanobead may be a polymer material to contain a large amount of the fluorescent compound without a separate binder.

In one embodiment, the nano-beads may be formed by embedding the compound in a polymer matrix.

The nano-beads made of the polymer material have an advantage in that they can contain a large amount of fluorescent material due to the swelling property of the polymer. For example, when the fluorescent compound is mixed with a solution containing polymer beads, a large amount of the fluorescent compound may be embedded in the polymer matrix while the polymer swells. Accordingly, the fluorescence sensitivity can be excellently improved by binding the antibody to nanobeads containing a large amount of fluorescent material, compared to the case in which a conventional 1:1 binding of a fluorescent material and an antibody is used in a diagnostic kit.

The polymer matrix is not limited thereto, for example, polyolefin-based polymers such as polystyrene, polymethyl methacrylate, polyethylene terephthalate (PET), high density polyethylene (HDPE), polyvinyl alcohol ( Polyvinyl alcohol, PVA), bio polyethylene (Bio-PE), bio polyethylene terephthalate (Bio-PET), bio polytrimethylene terephthalate (Bio-PTT), bio polyamide (Bio-PA), bio polypropylene (Bio) -PP), polylactic acid (PLA), thermoplastic starch (TPS), aliphatic polyester (AP), polycaprolactone (PCL), polyglycolic acid, PGA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), polyhydroxy alkanoate (PHA) and mixtures thereof There may be more than one type.

In one embodiment, the polymer matrix may include polystyrene.

The nano-beads may be formed by embedding the compound in a polymer matrix, wherein the nano-beads contain 0.01 to 10 mg/mL of the compound (fluorescent material), such as 0.1 to 5 mg/mL or 0.25 mg/mL to 2 It may be formed by mixing with the polymer matrix in a solution having a concentration of mg/mL.

The present invention according to another aspect provides a diagnostic kit comprising the above-described nano-beads.

In one embodiment, for use in various diagnostic kits, the nano-beads may be bound to an antibody to a detection target.

The antibody can be bound according to a known method for binding an antibody to an antibody-binding substance, and the method is not particularly limited. For example, the antibody may be functionalized with a carboxyl group on the surface of the nanobead and bound by an amide bond using an EDC-NHS coupling reaction with an amine group in the antibody.

The object to be diagnosed using the nano-bead is, for example, diseases such as tsutsugamushi, malaria, myocardial infarction (CK-MB (creatin kinase)), infection with viruses such as Zika, influenza, diagnosis of premature birth, etc., However, the present invention is not limited thereto.

The present invention according to another aspect provides a method of providing data for medical diagnosis using the above-described nano-beads.

The method of providing the data may be to use an apparatus including a light source and a filter illustrated in FIG. 7 .

As the light source, a light in a wavelength band capable of excitation of electrons is used so that the fluorescence exhibited by the nano-beads, specifically, the fluorescence exhibited by the nano-beads in the presence of a target material is expressed.

As the filter, a filter capable of removing background fluorescence in addition to the fluorescence to be detected is used.

Specifically, the method may include performing a test using a sample obtained from a subject using the nano-beads or a diagnostic kit including nano-beads bound to the nano-beads with an antibody to the substance to be diagnosed. can

The method includes irradiating light to the diagnostic kit using the light source after performing the test. When light is irradiated to the diagnostic kit including the nano-beads, emission of fluorescence, a detection target from which background fluorescence is removed through a filter, can be induced.

Next, the method includes obtaining an image of the emitted fluorescence when the fluorescence is emitted. For example, obtaining the fluorescence image may be obtaining an image of emitted fluorescence using a mobile phone camera.

At this time, by measuring the intensity of fluorescence from the image, by substituting the measured fluorescence intensity into the fluorescence intensity-antigen amount data prepared using a standard reagent, it is possible to quantify the diagnostic object (antigen) in the subject. can

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention.

[Example 1 - Preparation of Fluorescent Compound]

According to the synthetic route shown in FIG. 2, a fluorescent compound was prepared as follows.

1. Preparation of compound 1b

First, sodium dissolved in 15 mL of methanol was added to 500 mg of 2,5-diamine-1,4-dithiolbenzene dihydrochloride (Compound 1a, 2,5-diamino-1,4-benzenedithiol dihydrochoride). and stirred at room temperature for 30 minutes. After the addition of 187 mg of 1-azido-3-iodo-propane (1-azido-3-iodo-propane), the mixture was refluxed for 5 hours. The reaction was terminated with an aqueous solution of 10 wt% NaOH (10 mL) and filtered with methanol. 298 mg of 1,4-diamine-2,5-(3-azidopropylsulfide)benzene (1,4-diamine-2,5-(3-azidopropylsulfide)benzene) was obtained. (Compound 1b, yield 30%).

2. Preparation of compound 1c

A mixture of 100 mg of compound 1b and 0.41 mL triethyl amine, 0.28 mL acetic anhydride and dichloromethane (CH ₂ Cl ₂ , 10 mL _{) was stirred under nitrogen (N 2)} at room temperature for 24 hours. The reaction was then quenched with water (10 mL) and the precipitated product was filtered with water (20 mL). The residue was dried in vacuo and the filtrate _{was extracted with CH 2} Cl ₂ (20 mL), washed with water (10 mL) and brine (10 mL) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure to give compound 1c as colorless crystals (125 mg, 63%).

3. Preparation of compound 1d

A mixture of 78 mg of compound 1c and CH ₂ Cl ₂ (10 mL) was stirred at room temperature. Then, 319 mg of a colorless suspension of m-chloroperbenzoic acid was added and stirred for 32 hours. The resulting mixture _{was quenched with saturated Na 2} SO ₃ (aq) (10 mL), and dichloromethane (CH ₂ Cl _{2 )} was removed under reduced pressure. (Compound 1d, 90 mg, 100% crude).

