KR101090340B1 - Nanohybrid-driven Optical DNA Identification System - Google Patents

Abstract

The present invention relates to a system for encoding and reading information using a gene, and specifically, an optical gene using an optical gene code identification composition comprising a nanocomposite in which an optical gene with a phosphor is encapsulated with a cationic nanomaterial. A code identification system. The optical genetic code identification system of the present invention can be constructed as a discrimination system that provides information with high reliability and that cannot be damaged or duplicated in authenticity determination and history tracking.

Optical Genetic Code Identification System, Optical Synthetic Gene, Cationic Nanomaterial, Nanohybrid, Biosensor

Description

Nanohybrid-driven Optical DNA Identification System using Nanohybrid Technology

The present invention relates to a system for encoding and reading information using genes.

In general, barcode is the most commonly used information and encryption code recognition technology. Bar codes are black and white bar symbols that combine letters or numbers and are used to enter and read data quickly. Watermark is a code system that is often used to determine the authenticity of banknotes.Biometric recognition technology uses fingerprint authentication to extract and fingerprint different fingerprint information for each person, and uses eye iris information with unique characteristics for each person. There is an iris recognition. In addition, a radio frequency identification system, which is a contactless recognition system that transmits and processes information of objects and main environmental information using a small semiconductor chip, is also a code recognition system. Code and cryptosystems are not globally standardized, and they may have difficulty in identifying and proving authenticity due to damage, duplication, and forgery, and may cause unexpected human rights violations.

These problems can be overcome by molecular technology. Invisible micro-sized nano-identifiers have the advantage that they are not easily detected because they are evenly detected by the object, and can be tampered with and not replicated or counterfeited. As the molecular level code, the most appropriate is to use the nucleotide sequence of a gene as an information unit. The nucleotide sequence of a gene can contain all the genetic information of a living organism, which is very efficient and can be widely used worldwide. There is a criterion to judge, and the reliability is very high. Therefore, intentionally gene synthesis technology, design have been studied are actively in the field of bioinformatics, medical diagnostics, forensic science with a detection method that can explore multiple combination in a very small amount (Condon A, Nat. Rev. Genet. 7: 565-575, 2006). Inorganic nanoparticles are also used in long-lasting labeling technology because of their robustness and complex information coding (Han M, Nat. Biotechnol. 19: 631-635, 2001). In line with this, micro-electromechanical systems (MEMS) or nano-electromechanical systems (NEMS), which can detect single molecules quickly and accurately, miniaturize all pretreatment and analysis steps for genetic samples such as dilution, mixing, reaction, separation, and quantification. Advances in the development of lab on a chip or lab on a disk that can be performed in biosensors (Harold C, Nature 442: 387-393, 2006; Cho YK et al . , Lab Chip 7: 565-573, 2007).

Recently, a nano DNA barcode system using a DNA base pair sequence as a code unit of information has been proposed (Korean Patent No. 10-0849465; Choy JH, Adv . Mater . , 16, 1181-1184, 2004). The system inserts DNA between the layers of inorganic nanoparticles, stabilizing DNA that can be easily destroyed and modified by various factors (enzyme, pH, heat) when exposed to the external environment, and polypyrrole-margeite After collecting DNA using (polypyrrole-Maghemite), PCR (Polymerase chain reaction) method was used as a technique for reading the DNA information code. However, the system has several problems in terms of practical use. 1) There is no probe component in the DNA code that enables field detection; 2) there is a weakness of the synthesis method that cannot encapsulate inorganic nanoparticles even large DNA molecules; 3) There are limitations such as the use of PCR method which requires long time consumption and small amount of DNA. Therefore, in order for DNA to be actually used as a practical identification system such as a barcode, a breakthrough in the generation of DNA information codes and an analysis method thereof is required.

Among bio-active molecules, genes, or DNAs, are ideal molecules containing various information, and they can design base sequences and structures to have new functions, and thus design synthetic genes with organic or inorganic substances, diagnosis and treatment of disease-causing gene expression. Research has been actively conducted in the fields of synthetic chemistry, biotechnology, and medicine where genes are applied, such as treatment through intracellular delivery and detection of very small amounts of macromolecules. However, since the gene itself can be easily destroyed and modified by various factors (enzyme environment, chemical, physical environment, etc.) when exposed to the external environment, there is a limit to the effectiveness without proper stabilization measures.

Therefore, in order to overcome the unstable problems of the gene itself and to increase the storage, storage, or delivery effect, layered inorganic nanoparticles LDH (Layered double hydroxide) as a inorganic carrier that can stabilize the gene has received considerable attention. The research on this has also continued. One of the metal double layer hydroxides, called LDH, is called anionic clays, which consist of a layer of positively charged metal hydroxides and anion and water that will offset this positive charge between layers.

