KR20130114932A - Sensor for detecting neurotransmission-related substance - Google Patents

Abstract

PURPOSE: A sensor for detecting neurotransmission-related substances is provided to have marked sensitivity in comparison with a conventional polymerase chain reaction (PCR) method. CONSTITUTION: A sensor for detecting neurotransmission-related substances comprises: a microarray chip board in which first antibodies against neurotransmission-related substances are combined; gold nanoparticles in which second antibodies against the neurotransmission-related substances and bar code DNA are combined; and a metal nano substrate including metal nano patterns in which complementary DNA (cDNA) having a sequence complementary to the bar code DNA is fixed on the surface.

Description

Sensor for Detecting Neurotransmission-related substance

The present invention relates to sensors for detecting neurotransmitter-related substances.

Dopamine is a member of the catecholamine family and plays an important physiological role in the body of animals. Dopamine is composed of a catechol structure linked to an amine group has a dihydroxyphenylalanine (dihydroxyphenylalanine) structure. In the brain, dopamine is a neurotransmitter secreted by neurons and delivered to other neurons. The human brain has five types of dopamine receptors consisting of D1, D2, D3, D4, and D5. Dopamine is produced in several areas of the brain, including black matter, ventral skin. Dopamine can be used as an intravenous drug that acts on the sympathetic nervous system, which functions to increase heart rate and blood pressure. However, dopamine cannot penetrate the blood brain barrier and therefore cannot directly affect the central nervous system as a drug.

Tyrosine Hydroxylase (TH) is the first enzyme and rate regulator in the synthesis of catecholamines in the central and peripheral nervous systems. Tyrosine hydroxylase is expressed in melanocytes, ventral epidermal region, hypothalamus, dorsal neurons of dorsal neurospheres, and non-adrenergic neurons of the alveolar and lateral epidermis, and adrenergic neurons of the brain stem. Diagnosis of tyrosine hydroxylase after cerebrospinal fluid neurotransmitter and DNA analysis in children with severe axial hypotension and motor dysfunction associated with tension dysfunction and ballistic exercise.

Conventional neurotransmitters were measured by liquid chromatography and ELISA method. In particular, the presence of a small amount in the body fluid is required to improve the detection sensitivity.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop a sensor for detecting a highly sensitive neurotransmission-related substance in order to prevent brain diseases such as Parkinson's disease. As a result, the present invention has been completed by developing a sensor for detecting a neurotransmission-related substance having an easy detection method and an excellent sensitivity compared to the conventional ELISA (Enzyme-linked Immunosorbent Assay) or liquid chromatography method.

Accordingly, it is an object of the present invention to provide a sensor for detecting neurotransmitter-related substances which has a rapid and improved detection sensitivity.

Another object of the present invention is to provide a method for detecting neurotransmission-related substances.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention provides a sensor for detecting neurotransmitter-related substances comprising:

(a) a microarray chip plate having a first antibody bound to a neurotransmitter-related substance;

(b) gold nanoparticles bound to a second antibody and a barcode DNA to the same neurotransmitter-related substance as the neurotransmitter-related substance; And

(c) a metal nano substrate having a metal nano pattern in which complementary DNA having a sequence complementary to the barcode DNA is immobilized on a surface thereof.

The present inventors have tried to develop a sensor for detecting a highly sensitive neurotransmission-related substance in order to prevent brain diseases such as Parkinson's disease. As a result, a sensor for detecting a neurotransmission-related substance having an easy detection method and an excellent sensitivity has been developed as compared with a conventional ELISA or liquid chromatography method.

As used herein, the term 'neural shear-related substance' is preferably dopamine, tyrosine hydroxylase, adrenaline, noradrenaline, glutamate, sirrotin ), γ-Aminobytyric acid, acetylcholine, DOPA dihydroxyphenylalanine decarboxylase, aromatic L-amino decarboxylase, dopamine β- Dopamine β-hydroxylase or Phenylethanolamine N-methyltransferase, and more preferably dopamine or tyrosine Hydroxylase.