4. Preparation of compound 1

To 90 mg of compound 1d was added THF (10 mL) and 6M NaOH (aq) (15 mL), and the mixture was stirred at reflux for 16 h. THF was removed under reduced pressure. The precipitated product was filtered with water (20 mL). The residue was purified by recrystallization from acetonitrile to give as pale yellow crystals. (Compound 1, 63 mg, 85%).

^{3 shows 1} H-NMR results of Compound 1 of Example 1 prepared above.

[Example 2 - Preparation of nano-beads]

According to the manufacturing method of the nano-beads shown in Figs. 4 (1), (2), nano-beads were prepared as follows.

First, polystyrene (PS) beads (average particle diameter (D ₅₀ ) 150 mm) and 0.15 mL of 2% Pluronic F-127 were mixed in 0.15 ml of distilled water. 1 mg/mL of the fluorescent compound of Example 1 was added to this solution. The THF in the solution was evaporated while stirring the solution on a 55°C hotplate for 8 hours so that the fluorescent compound was supported in the swollen PS beads. Next, the supernatant was removed by centrifugation and washed to obtain PS beads carrying a fluorescent compound.

[Example 3 - Preparation of diagnostic kit]

According to the manufacturing method of the diagnostic kit (LFA) shown in FIG. 4(3), a diagnostic kit was prepared as follows.

The carboxyl (-COOH) group functionalized on the surface of the fluorescent nanobead of Example 2 prepared above and the amine (-NH ₂ ) group on the surface of the antibody were formed using EDC-NHS coupling reaction to form amide bonding.

After binding the antibody in this way, coating/dry was applied to the conjugate pad (GFDX203000, Glass fiber diagnostic pad, Company: Millipore). Nitrocellulose membrane (HF135MC100, Company: Millipore)) is dotted with control line (Anti-Mouse IgG antibody produced in goat) and test line, respectively, and the remaining pads (absorbance pad (Company: Millipore), sample pad (Company: Millipore), conjugate pad) to prepare an LFA diagnostic kit.

In the test line, each malaria antibody produced in mouse or CK-MB antibody was used, and the experiment was performed using antigen/antibody reaction against malaria and CK-MB, respectively.

[Experimental Example 1 - Detection of Malaria in Blood Using LFA Membrane]

0.25 mg/mL of the fluorescent material of Example 1, 0.005 mL of blood, and flow buffer were applied on the membrane of the LFA diagnostic kit prepared above, and the fluorescence was detected using a UV lamp, and the results are shown in FIG. shown.

5, it was confirmed that there is no effect of auto-fluorescence emitted from the blood itself, and the quenching does not occur even when the fluorescent material meets the buffer.

[Experimental Example 2 - Evaluation of Fluorescence Sensitivity According to Concentration of Fluorescent Material]

According to the preparation method of Example 2, nanobeads were prepared so that the fluorescent compound of Example 1 was contained at concentrations of 0 μg, 1 μg, 100 ng, 10 ng, 1 ng, 100 pg, 10 pg and 1 pg, respectively. prepared.

The results of checking the fluorescence sensitivity according to the method of Experimental Example 1 after preparing an LFA diagnostic kit according to the preparation method of Example 3 using each of the prepared nanobeads are shown in FIG. 6 .

6, when using the compound of Example 1, a small amount, for example, 1 ng/mL to 100 pg/mL of a target material (antigen) can be detected when using fluorescent nano beads, developed through this study It was confirmed that the used diagnostic kit can exhibit excellent fluorescence sensitivity.

Claims (12)

As a compound represented by Formula 1,
[Formula 1]
N ₃ -[A]-N ₃
In Formula 1, A is a compound represented by Formula 2 below:
[Formula 2]

(In Formula 2,
R ¹ and R ² are the same as or different from each other, and independently of each other are substituted or unsubstituted C ₁ to C ₃ alkylene,
X ¹ and X ² are each independently —NH ₂ ,
Y ¹ and Y ² are each independently —SO ₂ —).
delete delete delete The method according to claim 1,
The compound is a compound represented by the following formula (3).
[Formula 3]

Nanobeads comprising the compound according to any one of claims 1 and 5.
7. The method of claim 6,
The nano-beads are nano-beads formed by embedding the compound in a polymer matrix.
8. The method of claim 7,
The polymer matrix is polystyrene, polymethyl methacrylate, polyethylene terephthalate (PET), high density polyethylene (HDPE), polyvinyl alcohol (Polyvinyl alcohol, PVA), bio polyethylene (Bio-PE), Bio-polyethylene terephthalate (Bio-PET), bio-polytrimethylene terephthalate (Bio-PTT), bio-polyamide (Bio-PA), bio-polypropylene (Bio-PP), poly lactic acid (PLA), Thermoplastic starch (TPS), aliphatic polyester (AP), polycaprolactone (PCL), polyglycolic acid (PGA), polybutylene succinate (PBS) ), polybutylene adipate terephthalate (Poly butylene adipate terephthalate, PBAT), polyhydroxyalkanoate (polyhydroxyalkanoate, PHA), and nano-beads comprising at least one selected from the group consisting of mixtures thereof.
A diagnostic kit comprising the nano-beads according to claim 6.
10. The method of claim 9,
The diagnostic kit, wherein the nano-beads include an antibody bound thereto.
A method for providing data for medical diagnosis, comprising measuring the fluorescence intensity of the nanobeads according to claim 6 .
12. The method of claim 11,
The method of claim 1, wherein the diagnosis target is the presence or absence of at least one infection selected from the group consisting of Tsutsugamushi, malaria, Zika virus, influenza virus, myocardial infarction, and premature birth.

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