Until now, intercalation chemistry of ion exchange reaction and coprecipitation reaction was used to support bioactive molecules in the LDH, and as a result, layered bio-inorganic nanocomposites were prepared. The gene-LDH nanocomposite in which the gene is inserted into the LDH is also introduced by the strong electrostatic attraction between the LDH layers having the cationic layer charge through the ion exchange reaction and the coprecipitation reaction. A bio-inorganic nanocomposite having a two-dimensional layered structure was prepared. Genes encapsulated and stabilized in inorganic nanoparticles as described above are not denatured even in the enzymatic environment, and under acidic conditions, LDH is reversibly easily dissociated and functions are gradually exerted as the gene is gradually released (Korea Patent No. 10-0359716). No. Choy JH et al ., J. Am . Chem. Soc . 121 : 1399-1400, 1999; Choy JH et al ., Angew . Chem . Int . Ed . 39: 4041-4045, 2000; Korean Patent No. 10-0849465; Choy JH, Adv . Mater . , 16, 1181-1184, 2004).

However, LDH, which is a layered inorganic nanoparticle introduced to stabilize bioactive molecules such as conventional genes; Conventional ion exchange methods and coprecipitation methods for preparing two-dimensional multilayer bio-inorganic nanocomposites or gene-LDH nanocomposites have the following problems. 1) Since the interlayer spacing of LDH has a limit of expansion, it is possible to carry genes of three-dimensional huge or specific structure such as plasmid gene, gene with nanoparticle with quantum effect, gene with organic phosphor attached, etc. Can not; 2) the length or size of genes that can be stabilized depending on the particle size of LDH even if inserted between LDH layers (Oh JM., J. Phys . Chem . Solids . 67: 1028-1031, 2006); 3) It is very difficult to carry anionic genes to which organic and inorganic substances are attached and contain some positive charges; 4) Genetic-LDH nanocomposite of a single phase (Single phase) there is an economic problem that takes an excessive gene and a long time in the chemical reaction step.

Thus, the present inventors

1) the genes of a specific base sequence to which phosphors having different fluorescent properties are attached are encapsulated with exfoliated metal double layer hydroxide (LDH), thereby stably storing the gene;

2) primary reading is possible without destroying the nanocomposite by detecting fluorescence of the media or medium containing the opto-LDH nanocomposite;

3) by secondary reading the genetic code preserved in the nanocomposite using a biosensor;

The present invention has been completed by confirming that the optical genetic code identification system of the present invention can be constructed as a discrimination system that provides information with high reliability and cannot be damaged or duplicated in authenticity determination and history tracking.

It is an object of the present invention to provide a composition for optical gene code identification.

It is an object of the present invention to provide an ink for an optical genetic code identification system comprising the composition and paint.

It is another object of the present invention to provide a label for an optical genetic code identification system containing the composition.

Another object of the present invention is to provide a biosensor for optical gene code identification.

Another object of the present invention is to provide a code identification method using the label and the biosensor.

In order to achieve the above object, the present invention

i) a shell, the nanomaterial having a cationic charge; And,

ii) a core having a sense single-stranded optical gene labeled with a primary phosphor and a nucleotide sequence complementary to the sense single-stranded optical gene, and labeled with a secondary phosphor having a different fluorescence property than the primary phosphor Double-stranded optical genes formed by complementary binding of antisense single-stranded optical genes;

It provides an optical genetic code identification composition comprising a nanocomposite consisting of.

The present invention also provides an ink composition for an optical genetic code identification system comprising the composition and paint.

The present invention also provides a label for an optical genetic code identification system comprising the composition.

In addition,

(i) a sense single strand gene probe in which base sequences complementary to the sense single strand optical gene are sequentially connected; or,

(ii) a sense single stranded gene probe sequentially linked to a base sequence complementary to the antisense single stranded optical gene;

Provides a biosensor for optical gene code identification, dipped in a solid substrate.

In addition,

1) detecting fluorescence of the label for the optical genetic code identification system;

2) if the fluorescence is not detected, it is determined as a forgery; if fluorescence is detected, treating the label with an acidic solution to decompose the capsule formed of the exfoliated metal double layer hydroxide;

3) extracting only the optical gene from the label in which the capsule was digested;

4) denature the extracted optical gene, and hybridize with the biosensor;

5) washing the hybridized biosensor to remove unbound optical genes;

6) after drying the washed biosensor, detecting an optical image; And,

7) provides a code identification method using the biosensor for identifying the optical genetic code, comprising the step of analyzing the optical image detected in step 6) to determine whether the forgery or genuine.

Hereinafter, the present invention will be described in detail.

The present invention

i) a shell, the nanomaterial having a cationic charge; And,

ii) a core having a sense single-strand optical gene labeled with a primary phosphor at the 5 'end and a base sequence complementary to the sense single strand optical gene, and having different fluorescence properties at the 5' end than the primary phosphor A double-stranded optical gene formed by complementarily binding an antisense single-stranded optical gene labeled with a secondary phosphor;

It provides an optical genetic code identification composition comprising a nanocomposite consisting of.

In a specific embodiment of the present invention, single-stranded genes do not form hairpin loops or self dimers, and have different colors at 5 'ends of complementary sequences of about 35 bp in length. Fluorescent double-stranded optical genes were designed and synthesized. Specifically, the single-stranded optical gene (Cy3-A) described in SEQ ID NO: 1 of 35 bp in length with a green phosphor attached to the 5 'end; And a single stranded optical gene (Cy5-B) described in SEQ ID NO: 2, complementary to SEQ ID NO: 1, to which a red phosphor was attached, was hybridized to synthesize a double stranded optical gene.