The dopamine is one of the catecholamine families, and is a neurotransmitter secreted from neurons of the brain, and serves to transmit signals to other neurons, and the structure of dopamine is as follows:

The tyrosine hydroxylase catalyzes the reaction to convert the amino acid L-tyrosine into L-3,4-dehydroxyphenylalanine (L-DOPA). The L-3,4-dihydroxyphenylalanine is a precursor of dopamine, a precursor of noradrenaline (norepinephrine) and adrenaline (epinephrine), and the reaction formula is as follows:

One of the main constitutions of the present invention is a microarray chip plate incorporating a first antibody against a neurotransmission-related substance. The microarray chip plate has a form in which a first antibody that specifically binds to a neurotransmission-related substance is bound.

The microarray of the present invention is a microarray immobilized with an antibody which is not a form in which DNA or RNA commonly used in the art is immobilized on a substrate. Preferred substrates include suitable rigid or semi-rigid supports, such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.

Antibodies that can be used in the present invention are polyclonal or monoclonal antibodies that specifically bind neurotransmitter-related substances, preferably monoclonal antibodies. The antibody comprises an antigen-binding fragment of an antibody molecule as well as a complete antibody form. Antibodies to neurotransmitter-related substances of the present invention may be prepared by methods conventionally practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. . Mol Biol, 222:.. 58, can be prepared by 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D. Using Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies : A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. The polyclonal antibody can be obtained by injecting a suitable animal with a sirtuin protein antigen, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked by a disulfide bond with a heavy chain. The heavy chain constant region has gamma (gamma), mu (mu), alpha (alpha), delta (delta) and epsilon (epsilon) types and subclasses gamma 1 (gamma 1), gamma 2 ), Gamma 4 (gamma 4), alpha 1 (alpha 1) and alpha 2 (alpha 2). The constant region of the light chain has the kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

An antigen binding fragment of an antibody molecule means a fragment having an antigen binding function and includes Fab, F (ab ') 2, F (ab') ₂ and Fv. Fabs in the antibody fragment have one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (C _H1 ) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain C _H1 domain. The F (ab ') ₂ antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for producing Fv fragments with minimal antibody fragments having only heavy chain variable regions and light chain variable regions are described in PCT International Publication Nos. WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086, WO 88/09344. The double-chain Fv is a non-covalent bond, and the variable region of the heavy chain and the light chain variable region are connected to each other. The single-chain Fv generally shares the variable region of the heavy chain and the variable region of the short chain through the peptide linker Or directly connected at the C-terminus to form a dimer-like structure like the double-stranded Fv. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, a Fab can be obtained by restriction of the whole antibody to papain, and F (ab ') ₂ fragment can be obtained by cleavage with pepsin) Can be produced through recombinant DNA technology.

According to a preferred embodiment of the present invention, in order to prepare a microarray chip plate in which the first antibody to the neurotransmission-related substance of the present invention is bound, a reaction for binding the microarray chip plate and the antibody is performed.

One of the main constitutions of the present invention is gold nanoparticles in which a second antibody and barcode DNA are bound to the same neurotransmitter-related substance as the neurotransmitter-related substance. The second antibody specifically binds to the same neurotransmitter-related substance as the first antibody, and the epitopes of the neurotransmitter-associated substance to which the first and second antibodies bind are different.

The barcode DNA is a nucleic acid sequence consisting of 10-100 bases designed based on the nucleic acid sequence of the neurotransmitter-related material, preferably the first sequence or the third sequence of the sequence listing. The first sequence is a barcode DNA specific to dopamine, and the third sequence is a barcode DNA specific for tyrosine hydroxylase.

Nucleic acid sequences used in the present invention are to be interpreted to include, in addition to the above-mentioned sequences, nucleic acid sequences showing substantial identity to the nucleic acid sequences. This substantial identity is at least 80% of the phases when the nucleic acid sequence of the present invention is aligned with the maximal correspondence of any other sequence and the aligned sequence is analyzed using algorithms commonly used in the art. By nucleic acid sequence is shown that shows homology, more preferably at least 90% homology, and most preferably at least 95% homology. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv . Appl . Math . 2: 482 (1981) ; Needleman and Wunsch, J. Mol . Bio . 48: 443 (1970); Pearson and Lipman, Methods in Mol . Biol . 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc . Acids Res. 16: 10881-90 (1988); Huang et al., Comp . Appl . BioSci . 8: 155-65 (1992) and Pearson et al., Meth. Mol . Biol . 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol . 215: 403-10 (1990)) is accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to a preferred embodiment, a thiol group is synthesized at the terminal of the barcode DNA of the present invention to bind to gold nanoparticles. The thiol-group barcode DNA and gold nanoparticles are reacted at room temperature for 30 minutes in a buffer of pH 8, and dopamine (or tyrosine hydroxylase) is added thereto and reacted with vibration at 4 ° C. for 16 hours.

