KR20070033091A - Method for manufacturing nanohybrid particle using for biomolecule detection, biomolecule detection system, biomolecule detection method, and analysis apparatus using for biomolecule detection using nanohybrid particle - Google Patents

Abstract

Provided are a method for manufacturing nano-hybrid particle used for detecting a biomolecule, and a biomolecule detection system and method for detecting a biomolecule in a convenient manner at low costs. The nano-hybrid particle is manufactured by preparing core particles, and nano-particles to be coated around the core particles; measuring zeta potential for pH; examining a pH section with zeta potentials of opposite symbols; titrating pH of a mixed solution of the core particles and the nano-particles; forming nano-hybrid particles where the nano-particles are coated around the core particles; collecting the nano-hybrid particles by using a magnet; and drying the collected nano-hybrid particles to obtain nano-hybrid particle powder. The analysis apparatus for biomolecule detection comprises a magnetic force generation wire(230a), a magnetic force change sensing wire(230b), a power distribution unit(240a), a power distribution sensor(240b), a magnetic force change sensor(250), and a power generator(260).

Description

Methods for manufacturing nanohybrid particle using for biomolecule detection, biomolecule detection system, biomolecule detection method , and analysis apparatus using for biomolecule detection using nanohybrid particle}

1A and 1B are diagrams schematically showing nanohybrid particles according to a preferred embodiment of the present invention, and FIGS. 1C and 1D are diagrams schematically showing biomolecules fixed to nanohybrid particles.

2 is a graph showing zeta potential according to pH of gold (Au) and γ-iron oxide (γ-Fe ₂ O ₃ ).

FIG. 3 is a diagram illustrating a state in which gold (Au) and γ-iron oxide (γ-Fe ₂ O ₃ ) react to form nanohybrid particles (Au / γ-Fe ₂ O ₃ ).

FIG. 4 is a graph measuring absorbance according to wavelength.

5 is a graph showing X-ray diffraction patterns.

6A to 6C are transmission electron microscope (Transmission Electron Microscope) photographs of gold / γ-iron oxide (Au / γ-Fe ₂ O ₃ ) nanohybrid particles.

FIG. 7 is a diagram showing a biomolecule having a thiol group and nanohybrid particles (Au / γ-Fe ₂ O ₃ ) reacting to form a biocomposite particle.

FIG. 8 is a view illustrating a biomolecule, 3-aminopropyltriethoxysilane, reacting with nanohybrid particles (SiO ₂ / γ-Fe ₂ O ₃ ) to form biocomposite particles.

9 is a graph measuring absorbance according to the concentration of glutathione.

10A to 10D are diagrams schematically showing an apparatus for detecting biomolecules.

<Explanation of symbols for the main parts of the drawings>

100: nanohybrid particle 110: core particle

120: nanoparticle 130: biomolecule

200: biological composite particles

210: substrate 220: biomolecule carrying cell

230a: magnetic field generating line 230b: magnetic field change detecting line

240a: electric classification means 240b: electric classification detection means

250: magnetic change detection device 260: electricity generating means

The present invention relates to a method for producing biomolecule detection nanohybrid particles, a biomolecule detection system, a biomolecule detection method and an analysis device for biomolecule detection, and more particularly, around the core particles and the core particles in response to magnetic force. The present invention relates to a method for producing biomolecule detection nanoparticles for detecting biomolecules using nanoparticles coated on the substrate, a biomolecule detection system using nanohybrid particles, a biomolecule detection method, and an analysis device for biomolecule detection.

The field of molecular biology for observing and studying the components of animals and plants is based on the analysis of the characteristics and quantification of individual proteins or complex biological molecules, and the prevention or diagnosis of diseases, the detection of the presence of pathogens, etc. It is applied to a wide range of medical fields.