By adding the single-stranded optical gene to a layered LDH (Layered Double Hydroxide) nanoparticle colloid solution exfoliated in formamide, an optical gene inorganic capsule having a nanohybrid identifier was synthesized. At this time, the length of the gene is not limited to 35 bp, it may be a length of about 10 to 3000 bp. When encapsulated 2500 bp or 35 bp long genes with carriers of LDH exfoliated on formamide (see Fig. 2), they formed a gene-LDH nanocomposite of amorphous or core-cell structure (Figs. 3 and 4). Stability of the external environment (see FIG. 7), thereby enabling storage, delivery and increased function of the gene. In addition, the gene labeled with the phosphor at the 5 'end was also encapsulated by LDH exfoliated in formamide (see FIGS. 5 and 6), and it was confirmed that the gene was stably stored (see the right panel of FIG. 7B). As described above, the optical gene-LDH nanocomposite having a stable capsule structure can be secured with excellent dispersibility in any medium, thereby maximizing the application field and maintaining stability in the long term / periodically.

When the nanomaterial encapsulates an optical gene having an anionic charge, it is preferable to have a cationic charge, and the nanomaterial having a cationic charge may be a polymer, a biodegradable polymer, liposomes, and inorganic nanoparticles. . The inorganic nanoparticles may be silica or cationic metal, and the like, and it is preferable to use cationic layer charge exfoliated metal double layer hydroxide (LDH), and cationic layer charge exfoliated LDH. Recombination through self-assembly due to anionic optical genes and strong electrostatic attraction can maximize stability by encapsulating optical gene molecules.

Specifically, the exfoliated LDH is a precursor (pristine) of the layered LDH of the formula (1),

_{^{[M 2 + (1-x}} ) N 3 + x (OH) 2] [A n -] z and yH ₂ O

(In the formula 1,

M ^{+ 2} is a divalent metal cation,

N ^{3 +} is a trivalent metal cation,

A is an anionic guest species,

n is the number of charges of the anion of A,

x is a number greater than 0.1 and less than 0.4,

z and y are positive numbers greater than zero).

M is any one selected from the group consisting of magnesium (Mg ^{2 +} ), nickel (Ni ^{2 +} ), copper (Cu ^{2 +} ) and zinc (Zn ^{2 +} ),

N is characterized in that any one selected from the group consisting of aluminum (Al ^{3 +} ), iron (Fe ^{3 +} ), vanadium (V ^{3 +} ), titanium (Ti ^{3 +} ) and gallium (Ga ^{3 +} ).

The single-stranded gene can be used as long as it is designed not to form self hairpin loops and self dimers, and the base sequence has a length of 10 to 3000 bp wide. Ranges are also available. However, since the nanocomposite composed of the double-stranded optical gene of the present invention is preferably 'nano' size that cannot be discerned by the eye, the gene is 10 to 10 to prepare a complex corresponding to the nanounit (generally 500 nm). It is preferably about 1500 bp, more preferably 15 to 1000 bp, even more preferably 20 to 500 bp, most preferably about 25 to 300 bp. For reference, in the case of the double-stranded gene, 1 bp is 0.34 nm, so the length of the 1500 bp gene is 510 nm, and in general, a probe having a length of at least 10 bp or less is not produced because it is not selective.

However, in order to apply to the genetic code identification system, 10 to 300 bp is preferred, more preferably 20 to 200 bp, most preferably 25 to 50 bp.

The double stranded gene may also have the form of a hybrid of several short single stranded genes and a long single stranded gene. Complementary sequences of several short-length single-stranded genes can be used to encode unique, multi-stranded genes with secondary and tertiary structures, and plasmid and engineered DNA can also be informed. have. In addition, RNA, PNP (Peptide Nucleic Acid), which is an artificial DNA analog, may be used. Such multiple encodings can design and produce a vast amount of information / password codes using a combination code of a specific base sequence and a structure code, such as the structure / color / number of phosphors.

The phosphor may be used as long as it is stable from external conditions such as temperature, pH change, heat, and the like, and organic phosphor or inorganic phosphor having a quantum effect may be used. Specific examples of the organic phosphor include CY ^TM , Fluorescein, Alexa Flur ^® , Bodipy ^® , CAL Fluor ^® , Oregon Green, Oyster ^® , Rhodamine, TAMRA ^TM and WellRED, and inorganic phosphors are also gold nanoparticles. , ZnS, CdSe, CdTe, CdS and various, and can be attached to the 5 ', 3' or in the middle by using a direct covalent bond or spacer (Spacer). At this time, the phosphor itself can be used as a unit of the code, and the combination of the primary phosphor and the secondary phosphor to have different fluorescence properties (eg, different components, structures and colors) is possible to design a variety of codes, ① in the phosphor The light emitting optical signal can be used to determine whether the nanocomposite composed of the optical gene is inserted; Or (2) can also serve as molecular sensor elements and probes used to read sequences of optical genes.