One of the main constitutions of the present invention is a metal nano substrate having a metal nano pattern in which complementary DNA having a sequence complementary to the barcode DNA is immobilized on a surface.

Complementary DNA immobilized on the metal nano substrate is a sequence complementary to the barcode DNA. Preferably, the complementary DNA is SEQ ID NO: 2 or SEQ ID NO: 4.

According to a preferred embodiment, a thiol group is synthesized at the 5 'position of the complementary DNA and immobilized on a metal nano substrate.

The metal which can constitute the metal nano substrate of the present invention is preferably gold (Au), silver (Ag), barium (Ba), chromium (Cr), cobalt (Co), manganese (Mn), iron (Fe). ), Nickel (Ni), copper (Cu), zinc (Zn), niobium (Nb), palladium (Pd), platinum (Pt), terbium (Tb), gadolinium (Gd), dysprosium (Dy), holmium (Ho) ), Erbium (Er), samarium (Sm) and neodymium (Nd), one or more metals, more preferably gold or silver.

According to another aspect of the present invention, the present invention provides a method for detecting a neurotransmitter-related substance comprising the following steps:

(a) a microarray chip plate to which (i) a neurotransmitter first antibody is bound, and (ii) a gold nanoparticle to which the second antibody and barcode DNA are bound to the same neurotransmitter-related material as the neurotransmitter-related material. Contacting the sample to be analyzed;

(b) separating the gold nanoparticles to which the second antibody and the barcode DNA are bound from the result of step (a);

(c) contacting the isolated gold nanoparticles with the separated second antibody and barcode DNA to a substrate having a gold nanopattern to which complementary DNA is immobilized; And

(d) analyzing the hybridization of the barcode DNA and the complementary DNA.

Since the method of the present invention uses the sensor for detecting neurotransmitter-related substances, the contents common between the two are omitted in order to avoid excessive complexity of the present specification.

Samples that can be applied to the neurotransmitter-related substance detection method of the present invention include both cell biological and clinical samples.

Serine proteases (eg, protease K), in order to separate the barcode DNA-bound gold nanoparticles from the microarray chip plate-neuronal transfer material-gold nanoparticle complexes to which the first antibody of the present invention is bound, Threonine proteases, Cysteine proteases, Aspartate proteases, Metalloproteases or Glutamic acid proteases can be carried out, preferably, protease To process K.

Whether the barcode DNA and the complementary DNA are hybridized by contacting the barcode DNA-bonded gold nanoparticles to a metal nano substrate having a complementary DNA having a sequence complementary to the barcode DNA is immobilized on a surface thereof. For analysis, it is preferably carried out using a surface enhanced Raman spectrometer.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a sensor for detecting a neurotransmission-related substance.

(b) The present invention provides a sensor for detecting a neurotransmission-related substance having a high sensitivity compared to a conventional PCR method.

(c) The present invention provides a sensor for detection having a specific result for various neurotransmission-related substances.