Biochip is a biological material such as nucleic acid is fixed on the substrate (substrate), refers to a material or device that is aligned with the probe (probe) used for biochemical analysis for biomolecules including DNA and the like. DNA chips, which are a type of biochip, have DNA fixed on a substrate, and protein chips have a protein fixed on a substrate. The principle of such biochips is based on the interaction between probe molecules and target molecules immobilized on the substrate. Biochips can be used to search for nucleic acids, proteins, and other substances that can bind to nucleic acids, proteins, etc., immobilized on a substrate, and can also be used to analyze whether a target molecule that binds to a probe molecule exists in a sample. Do. As such, biochips are widely used in biological research, medical diagnosis, drug discovery, and forensics.

Biomolecule detection methods using biochips include blot hybridization method, radioactive photograph, laser induced fluorescence, surface plasma resonance, and electrochemical method. ).

The blot hybridization method requires a lot of labor, time and huge resources. For example, in the case of a DNA fragment consisting of 10 nucleotide sequences, since there are 4 ¹⁰ possible kinds of molecules, there is a problem in that it takes a lot of time and effort to detect using the blot hybridization method.

Radiophotographic analysis takes several hours to one day, but has a resolution of only 0.1 μm to 10 μm order, and there is a problem of stability of radioisotopes.

Laser-induced fluorescence is complicated by the process of labeling and separating and purifying the DNA of a sample with fluorescent material before measurement, and requires expensive equipment such as a laser and an optical measurement accessory and an image scanner.

Surface plasma resonance method has a problem that the sensitivity is not good because it can be detected only if the probe DNA of about 10 ¹¹ is immobilized on the surface of 1 cm 2.

The electrochemical method has a problem that the detection method is simple, uses a low-cost measuring device and the detection method is simple, but the sensitivity is not good.

Prior art publications on DNA detection methods include methods using photochemical reactions with DNA probes (see PCT Publication No. WO 9984813 A1 19990826).

An object of the present invention is to provide a method for producing nanohybrid particles used to detect biomolecules.

Another technical problem to be achieved by the present invention is to provide a biomolecule detection system using nanohybrid particles that are simple to manufacture and can easily detect biomolecules at a low time and cost.

Another technical problem to be achieved by the present invention is to provide a biomolecule detection method using nanohybrid particles that can easily detect the biomolecules in a small time and cost.

Another technical problem to be achieved by the present invention is to provide an analysis device for detecting biomolecules using nanohybrid particles, which is simple to manufacture and can easily detect biomolecules at low time and cost.

The present invention comprises the steps of preparing the core particles and the nanoparticles to be coated or adsorbed around the core particles in response to the magnetic force, and measuring the zeta potential of the pH between the core particles and the nanoparticles in the global range; And irradiating a pH section having zeta potentials having opposite signs on the core particles and the nanoparticles, and allowing the core particles to have a pH section having zeta potentials opposite to each other. And titrating a pH of a solution to which the nanoparticles are mixed, and mixing the core particles and the nanoparticles into the solution to form nanohybrid particles coated or adsorbed around the core particles. Collecting the nanohybrid particles using magnetic force, and drying the collected nanohybrid particles It provides a process for the preparation of nanohybrid particles for biomolecule detection, comprising the step of obtaining the bleed particle powder.

In addition, the present invention includes a nano-hybrid particles comprising a core particle reacts to magnetic force and nanoparticles coated or adsorbed around the core particle, the nanohybrid particles react with a biomolecule to hybridize the biomolecule The present invention provides a biomolecule detection system using nanohybrid particles having a property of reacting with a magnetic body, wherein the biocomposite particles are formed.

In addition, the present invention is to prepare a nano-hybrid particles comprising a core particle reacts to magnetic force, and nanoparticles coated or adsorbed around the core particles, and to react and hybridize biomolecules to the nanohybrid particles And extracting the nanohybrid particles mixed with the biomolecules using magnetic force, and analyzing the extracted nanohybrid particles with the hybridized material using an analysis device using magnetic force. Provided is a method for detecting biomolecules using particles.