The spacer may further contain a spacer between the nucleotide sequence of the double-stranded optical gene and the phosphor, and the spacer may be any molecule capable of linking the gene and the phosphor without affecting hybridization of the optical gene, for example, C- 3 linker, C-6 linker, C-6 TFA linker, C-5 amino modifier, C-12 linker, amino dT C2 linker, amino dT C6 linker, 5 ′ Thiol C- 2 linker, 5 'thiol C-6 linker, 5' thiol C-6 SS and the like.

The nanocomposite composed of the double-stranded optical gene of the present invention can stably store information and optical probes encoded in the nucleotide sequence, which can be applied to security technology, product identification technology, authenticity identification technology, traceability system, anti-counterfeiting technology, and the like. It can be usefully used as a nanohybrid identifier. Specifically, the composition of the present invention i) ID card, passport, etc., easy to counterfeit and duplicate security products; ii) high-priced products with the threat of theft, such as authentic and luxury goods; iii) products requiring quality control in the distribution process where product history can be tampered with; It is preferable to disperse and apply physically and chemically to various media and media, such as these. For example, the composition of the present invention is mixed with machine readable materials, micro tracers, new materials for coins, special inks, pigments, paints, paints, etc., for the manufacture of security products. Labeling by using it, dispersing and dispersing it, inserting it directly into special printing security paper, special paper for paper, durable papers, and applying it directly to objects and packaging by physical and chemical adsorption. Can be used.

The present invention also provides an ink composition for an optical genetic code identification system comprising the composition and paint.

The paints include special inks for preventing forgery and alteration, commercial inks, pigments, dyes and paints, and the like.

The present invention also provides a label for an optical genetic code identification system comprising the composition.

The composition may be applied to the label, added during the manufacture of the label, or printed on the label. Examples include ① dispersing in a solvent and applying it to a label, ② directly injecting the product into a paper or the like, and ③ mixing a paint or a paint.

In addition,

(i) an antisense single stranded gene probe sequentially linked to a base sequence complementary to the sense single stranded optical gene; or,

(ii) a sense single stranded gene probe sequentially linked to a base sequence complementary to the antisense single stranded optical gene;

Provides a biosensor for optical gene code identification, dipped in a solid substrate.

The composition of the present invention can be read first through the detection of phosphors. In a specific embodiment of the present invention, the nanohybrid identifier is mixed with black ink, and then the nanohybrid ink including the nanohybrid identifier is stamped with a [S] letter on a glass slide, respectively, to obtain an image with an optical scanner. As a result, [S] stamped with pure ink did not contain a fluorescent material (see FIG. 8), and [S] stamped with nanohybrid ink showed a yellow [S] image in which green and red phosphors were merged. (See Figure 9). Thus, the detection of the yellow [S] image, it was confirmed that the nanohybrid identifier can be read first without removing the nanocapsules.

In addition, the nanohybrid identifiers of the present invention can be read secondary through detection of hybridization reactions with the gene and complementary gene sequences. In a specific embodiment of the present invention, a biosensor having a base sequence complementary to the optical gene and comprising 12 thymines attached with an amine at the 5 'end was properly loaded with a probe gene. At this time, by appropriately adjusting the spot positions of the two types of optical genes, the read image is designed to be a red ⓢ graphic on a green background. Subsequently, the hybridization reaction was performed by extracting only the optical gene from the glass slide in which the nanohybrid ink was stamped with the [S] letter. As a result, an optical genetic code was quickly and precisely obtained by obtaining a red ⓢ graphic on a green background as an optical image. It could be read (see FIG. 10).

Accordingly, the optical genetic code identification system of the present invention can be constructed as a highly accessible identification system for authenticity determination and history tracking by going through two-step reading process of multi-color optical detection and gene code reading.

In the present invention, a "biosensor" used as a reader is similar to a biochip utilizing a unique function of a biomolecule. Biosensors are manufactured by integrating, arranging, and attaching thousands or tens of thousands of genes at high density onto the surface of glass slides on solid substrates. It is a revolutionary way to effectively replace the existing "PCR technology" for detecting and analyzing them.

The biosensor was modified by using an Aldehyde (-CHO) on a glass slide in a microarray format and combining a probe gene probe having an amine (Amine; NH ₂ ) attached thereto.

The spacer may further contain a spacer between the probe and the solid substrate of the present invention, which may be any molecule capable of linking the probe and the solid substrate without affecting hybridization, eg, amine-oligo dT Linker, C-3 linker, C-6 linker, C-6 TFA linker, C-5 amino modifier, C-12 linker, amino dT C2 linker, amino dT C6 linker, 5 'Thiol C-2 linker, 5' thiol C-6 linker, 5 'thiol C-6 SS and the like.

The solid substrate allows for optical detection and does not severely interfere with signal emission. The solid substrates include, but are not limited to, plastic materials, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, teria sol, carbon graphite, titanium dioxide, latex or cross-linked dextran such as sepharose, cellulose, Nylon, crosslinked micelles and Teflon can all be used. The surface of the solid substrate can be modified to allow attachment of the probe at individual spots. Specifically, the addition of patterns of chemical functional groups such as amino groups, carboxyl groups, oxo groups and thiol groups can generally be used to covalently link probes comprising the corresponding reactive functional groups or linker molecules. In a specific embodiment of the present invention, the surface of the substrate was modified with aldehyde so that probes prepared with amine groups readily bound to the substrate.