1 shows a sensor chip fabrication for detecting neurotransmitters.
2 shows a schematic diagram of a nanopattern technique using nanoporous alumina.
3 shows the pupil of the aluminum mast in a scanning electron microscope (SEM) image.
4 shows an SEM image of a gold (Au) nano substrate.
5 is a graph showing a surface enhanced Raman Spectroscopy (SERS) measurement of the gold nano-substrate.
6 shows SEM images of silver (Ag) nano substrates.
7 is a graph showing SERS measurements of gold nano substrates.
8 shows an absorbance graph of complementary DNA of dopamine immobilized on gold nano substrates.
9 shows Raman peaks of complementary DNA of dopamine immobilized on gold nano substrates.
10 shows Raman peaks of complementary DNA of dopamine immobilized on silver nano substrates.
FIG. 11 shows Raman peaks of complementary DNA of tyrosine hydroxylase immobilized on gold nano substrates.
12 shows Raman peaks of complementary DNA of tyrosine hydroxylase immobilized on silver nano substrates.
13A and 13B show absorbances of gold nanoparticles to which barcode DNA is bound. FIG. 13A illustrates gold nanoparticles in which barcode DNA is not bound, and FIG. 13B illustrates gold nanoparticles in which barcode DNA is bound.
14 shows the results of SERS analysis of bound barcode DNA after treatment of protease K to an antibody-bound antibody substrate-dopamine-gold nanoparticle complex.
FIG. 15 shows SEM images of metal nano substrates bound with complementary DNA immobilized on metal nano substrates and barcode DNA isolated from magnetic particle-neural transport related material-gold nanoparticle complexes.
Figure 16 shows the change in SERS peak with the concentration of tyrosine hydroxylase. As the concentration of tyrosine hydroxylase increases, the SERS peak also increases.
17 shows Raman peak upon binding of barcode DNA to complementary or non-complementary DNA bound on gold nano substrates.
FIG. 18 is a DNA sensor chip for neurotransmitter detection, consisting of a blood inlet, an antibody inlet, and a buffer solution inlet in a sample preparation step, and a microarray chip incorporating a primary antibody; hybridization of the barcode DNA and the complementary DNA The sensor chip for detecting neurotransmission was constructed through the step of analyzing whether or not.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

Nano Substrate Fabrication for DNA Sensor Fabrication

Nano-pattern substrate is produced by thermally depositing gold using nano-porous alumina and then electroplating gold to melt alumina to obtain a substrate with uniform nanorods. And silver nano-patterns were produced (FIG. 1).

The well-arranged alumina mask was prepared through a two-step anodization process using alumina having a thickness of 100 μm with a purity of 99.99%. In the first anodic mythology, a 40 V voltage was applied in a 0.3 M oxalic acid electrolyte solution using aluminum as the anode and a platinum electrode as the cathode using two electrode systems, and the temperature was maintained at 4 ° C. during the anodic oxidation.

After the first anodic oxidation, a second anodization was performed. In order to melt the barrier layer of the alumina mask having a structure at both ends, and to increase the diameter of the pupil, treatment was performed at 65 ° C. for 5 hours using 0.4 M phosphoric acid and 0.2 M chromic acid. In order to separate the aluminum layer, a second anodization process was performed in a 5 wt% phosphoric acid solution at 30 ° C. for 15 minutes to prepare an alumina mask. The prepared alumina mask was attached to an indium tin oxide (ITO) plate and then deposited. After deposition, a gold nano substrate having a constant pattern of 60-70 nm was prepared. An image of the nano substrate was observed with a scanning electron microscope (SEM).

DNA production

To detect dopamine, dopamine barcode DNA (5'-CCACACTGCCG-GATGTGGATTTAACCTTTCAGCAAATGTCTT- (A) _10- (CH ₂ ) ₃ -SH-3 '), complementary DNA (5'-SH-AAG ACA TTT GCT GAA AGG TTA) Forward primer (5'-AAG ACA TTT GCT GAA AGG TTA AAT CCA CAT-3 ') and reverse primer (5'-ATG TGG ATT TAA CCT TTC AGC AAA) for AAT CCA CAT CCG GCA GTG TGG-3') and PCR TGT CTT-3 ').

To detect Tyrosine Hydroxylase (TH), tyrosine barcode DNA (5'-TTT GGG GAA CAC CGT GGA GGG GCA TAG CAT CTC-(A) _10- (CH ₂ ) ₃ -SH-3 '), Complementary DNA (5'-SH-AAA CCC CTT GTG GCA CCT CGT ATC GTA-3 ') and forward primer (5'-GAT GGA TGC CTC ATT GGG G-3') and reverse primer (5'-) for PCR GCA GCT CCA GGC CCC CCA GC -3 ').

Fabrication of gold self-assembled microarray plate with neurotransmitter antibody

A amide, carboxyl, and aldehyde group coupling reagent using MUA (Mercaptoundecanoic Acid) was used as a self-assembled monolayer on the gold self-assembled monolayer. Secondary dopamine antibody (abcam, monoclonal, USA) was coupled to prepare a microarray chip stably bound antibody.