In addition, the present invention, so that the biomolecule hybridized biocomposite particles or biomolecules can be contained in the nano-hybrid particles including the substrate, the core particles react to the magnetic force and the nanoparticles coated or adsorbed around the core particles A plurality of biomolecule supporting cells arranged on a front surface of the substrate and arranged in a matrix in a horizontal and vertical direction, a magnetic force change sensing wiring arranged in a horizontal or vertical direction on a front surface of the substrate, and a rear surface of the substrate And a magnetic field generating line arranged in a direction perpendicular to the magnetic field change detecting line, wherein an area where the magnetic field change detecting line and the magnetic field generating line cross each includes nanohybrid particles provided to correspond to the biomolecule supporting cell. Provided is an analysis device for detecting biomolecules.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following embodiments are provided to those skilled in the art to fully understand the present invention, and may be modified in various forms, and the scope of the present invention is limited to the embodiments described below. It doesn't happen. Like numbers refer to like elements in the figures.

The detection and analysis of biomolecules is very important for biological research, medical diagnosis, reagent search, forensics, etc., such as searching for specific genes and sequencing, and determining whether viruses or pathogenic microorganisms are infected.

Conventional biomolecule assays are not only time-consuming but also require a lot of manpower and require expensive equipment, and thus new biomolecule assays are required.

In a preferred embodiment of the present invention, the target biomolecules are immobilized on iron (Fe) oxides coated with (or adsorbed) nanoparticles to measure the magnetic properties of the magnetic material, thereby simplifying the biomolecule analysis process and in a short time. The present invention provides a method for preparing nanohybrid particles for biomolecule detection, a biomolecule detection system using nanohybrid particles, a biomolecule detection method, and an analysis device for biomolecule detection.

1A and 1B are diagrams schematically showing nanohybrid particles according to a preferred embodiment of the present invention, and FIGS. 1C and 1D are diagrams schematically showing biomolecules fixed to nanohybrid particles.

1A to 1D, a biomolecule detection system according to a preferred embodiment of the present invention includes a nanohybrid particle 100, and the nanohybrid particle 100 includes a core particle 110 reacting to a magnetic force. , Nanoparticles 120 coated or adsorbed around the core particles 110. The nanohybrid particle 100 reacts with the biomolecule 130 to form the biocomposite particle 200 in which the biomolecule 130 is hybridized, and the biocomposite particle 200 has a characteristic of responding to electromagnetic force. . The biomolecule 130 is immobilized or hybridized to the nanoparticle 120. The core particle 110 may be γ-Fe ₂ O ₃ or Fe ₃ O ₄ , which is an iron oxide-based material reacting with a magnetic body. The nanoparticles 120 may be metal nanoparticles such as gold (Au), silver (Ag), platinum (Pt), palladium (Pd), copper (Cu), nickel (Ni), or zinc (Zn) or silicon oxide. (SiO ₂ ) and the like. FIG. 1A is a diagram illustrating a metal nanoparticle 120 such as gold (Au) adsorbed around a core particle 110, and FIG. 1C is a diagram illustrating a biomolecule fixed to the metal nanoparticle 120. to be. Figure 1b is a view of silicon oxide (SiO ₂₎ (120) showing the appearance coating around the core particle 110, Fig. 1d is a view showing a state of a biomolecule fixed to the silicon oxide (SiO ₂₎ (120) to be.

Hereinafter, metal nanoparticles such as gold (Au), silver (Ag), platinum (Pt), palladium (Pd), copper (Cu), nickel (Ni), and zinc (Zn) are patent applications. 10-2003-0037065 may be prepared by the 'Method for producing metal nanoparticles using an electrolysis method,' or may be used for commercially available metal nanoparticles.

Hereinafter, a method of manufacturing nanohybrid particles having a form in which nanoparticles are coated or adsorbed around core particles will be described.