The biosensor of the present invention can be made with spots in which probes are deposited at very high, high, medium, low or very low density. The range for very high-density biosensors is from about 10,000,000 to about 2,000,000,000 spots per biosensor. The range of high-density biosensors has from about 100,000 to about 10,000,000 spots. The medium density biosensor ranges from about 10,000 to about 50,000 spots. Low-density biosensors are generally less than 10,000 sites. Very low-density biosensors have less than 1,000 spots. Probe spots may include a pattern, that is, a regular design or configuration, or may be randomly distributed. Regular pattern spots can be used to be specified on the X-Y coordinate plane. Therefore, the biosensor for optical genetic code identification is designed to randomly designate the position where the complementary nucleotide sequences of the optical genetic code are hybridized in advance, so that the reading result is read as an optical graphic image.

In addition,

1) detecting fluorescence of the label for the optical genetic code identification system;

2) if the fluorescence is not detected, it is determined as a forgery; if fluorescence is detected, treating the label with an acidic solution to decompose the capsule formed of the exfoliated metal double layer hydroxide;

3) extracting only the optical gene from the label in which the capsule was digested;

4) denature the extracted optical gene, and hybridize with the biosensor;

5) washing the hybridized biosensor to remove unbound optical genes;

6) after drying the washed biosensor, detecting an optical image; And,

7) provides a code identification method using the biosensor for identifying the optical genetic code, comprising the step of analyzing the optical image detected in step 6) to determine whether the forgery or genuine.

Step 2) The capsule decomposition is preferably dissolved for 2 to 6 hours, preferably 5 hours using an acidic buffer solution of pH 4 or less. After dissolving only the metal double layer hydroxide selectively so that the genetic code is not modified, EDTA (Ethylene diamine tetra acetic acid) can be added to easily remove the capsule and safely extract the optical genetic code. When double-stranded genes are dissolved in a strong acid solution for a long time, structural modification occurs as the protonation of base pair arrays (A, T, C, G) occurs. In the presence of a metal cation, the base pair of the double-stranded gene and the metal cation are transformed into a single strand, or they are collected by forming a complex by interacting with a phosphate group to make the gene unstable in an insoluble form (Muntean CM et. al . , J. Raman Spectrosc . 36: 1047-1051, 2005; Duguid J et al . , Biophys . J. 65: 1916-1928, 1993). Therefore, by selectively dissolving only the metal bilayer hydroxide in an acidic buffer solution for a suitable time, and then adding EDTA, a chemical known to bind strongly to divalent / 3 divalent metal cations to form chelate compounds, the metal cations dissolved and formed in the acidic solution It can be easily removed by forming chelates so that optical genes can be safely extracted from the threat of modification / degeneration.

In the step 4), hybridization of the biosensor and the optical gene was performed for "1 second" to obtain a hybridized result image. Through this, the complementary genes were hybridized at one moment to emit a fluorescence signal. Through the presence or absence of an image detection, it is possible to determine whether or not the information is matched by visually confirming / recognizing. Therefore, the biosensor of the present invention not only functions as a multi-color image code in which a biological signal is converted into an optical signal and transmitted when the optical genetic code is accurately and quickly matched with a previously given information / password. Without any additional explanation, the reader can easily and simply recognize the reading information in the blink of an eye and use it to intuitively determine whether the match is intuition.

The optical genetic code identification system of the present invention can be constructed as a discrimination system that provides information with high reliability and that cannot be damaged or duplicated in authenticity determination and history tracking.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the present invention, the contents of the present invention is not limited to the following examples.

< Example  1> Giant Length (~ 2500 bp Nanocomposite synthesis of genes

<1-1> Exfoliated  metal Double layer  Synthesis of Hydroxide

A 100 nm metal bilayer hydroxide was synthesized by coprecipitation and hydrothermal synthesis. 0.3 M Mg (NO ₃ ) ₂ · 6H ₂ O and 0.15M Al (NO ₃ ) ₃ · 9H ₂ O are dissolved in tertiary distilled water from which carbonates have been removed, and 0.5 M NaOH until the pH is between 10 and 11. After titration, the mixture was vigorously stirred at room temperature for 30 minutes to obtain metal layered double hydroxide (LDH) crystals. The co-precipitated LDH was placed in a hydrothermal synthesis vessel, stirred at 100 ° C. for 12 hours, separated by a centrifuge, and the unreacted salt was removed by washing to obtain LDH with a uniform size of 100 nm. The synthesized LDH was dispersed in formamide at 0.05% by weight, and vigorously stirred at room temperature for 2 days to obtain exfoliated LDH nanosheets. The whole process was carried out in a nitrogen atmosphere to prevent the formation of carbonate ions (CO ₃ ^{2 −} ) by carbon dioxide in the air.