Second antibody binding to barcode DNA-bound gold nanoparticles

Thiol-olated oligo barcode DNA was dissolved in 50 μl 0.1 M Tris buffer and 50 μl of 0.1 N DDT (Dichlorodiphenyltrichloroethane, pH 5.2) solution was added and treated at 60 ° C. for 30 minutes. Ethyl acetate (ethyl acetate) solution was added and mixed, centrifuged, the supernatant was discarded, and ethyl acetate solution was added three times. Dry for 30 minutes in the hood. In addition, 1 ml of a gold colloidal solution (60 nm of nanoparticles) was taken and centrifuged and redispersed in 400 µl PBS solution. After adding the DDT-treated oligo DNA barcode to the PBS-containing gold nanocolloid solution, it was reacted for 2 hours at 4 ° C., bound to the dopamine 2nd antibody, and stirred for 16 hours at 4 ° C. for the oligonucleotide and The second antibody dopamine was bound. Barcode DNA was added to the solution containing the oligonucleotide having the thiol group and the gold nanoparticles bound to the antibody and allowed to stand at room temperature for 4 hours, followed by centrifugation to obtain precipitated gold nanoparticles, which were suspended in PBS and stored at 4 ° C. It was used for the experiment while.

barcode DNA Through SERS ( Surface - Enhanced Raman Scattering ) Measure

After binding the neurotransmitter by concentration to the gold self-assembled microarray plate in which the first antibody is bound, the complexes in which the gold nanoparticle-barcode DNA-second antibody is bound are obtained by sandwiching the first antibody-antigen-second antibody. Induced binding. 1 μl of protease K, 0.5 M EDTA, 0.1 M Tris buffer was used for 1 hour at 50 ° C., and gold nanoparticle-barcode DNA was isolated and centrifuged to confirm separation of barcode DNA. In order to induce binding of gold nanoparticles with barcode DNA to gold nano substrates with complementary DNA, 16 hours at 4 ° C was used, and the complementary DNA and barcode DNA complexes on the substrate were used as surface enhancement Raman spectrometer. It was confirmed that the complementary DNA binding is increased according to the concentration of the neurotransmitter.

With Raman spectrometer (NTEGRA spectra, NTMDT, Russia) the maximum scanning range is XYZ 100 μm X100 μm X6. The resolution of the Raman spectrometer was measured with an XY plane of 200 μm, the laser wavelength was 785 nm, the scan speed was 10 s, and the scan range was 0-3000 cm ⁻¹ . Recorded. Raman's scattering process causes a change in the vibration potential of the molecule. However, as in the case of infrared spectroscopy, how much energy can be measured directly and the scattered light lost energy compared to Rayleigh scattering? It is known to measure vibrational energy by observing whether or not it has been obtained. Therefore, the peak value according to the wavelength of the Raman spectrometer was observed.

result

Nano Substrate Fabrication

The process of nanopatterning an aluminum mask on an ITO plate is shown in Figure 1: SEM image of the aluminum mask prior to depositing gold (Figure 3). As a result of measuring the pupil of the aluminum mask, a diameter of 71-74 nm was shown. These results are thought to be the size of the primary anodization process. In preparing the nanoporous alumina mask, it is very important to make the mask thin and to completely dissolve the alumina barrier layer.

The growth rate of the alumina film was 2.9 m / hr when anodic oxidation was carried out by applying 40 V oxidation process in 0.3 M oxalic acid solution. However, since the surface of the alumina shown in the first anodization is irregular, the surface was smoothly manufactured by etching. In addition, it was confirmed that hexagonal dense cells are arranged in a regular order during the second anodization.

After the deposition of gold or silver, the mask removal process confirmed that the nano array plate with the gold nano patterns regularly arranged. Nano gold nano array on which gold was deposited confirmed a pattern shape of 70 mean ± 5 nm in diameter and 45 ± 5 nm in height (FIG. 4). In addition, it was measured by Raman to confirm the gold nano peak of the gold nano-array equipped with gold (FIG. 5). Gold peaks were seen at 1240 cm ⁻¹ , 1362 cm ⁻¹ and 1418 cm ⁻¹ at Raman shift (cm ⁻¹ ).

In addition, the diameter of the nano-substrate equipped with silver was confirmed to have a pattern shape of 30 ± 5 nm and a height of 45 ± 5 nm (Fig. 6). In addition, as a result of measuring the silver nano-substrate with Raman, the Raman 974 cm ^-1 , 1125 cm ^-1 and 1244 cm ^-1 showed the same peak value as previously reported silver nano-peak (Fig. 7).