The particles dispersed or suspended in the solution are electrically charged to the cathode or the anode by dissociation of the surface polar groups on the particle surface and adsorption of ions. Therefore, in order to neutralize the interfacial charge, ions with excessively opposite counterions and ions with a small number of eastern arcs are diffusely distributed around the particles, exhibiting an electrical potential from the interface and having a gentle potential gradient. Thus, the charged particles are adsorbed with counter ions to form a relatively non-moving layer, and the counter ions and polar liquid molecules form a steady-state configuration in the diffused layer. Have a concentration gradient. The charged particles in the solution move at a constant speed in response to an externally applied electric field, which is called the electrophoretic velocity. This electric field results in a hydrodynamic plane of slipping in the double layer during electrophoresis of the particles, ie when the particles move, the compressed layer moves like particles, but the ion diffusion layer is a liquid phase. Move like this A sliding surface occurs at this shear plane, and the electrical potential at this interface is called zeta-potential.

Particle mobility and zeta potential can be measured by measuring the drift velocity of the resulting particles as an electric field is applied to a portion of the solution. Zeta potential is a function of pH and concentration of electrolyte, and is generally reduced by increasing pH and electrolyte concentration, and is affected by the addition of substances such as surfactants. As the pH increases, the zeta potential generally decreases in value and eventually becomes negative. The pH at which the zeta potential is zero is called an isoelectric point (iP). If the zeta potentials between the particles and the surface or between the particles are of the same sign, the repulsive force acts on each other.

2 is a graph showing zeta potential according to pH of gold (Au) and γ-iron oxide (γ-Fe ₂ O ₃ ).

Referring to FIG. 2, the pH at which the zeta potential of gold (Au) becomes zero, that is, the isoelectric point is pH 2.1, and at pH 2.1 or less, the zeta potential has a positive value. Zeta potential of γ-iron oxide (γ-Fe ₂ O ₃ ) is negative until pH 1.6. Therefore, below pH 2.1, since the zeta potentials of gold (Au) and γ-iron oxide (γ-Fe ₂ O ₃ ) have opposite signs, attraction may occur and hybridization may occur.

Hereinafter, a method of preparing nanohybrid particles using pH and zeta potential will be described in more detail.

First, the zeta-potential of the global pH is measured for the core particles and the nanoparticles to be coated (or adsorbed). By investigating the zeta potential for the entire pH range, it is to identify the region (pH section) where the reaction is possible between the core particles and the nanoparticles to be coated. The core particle may be γ-iron oxide (γ-Fe ₂ O ₃ ), iron oxide (Fe ₃ O ₄ ), or the like, which reacts with the magnetic material, and the nanoparticles to be coated are gold (Au), silver (Ag), platinum (Pt), Palladium (Pd), copper (Cu), nickel (Ni), zinc (Zn), silicon oxide (SiO ₂ ), and the like.

When the core particles are γ-iron oxide (γ-Fe ₂ O ₃ ), and the nanoparticles to be coated are gold (Au), γ-iron oxide (γ-Fe ₂ O ₃ ) has a pH as shown in FIG. 3. It has a negative zeta potential value when greater than 1.6, and gold (Au) has a negative zeta potential value when pH is greater than 2.1 and a positive zeta potential value when pH is less than 2.1.

For the core particles and the nanoparticles to be coated, the pH ranges in which the zeta potentials have opposite signs are examined. Since the attraction of the core particles and the zeta potentials of the nanoparticles to be coated are opposite to each other, the core particles and the nanoparticles to be coated may react to form nanohybrid particles. If the core particles are γ-iron oxide (γ-Fe ₂ O ₃ ) and the nanoparticles to be coated are gold (Au), zeta of gold (Au) and γ-iron oxide (γ-Fe ₂ O ₃ ) is below pH 2.1. Potentials have opposite signs from each other.

The pH of the solution (eg, deionized water) to which the core particles and the nanoparticles to be coated are mixed is titrated so that the zeta potentials of the core particles and the nanoparticles to be coated have a pH section having opposite signs. The pH may be adjusted using hydrochloric acid (HCl), sodium hydroxide (NaOH), and the like, and the titration is performed such that the zeta potentials of the core particles and the nanoparticles to be coated have opposite pH signs. When the core particles are γ-iron oxide (γ-Fe ₂ O ₃ ) and the nanoparticles to be coated are gold (Au), hydrochloric acid (HCl), sodium hydroxide (HCl) so that the pH is below 2.1 so that the zeta potentials have opposite signs. NaOH) and the like.