<1-2> huge length (~ 2500 bp Encapsulation of genes

50 to 2500 bp double-stranded herring gene (Herring testis DNA) was mixed in a weight ratio of 1: 1 to the exfoliated LDH colloidal solution in Example 1-1, and stirred at room temperature for 1 hour. After completion of the reaction, the centrifuge was separated and the excess gene was completely removed by washing to obtain a core-cell gene-bilayer hydroxide nanocomposite (FIG. 2A). The whole process is a carbonate ion (CO3 ^{2 -)} according to the carbon dioxide in the air it was carried out in a nitrogen atmosphere in order to prevent the generation.

< Example  2> Synthesis of Nanocomposite Encapsulated Gene with Phosphor

<2-1> Optical Gene Coding

Single stranded optical gene (Cy3-A) attached with a green phosphor, cyanine 3, described by SEQ ID NO: 1 [5 '-(Cy3) ATT CGG ACG CTC ATC ATT CTG GTG TGG CTG CTG GC-3'], 35 bp in length ); And a single-stranded optical gene described by SEQ ID NO: 2 [5 ′-(Cy5) GCC AGC AGC CAC ACC AGA ATG ATG AGC GTC CGA AT-3 ′], to which the red phosphor cyanine 5 is attached and complementary to SEQ ID NO: 1 After designing (Cy5-B), the single-stranded optical gene was synthesized by requesting Integrated DNA Technologies (USA). At this time, each single-stranded optical gene was prepared such that hairpin loops or self dimers were not formed. Single-stranded optical genes were dissolved in STE buffer (10 mM Tris-HCl, pH 8.0, 50 nM NaCl, 1 mM EDTA), mixed at the same concentration, heated at 94 ° C for 2 minutes, and then cooled slowly at room temperature. Was synthesized with the optical gene.

<2-2> optical gene LDH  Nanocomposite Synthesis

After the exfoliated LDH colloidal solution was added to the optical gene solution prepared in Example 2-1 (mass ratio = 0.5 to 1.5, boolean = 1 to 2), the resultant was stirred at 15 ° C for 1 hour for the optical gene as a nanohybrid identifier. Inorganic capsules were synthesized (FIG. 2B). The whole process was carried out under a nitrogen atmosphere to prevent the formation of carbonate ions (CO ₃ ^{2 –} ) by carbon dioxide in the air.

< Example  3> Characterization of Nanocomposites

<3-1> X-ray diffraction  analysis

X-ray diffraction (Rigaku, D / Max 2200, Japan) analysis was performed to confirm the crystal structure of the LDH and gene-LDH nanocomposites prepared in Examples 1 and 2.

As a result, as shown in FIG. 3, the interlayer spacing of LDH was 0.79 nm, and it was confirmed that the nitrate ion (NO ₃ ) is a typical structure, and the gene-LDH nanocomposite has a low angle of 2θ <15 °. Since there is no diffraction, it can be seen that the gene-LDH nanocomposite has an amorphous structure instead of the nanocomposite of the multi-layered structure.

<3-2> transmission electron microscope analysis

Transmission electron microscopy (JEOL JEM-2000EXII, Japan) analysis was performed to finely check the shape and crystal structure of the LDH and gene-LDH nanocomposites prepared in Examples 1 and 2. Each specimen was fixed to epoxy resin, cut to 50 nm thickness, and observed without staining.

As a result, it was confirmed that the LDH was a hexagonal particle having a uniform size of about 100 nm and a structure in which nitrate ions (NO ³ ) were inserted at an interlayer spacing of about 0.8 nm (FIG. 4A).

The large gene-LDH nanocomposite is a disordered encapsulation of a very thin exfoliated LDH nanosheet in a few μm size, thereby forming a core-cell nanocomposite having an amorphous structure close to the shape of a house of card. It was confirmed (FIG. 4B).

The optical gene-LDH nanocomposite formed an inorganic cavity about 10 nm thick as the exfoliated LDH nanosheets were recombined, and the gene-LDH nanocomposite having a perfect core-cell structure of about 150 nm size in which the gene was encapsulated therein. It was confirmed that (Fig. 4c).

< Example  4> Optical Gene LDH  Confirm encapsulation of nanocomposites

The optical genes, exfoliated LDH and optical gene-LDH nanocomposites prepared in Examples 1 and 2 were identified by transmission electron microscopy and ultraviolet spectrometer (Perkin Elmer Lambda 35, USA).

As a result, as shown in FIGS. 5 and 6, it was confirmed that the optical genes labeled with green (Cyanine 3) and red (Cyanine 5) phosphors were encapsulated with the carrier.

< Example  5> Gene LDH  Stability Evaluation Experiment of Encapsulation of Nanocomposite

The stability of the gene preserved in the gene-LDH nanocomposites prepared in Examples 1 and 2 was examined by an enzyme treatment reaction.

10 μg of the gene-LDH nanocomposite was dispersed in 10 μl of TE (Tris-EDTA) buffer solution, and then 20 μl of DNAase, DNase I (1000 unit), and the reaction buffer (200 mM Tris-HCl, pH). 8.3 μl, 20 mM MgCl ₂ ) was added and incubated at 37 ° C. for 1 hour. Thereafter, 20 µl of the reaction stopping buffer (50 mM EDTA) was added, incubated at 70 ° C. for 10 minutes, and centrifuged to remove the enzyme and the buffer.