These results confirmed that the gold or silver is well mounted on the nano-substrate in the present invention compared with the Raman peak reported in the previous studies of gold and silver.

ITO  Complementary to the substrate DNA  Confirmation of the bond

Dopamine complementary DNA was synthesized by attaching a thiol group at the 5 'position. After treatment with 0.1 N DTT (Dithiothreitol) and then treated with ethyl acetate solution to remove the salt was mounted on the gold nano substrate and silver nano substrate. As a result of confirming that the complementary DNA is mounted on the gold nano substrate and the silver nano substrate by UV (Ultraviolet), the DNA peak was increased at 253 nm (FIG. 8). In addition, the results measured by Raman complementary DNA was mounted on the gold and silver nano substrates showed a peak value (Fig. 9).

Dopamine complementary DNA was attached to a 5'-SH-AAG ACA TTT GCT GAA AGG TTA AAT CCA CAT CCG GCA GTG TGG-3 'to attach to the gold nano substrate. The 134 cm ^-1 peak was measured the highest. Also guanine in 672 ㎝ ^-1 (Guanine; G) was a peak is detected, 725 ㎝ ^- the ^first adenine (adenine; A) was a peak is detected, the 1078 ㎝ ^-1 thymine (Thymine; T) and cytosine (cytosine; C ) Peaks were detected, adenine peaks were detected at 1314 cm ^-1 , and adenine, thymine and cytosine peaks were detected at 1482 cm ^-1 . In addition, peak values were also shown at 805 cm ⁻¹ and 857 cm ⁻¹ (FIG. 9).

However, complementary DNA mounted on silver nano substrates showed different values. The same DNA, but somewhat different depending on which substrate it is bound to. Guanine peaks were detected at 672 ㎝ ^-1, 785 ㎝ ^-1 was the adenine peak is detected, the 1101 ㎝ ^-1 thymine and cytosine was the detection, in the 1315 ㎝ ^-1 peak is detected was adenine, 1244 ㎝ ^-1 DNA Raman peak values were detected (FIG. 10).

The restriction enzyme involved in the dopamine biosynthesis process is reported to reduce tyrosine hydroxylase when dopamine is reduced during Parkin's disease. In the present invention, barcode DNA and complementary DNA of tyrosine hydroxylase were prepared. When tyrosine hydroxylase was fixed to the gold substrate and the Raman peak was confirmed, the DNA of the tyrosine hydroxylase showed a Raman peak different from that of the dopamine DNA (FIG. 11). Raman peak of tyrosine hydroxide enzyme DNA was guanine peak is detected at 672 ㎝ ^-1, adenine peak, was thymine and cytosine peak peak is detected at 785 ㎝ ^-1 at 720 ㎝ ^-1, check the gold peak in 1257 ㎝ ^-1 It was. In addition, adenine, thymine and cytosine peaks were detected at 1478 cm ^-1 , adenine peaks were detected at 1578 cm ^-1 , and peak values were also detected at 160 cm ^-1 , 228 cm ^-1 and 306, cm-1.

Complementary DNA of tyrosine hydroxylase was synthesized by attaching a thiol group at 5 'with 5'-SH-AAA CCC CTT GTG GCA CCT CGT ATC GTA-3'. Raman peaks of dopamine complementary DNA and tyrosine hydroxylase complementary DNA showed specific peaks at 672 cm ⁻¹ , but ATGC base values were commonly detected in both DNAs. . In the previous report, the peak value was observed due to the difference in the size of the nucleotide sequence, but the Raman peak value was observed even though the two DNA sizes were not different.

In addition, Raman results of tyrosine hydroxylase complementary DNA immobilized on the silver nano substrate are shown in FIG. 12. Tyrosine hydroxylase complementary DNA immobilized on a silver substrate was found to have a significant increase in Raman signal compared to gold nano substrates. The enzyme is tyrosine hydroxide complementary DNA in the nano was the adenine peak detection at 716 ㎝ ^-1, from 786 ㎝ ^-1 thymine and cytosine was detected peaks, 1250 ㎝ ^-1 was detected with the gold peak. In addition, adenine, thymine and cytosine peaks were detected at 1478 cm ^-1 , and adenine peaks at 1578 cm ^-1 showed the same peak value as that of the gold nano substrate. Peak values not observed in the gold substrate were shown at 1138 cm ⁻¹ and 1093 cm ⁻¹ .