Mix the core particles with the nanoparticles to be coated. Since the zeta potentials of the core particles and the nanoparticles to be coated are set to pHs having opposite signs by the pH titration, the core particles and the nanoparticles to be coated are attracted to each other to cause hybridization, and the nanoparticles to be coated are cores. The particles are coated or adsorbed around the particles. Gold (Au) 120 and γ-iron oxide (γ-Fe ₂ O ₃ ) 110 is shown in FIG. 3 to form nanohybrid particles (Au / γ-Fe ₂ O ₃ ).

Magnets are used to collect nanohybrid particles. The core particles have a property of reacting with the magnetic body, and the nanohybrid particles generated by hybridization of the core particles with the nanoparticles to be coated can still be collected by the magnetic body (magnet) because the core particles still retain their magnetic properties. . For example, when the core particle is γ-iron oxide (γ-Fe ₂ O ₃ ) and the nanoparticle to be coated is gold (Au), γ-iron oxide (γ-Fe ₂ O ₃ ) still retains its magnetic properties.

The nanohybrid particles collected by the magnet are dried to obtain nanohybrid particle powder.

By measuring the absorbance (wavelength) according to the wavelength (wavelength) by observing the degree to which the nanoparticles to be coated reacts to the core particles according to the pH is shown in FIG.

FIG. 4 is a graph measuring absorbance according to wavelength. FIG. 4 illustrates the case of using gold (Au) as a nanoparticle to be coated and using γ-iron oxide (γ-Fe ₂ O ₃ ) as a core particle, and gold (Au) and γ-iron oxide (γ-Fe ₂ O _3). After the reaction, the resulting nanohybrid particles (Au / γ-Fe ₂ O ₃ ) were removed and the UV-visible spectrometry of the remaining gold (Au) nanoparticles was measured. In FIG. 4, (a) is a case of reacting at pH 1.74, (b) is a case of reacting at pH 2.28, (c) is a case of reacting at pH 6.39, and (d) is a case of reacting at pH 9.66. .

Referring to FIG. 4, when the reaction is performed at pH 1.74, the absorbance has a value of almost zero, and as the pH increases to 2.28, 6.39, and 9.66, the absorbance increases. That is, in the pH region in which the zeta potentials of gold (Au) and γ-iron oxide (γ-Fe ₂ O ₃ ) have the same sign (in the case of (b), (c), and (d) in FIG. 4), gold (Au) is hardly coated (or adsorbed) on γ-iron oxide (γ-Fe ₂ O ₃ ), but the amount of gold (Au in FIG. 4) is in the region where the zeta potentials have opposite signs. It can be seen that) reacted.

5 is a graph showing X-ray diffraction patterns. In FIG. 5, (a) is γ-iron oxide (γ-Fe ₂ O ₃ ), (b) is gold (Au), and (c) is gold / γ-iron oxide (Au / γ-Fe ₂ O ₃ ) nanohybrid The X-ray diffraction pattern of the particles is shown respectively. Gold (Au) was obtained using the above-described electrolysis method, and γ-iron oxide (γ-Fe ₂ O ₃ ) was used as commercial powder of NanoPhase Tech. Co. It was.

5, a gold (Au) and γ- iron oxide (γ-Fe ₂ O ₃₎ gold / iron oxide γ- strength in the portion (intensity) that make up the peak (peak) of the _{(Au / γ-Fe 2 O} 3 It can be seen that the nanohybrid particles also form a peak.