After the enzymatic reaction was dispersed in hydrochloric acid buffer solution of pH 4, and dissolved for 5 hours at room temperature to selectively decompose only inorganic capsules. EDTA was then processed to minimize the interaction between metal cations and genes, thus extracting only pure optical genes. In the case of the optical gene, 5 µg of the extracted optical gene was dissolved in 15 µl of nuclease-free hybridization buffer solution (30% formamide, 5X SSC, 0.1% SDS, poly A), followed by heating at 95 ° C for 5 minutes. Made with strand optical genes. The extracted genes were electrophoresed on 1% or 5% agarose gel, respectively, and then scanned by ultraviolet and optical image analyzer (Typhoon 9400, USA) to obtain electrophoretic images.

As a result, it was confirmed that the large gene-LDH nanocomposite is protected from the DNA degrading enzyme to the gene of 50 to 2500 bp (FIG. 7A).

In addition, it was confirmed in the ultraviolet image (left panel of FIG. 7B) and the optical image that the optical gene-LDH nanocomposite was stably protected 35 bp of gene from DNA degrading enzyme (right panel of FIG. 7B).

Through this, it was confirmed that the core-cell gene-LDH nanocomposite of the present invention can protect genes and phosphors from DNA degradation conditions regardless of length and structure.

< Example  6> Nanohybrid Ink Preparation and First Reading Test

The nanohybrid identifier prepared in Example 2 was added to black stamp ink (Shachihata, Japan) at 1% by weight and dispersed to obtain a nanohybrid ink. Pure stamp inks and nanohybrid inks were stamped on the glass slides with the letter [S], respectively, and the images were scanned with an optical scanner (Typhoon 9400, USA).

As a result, it was confirmed that [S] stamped with pure ink did not contain fluorescent material (FIG. 8), and [S] stamped with nanohybrid ink showed a yellow [S] image in which green and red phosphors were merged. (FIG. 9). Thus, the detection of the yellow [S] image, it was confirmed that the nanohybrid identifier can be read first without removing the nanocapsules.

< Example  7> Manufacture of biosensor

A 47 bp long probe gene SEQ ID NO: 3 having nucleotide sequences complementary to the single-stranded optical genes Cy3-A and Cy5-B of Example 2, each comprising 12 thymines attached to an amine at the 5 'end A 'and B' described in SEQ ID NO: 4 were prepared. After modifying the glass slide surface with aldehyde, a biosensor similar to the conventional method was manufactured by dropping a total of 144 spots (12 × 12) in an area of 49 mm 2. At this time, one type of probe gene was used for one spot, and by appropriately adjusting the spot positions of the two types of probe genes, the read image was designed to have a red ⓢ graphic on a green background (see FIG. 10).

< Example  8> Optical genetic code readout

After the nanohybrid ink prepared in Example 6 was treated with acid buffer solution (pH 4) at room temperature for 5 hours on a glass slide stamped with [S], only pure optical genes were extracted by treating EDTA.

5 μg of the extracted optical gene was dissolved in 15 μl of nuclease-free hybridization buffer solution (30% formamide, 5X SSC, 0.1% SDS, poly A) and heated to 95 ° C. for 5 minutes to form a single stranded optical gene. .

15 μl of the solution containing the single-stranded optical gene was added to the biosensor prepared in Example 4, followed by hybridization for 1 second, and immediately after the first to third washing (first: SSC and SDS; second: SSC). And SDS; tertiary: SSC). The washed biosensors were dried and scanned with an optical scanner (Molecular Devices GenePix 4200B, USA).

As a result, as shown in FIG. 10, an optical gene was extracted from the nanohybrid identifier and a red ⓢ graphic was obtained on a green background with an optical image read by a biosensor, thereby enabling the optical gene code to be read quickly and accurately. Accordingly, the optical genetic code identification system of the present invention can be constructed as a highly accessible identification system in authenticity determination and history tracking.

1 is a flowchart showing the overall process of the present invention.

2 is a schematic diagram of the formation structure of the gene-LDH nanocomposite of the present invention:

a: macrogene-LDH nanocomposite; And,

b: optical gene-LDH nanocomposite.

Figure 3 is an X-ray diffractogram of the gene-LDH nanocomposite:

a: LDH;

b: macrogene-LDH nanocomposites; And,

c: optical gene-LDH nanocomposite.

4 shows a transmission electron microscope image of a gene-LDH nanocomposite:

[Left panel: morphology, right panel: cross-section analysis]

a: LDH;

b: macrogene-LDH nanocomposites; And,

c: optical gene-LDH nanocomposite.

5 shows transmission electron microscopy images of optical gene-LDH nanocomposites:

a: optical gene;

b: exfoliated LDH; And,

c: optical gene-LDH nanohybrid inorganic capsule.

6 is a diagram showing the ultraviolet spectrum results of the optical gene-LDH nanocomposites:

a: optical gene;

b: exfoliated LDH; And,

c: optical gene-LDH nanohybrid inorganic capsule.