Gold Nanoparticles and Barcodes DNA Combination of

As a result of analysis by UV spectrometer to confirm that the DNA is bound to the gold nanoparticles, when only the gold particles are worn, the peak value was shown only at 520 nm (Fig. 13a). In general, the absorbed light peak was observed because the free electrons in the gold nanoparticles oscillated near the wavelength of 520 nm by the electrical vibration of the gold incident tube. In addition, as a result of confirming the barcode DNA binding to the gold nanoparticles, the absorption absorbance peak was confirmed at 260 nm by the binding. The absorption absorbance peak at 260 nm confirmed that the oligonucleotides were well bonded to the gold nanoparticles (FIG. 13B). As a result of evaluating the binding force of the barcode DNA to the gold nanoparticles, it was confirmed that the DNA nanoparticles were sufficiently bound to about 0.8 OD DNA. Gold nanoparticles bound to barcode DNA were measured to have a mean of 65 ± 5 nm in diameter. In addition, as the concentration increases as shown in Figure 17 in the SERS Raman value according to the amount of barcode DNA Raman peak value also increased.

DNA  Fabrication of the sensor

After binding barcode DNA and antibody to gold nano substrate, antigen binding was induced. At this time, the antibody binding according to the concentration of the antigen is proportional to the concentration of the antigen and binds only the gold nanoparticles to which the barcode DNA is bound on the nano substrate to which the complementary DNA is bound. At this time, the non-complementary reaction is similar to the SERS peak of the substrate to which the complementary DNA is bound, but the distinct SERS Raman peak can be confirmed where the barcode DNA is bound. As a result of confirming the change of the signal according to the difference in the Raman signal of the surface-enhanced Raman spectrometer, the DNA showing the complementary reaction and the DNA showing the non-complementary reaction as shown in FIG. 14 are 672 cm ^-1 at the Raman peak of the substrate having the complementary DNA binding. from guanine were peak is detected, the peak of adenine, thymine, and cytosine was a peak is detected at 785 ㎝ ^-1 at 720 ㎝ ^-1, in 1478 ㎝ ^-1 adenine, thymine and cytosine, adenine peaks were detected peak at 1578 ^-1 is ㎝ Detected. Substrates with non-complementary DNA reactions showed peak patterns similar to gold nanosubstrates.

Combined  barcode DNA of SERS  analysis

After binding the antibody to the gold nanoparticles, the antigen was treated with different concentrations and the protein was removed using protease K. Barcode DNA was extracted and bound to the gold nano substrates with complementary DNA. The amount of barcode DNA also varies depending on the concentration of the antigen, and the change in Raman peak was confirmed through SERS (FIG. 14). FIG. 15 shows SEM images of metal nano substrates bound with complementary DNA immobilized on metal nano substrates and barcode DNA isolated from magnetic particle-neural transport related material-gold nanoparticle complexes. Particularly tyrosine hydroxide enzyme 627 ㎝ ^-1 is phenylalanine, 740 ㎝ ^-1 is the indole group of tryptophan, 810 ㎝ ^-1 is OPO structure, 941 ㎝ ^-1 is the CC structure of alpha-helix, 1001 ㎝ ^-1 and 1033 ㎝ ^{- 1} is phenylalanine, 1204 cm- ¹ represents phenylalanine and tyrosine, 1270 cm ^{- 1} is an amide III beta-sheet, 1340 cm- ¹ is a CH complex and 1640 cm ^{- 1} is an amide II peak. In addition, the Raman peak is high at 129 cm ^-1 , 474 cm ^-1 , 603 cm ^-1 , 1088 cm ^-1 , 1110 cm ^-1 , 1133 cm ^-1 , 1288 cm ^-1 , 1310 cm ^-1 and 1388 cm ^-1 . Appeared (FIG. 16).

In general, DNA values of Raman peaks related to proteins were observed rather than Raman peaks related to adenine, thymine, guanine, and cytosine bases. As a result, the protein Raman peak was detected because the antibody-antigen remained in the gold nanoparticles even after the protease K treatment.