Figure 7 is an electrophoretic image showing the results of the stability evaluation for the enzyme of the gene-LDH nanocomposite:

a: macrogene-LDH nanocomposite; And,

b: optical gene-LDH nanocomposite.

8 is an image of [S] stamped with pure ink:

a: photo; And,

b: optical image.

9 is an image of [S] stamped with nanohybrid ink to which optical gene-LDH nanocomposites were added:

a: photo; And,

b: optical image.

10 is a diagram showing the results of reading the optical gene code with the optical gene code identification system of the present invention.

<110> Ewha University-Industry Collaboration Foundation <120> Nanohybrid-driven Optical DNA Identification System <130> 9P-02-27 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Cy3-A <400> 1 attcggacgc tcatcattct ggtgtggctg ctggc 35 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Cy5-B <400> 2 gccagcagcc acaccagaat gatgagcgtc cgaat 35 <210> 3 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> A ' <400> 3 tttttttttt ttgccagcag ccacaccaga atgatgagcg tccgaat 47 <210> 4 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> B ' <400> 4 tttttttttt ttattcggac gctcatcatt ctggtgtggc tgctggc 47  

Claims (18)

i) As a shell, a peeled metal double layer hydroxide (LDH) with a cationic layer charge; And, ii) a core having a sense single-stranded optical gene labeled with a primary phosphor and a nucleotide sequence complementary to the sense single-stranded optical gene, and labeled with a secondary phosphor having a different fluorescence property than the primary phosphor Double-stranded optical genes formed by complementary binding of antisense single-stranded optical genes; Optical genetic code identification composition comprising a capsule-type nanocomposite consisting of. The composition of claim 1, wherein the optical genetic code identification is any one selected from the group consisting of security, product identification, authenticity determination, traceability, and counterfeiting. delete The method of claim 1, wherein the phosphor is an organic phosphor selected from the group consisting of CY ^TM Dyes, Fluorescein, Alexa Flur, Bodipy, CAL FluorDyes, Oregon Green Dyes, OysterDyes, Rhodamine Dyes, TAMRA ^TM Dyes and WellRED Dyes. Composition. The composition of claim 1, wherein the phosphor is an inorganic phosphor selected from the group consisting of gold nanoparticles, ZnS, CdSe, CdTe, and CdS. The composition of claim 1, wherein the gene has a length of 10 to 1500 bp. The composition of claim 1, wherein the gene comprises Plasmid DNA, Engineered DNA, RNA, and Peptide Nucleic Acid (PNP). delete delete The method of claim 1, wherein the exfoliated metal double layer hydroxide (LDH) is a precursor of the layered metal double layer hydroxide (LDH) of the formula (1), [Formula 1] [M ²⁺ _(1-x) N _{3 + x (} OH _{) 2] [} A _{n-] z} yH ₂ O (In the formula 1, M ²⁺ is a divalent metal cation, N ³⁺ is a trivalent metal cation, A is an anionic guest species, n is the number of charges of the anion of A, x is a number greater than 0.1 and less than 0.4, z and y are positive numbers greater than zero). M is any one selected from the group consisting of magnesium (Mg ²⁺ ), nickel (Ni ²⁺ ), copper (Cu ²⁺ ) and zinc (Zn ²⁺ ), N is any one selected from the group consisting of aluminum (Al ³⁺ ), iron (Fe ³⁺ ), vanadium (V ³⁺ ), titanium (Ti ³⁺ ) and gallium (Ga ³⁺ ). . The composition of claim 1, further comprising a spacer between the nucleotide sequence of the optical gene and the phosphor. An ink composition for an optical genetic code identification system, comprising the composition of claim 1 and a paint. The ink composition according to claim 12, wherein the coating material is selected from the group consisting of special inks for preventing forgery and alteration, commercial inks, pigments, dyes and paints. Label for an optical genetic code identification system comprising the composition of claim 1. The label of claim 14, wherein the composition is included on the label, added to the label, or printed on the label. (i) an antisense single stranded gene probe sequentially linked to a base sequence complementary to the sense single stranded optical gene according to claim 1; or, (ii) a sense single-stranded gene probe in which sequential sequences complementary to the antisense single-stranded optical gene according to claim 1 are sequentially connected; Biosensor for optical genetic code identification, in which the chemical is deposited on a solid substrate. 1) detecting fluorescence of the label for the optical genetic code identification system of claim 14; 2) if the fluorescence is not detected, it is determined as a forgery; if fluorescence is detected, treating the label with an acidic solution to decompose the capsule formed of the exfoliated metal double layer hydroxide; 3) extracting only the optical gene from the label in which the capsule was digested; 4) denaturing the extracted optical gene, and hybridizing with the biosensor of claim 16; 5) washing the hybridized biosensor to remove unbound optical genes; 6) after drying the washed biosensor, detecting an optical image; And, 7) Code identification method using the biosensor for identifying the optical genetic code of claim 16, comprising the step of determining whether the optical image detected in step 6) is forgery or genuine. 18. The method of claim 17, wherein step 3) optical gene extraction is performed by adding EDTA to form chelates of metal cations.

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