SERS measurements of barcode DNA after treatment of antigen (tyrosine hydroxylase) concentrations of 0.156 ng / ml, 0.312 ng / ml and 0.625 ng / ml resulted in increased Raman peak values of 129 cm ^-1 and 474 cm ^-1 Showed results. 941 cm ^-1, which represents the CC structure, showed an increased peak pattern with tyrosine hydroxylase concentration.

In addition, the structure of 777 cm ^-1 thymine and cytosine nucleic acid was observed, thymine and adenine at 1209 cm ^-1 , and SERS Raman peak associated with adenine and thymine at 1242 cm ^-1 (FIG. 17).

As can be seen from the above results, the SERS peak of the DNA with complementary binding was observed at the very low concentration with the change of the peak concentration for dopamine and tyrosine hydroxylase. These results suggest that DNA sensors using barcode DNA will be very useful for diagnosing diseases.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry-University Cooperation Foundation Sogang University <120> Devices for Detecting Neurotransmission-related substance <130> PN120175 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 42 <212> DNA <213> dopamine barcode DNA <400> 1 ccacactgcc ggatgtggat ttaacctttc agcaaatgtc tt 42 <210> 2 <211> 42 <212> DNA <213> dopamine complementary DNA <400> 2 aagacatttg ctgaaaggtt aaatccacat ccggcagtgt gg 42 <210> 3 <211> 33 <212> DNA <213> tyrosine hydroxylase barcode DNA <400> 3 tttggggaac accgtggagg ggcatagcat ctc 33 <210> 4 <211> 27 <212> DNA <213> tyrosine hydroxylase complementary DNA <400> 4 aaaccccttg tggcacctcg tatcgta 27

Claims (10)

Sensors for detecting neurotransmitter-related substances, including:
(a) a microarray chip plate having a first antibody bound to a neurotransmitter-related substance;
(b) gold nanoparticles bound to a second antibody and a barcode DNA to the same neurotransmitter-related substance as the neurotransmitter-related substance; And
(c) a metal nano substrate having a metal nano pattern in which complementary DNA having a sequence complementary to the barcode DNA is immobilized on a surface thereof.
The method of claim 1, wherein the neurotransmitter-related substance is dopamine (dopamine), tyrosine hydroxylase (Tyrosine Hydroxylase), adrenaline (adrenaline), noradrenaline (gluradmate), glutamate (serotonin), γ- Γ-Aminobytyric acid, acetylcholine, DOPA dihydroxyphenylalanine decarboxylase, aromatic L-amino decarboxylase, dopamine β-hydroxylase β-hydroxylase) or phenylethanolamine N-methyltransferase.
The sensor of claim 2, wherein the neurotransmitter-related substance is dopamine or Tyrosine Hydroxylase.
The sensor of claim 1, wherein the sequence of the barcode DNA is a first sequence or a third sequence of the sequence listing.
The sensor according to claim 1, wherein the complementary DNA is SEQ ID NO: 2 or SEQ ID NO: 4.
The metal composition of claim 1, wherein the metal is composed of gold, silver, barium, chromium, cobalt, manganese, iron, nickel, copper, zinc, niobium, palladium, platinum, terbium, gadolinium, dysprosium, holmium, erbium, samarium, and neodymium. At least one metal selected from the group.
7. The sensor of claim 6, wherein the metal is gold (Au) or silver (Ag).
Neurotransmitter-related substance detection method comprising the following steps:
(a) a microarray chip plate to which (i) a neurotransmitter first antibody is bound, and (ii) a gold nanoparticle to which the second antibody and barcode DNA are bound to the same neurotransmitter-related material as the neurotransmitter-related material. Contacting the sample to be analyzed;
(b) separating the gold nanoparticles to which the second antibody and the barcode DNA are bound from the result of step (a);
(c) contacting the isolated gold nanoparticles with the separated second antibody and barcode DNA to a substrate having a gold nanopattern to which complementary DNA is immobilized; And
(d) analyzing the hybridization of the barcode DNA and the complementary DNA.
9. The method of claim 8, wherein the separating of step (b) is carried out by treating protease K.
The method of claim 8, wherein the analysis of the conjugate of step (e) is characterized in that for detecting the hybridization using a Raman spectrometer.

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