6A to 6C are photographs of a transmission electron microscope (hereinafter referred to as 'TEM') of gold / γ-iron oxide (Au / γ-Fe ₂ O ₃ ) nanohybrid particles. 6A to 6C illustrate gold / γ-iron oxide (Au / γ-Fe ₂ O ₃ ) nanohybrid particles prepared at pH 1.74 using gold (Au) and γ-iron oxide (γ-Fe ₂ O ₃ ). TEM picture.

6A to 6C, the average particle size of gold (Au) was 7 nm, and the average particle size of γ-iron oxide (γ-Fe ₂ O ₃ ) was measured to be 20 nm. As shown in FIGS. 6a to 6c, it can be seen that gold (Au) is coated (or adsorbed) on γ-iron oxide (γ-Fe ₂ O ₃ ) particles which are core particles.

Hereinafter, a method of immobilizing (or hybridizing) biomolecules to nanohybrid particles will be described.

First, biomolecules are dissolved in a solution (eg, deionized water).

The biomolecule is glutathione (GSH) when the nanohybrid particles are gold / γ-Fe ₂ O ₃ nanohybrid particles or gold / iron oxide (Au / Fe ₃ O ₄ ) nanohybrid particles. It may be a biomolecule such as lectin, protein A, HIV virus, anthrax, and the like.

In addition, when the nanohybrid particles are silver / γ-iron oxide (Ag / γ-Fe ₂ O ₃ ) nanohybrid particles or silver / iron oxide (Ag / Fe ₃ O ₄ ) nanohybrid particles, the biomolecule is a urine protein (urine). protein) and the like.

In addition, the nanohybrid particles are silicon / γ- iron oxide _{(SiO 2 / γ-Fe 2} O 3) nanohybrid particles or silicon oxide / iron oxide _{(SiO 2 / Fe 3 O 4} ) the biomolecule, if one nanohybrid particles Lysozyme, bovine serum albumin, and the like.

In addition to the cases listed above, it is of course possible to include all biomolecules that can be immobilized (hybridized) by reacting with the nanohybrid particles.

Nanohybrid particles are mixed with deionized water in which biomolecules are dissolved. Biomolecules will hybridize to the nanohybrid particles. As an example, a biomolecule such as DNA generally includes a thiol group having a mercapto group (-SH), and the mercapto group (-SH) of the biomolecule dissolved in a solvent (solution) is It is reacted with gold (Au) coated around the core particles, which is shown in Scheme 1 below.

RSH (solv) + Au (s) ⇔RSAu (s) + H ₂ (solv)

R in Scheme 1 means a hydrocarbon group. DNA dissolved in deionized water undergoes a thiol reaction with gold (Au) to immobilize (or hybridize) to gold (Au) coated around the core particles. FIG. 7 is a diagram illustrating a state in which DNA and nanohybrid particles (Au / γ-Fe ₂ O ₃ ) react to form biological composite particles.

As another example, when the nanoparticles coated around the core particles are SiO ₂ , the biomolecule is a material containing a hydrocarbon group, and the biomolecule is Si, which is a surface group of silicon oxide (SiO ₂ ) coated around the core particles in a solution. It reacts with -OH, which is shown in Scheme 2 below.

SiOH (s) + R (solv) ⇔SiOR '(s)

R and R 'in Scheme 2 refer to the first and second hydrocarbon groups.

As a specific example when the nanoparticles coated around the core particles are SiO ₂ , the biomolecule is 3-aminopropyltriethoxysilane (hereinafter referred to as 'APTES'), and APTES is a nanohybrid particle (SiO). ₂ / γ-Fe ₂ O ₃ ) is schematically shown in Figure 8 to form a biological composite particles. Si-OH, which is a surface group of silicon oxide (SiO ₂ ), and APTES undergo a coupling reaction as shown in FIG. 8.

The particles hybridized with the biomolecules and the nanohybrid particles are collected using magnetic force. The core particles have a property of reacting with a magnetic body, and nanohybrid particles having a biomolecule fixed therein can still be collected by magnetic force since the core particles still retain their magnetic properties. For example, when the core particle is γ-iron oxide (γ-Fe ₂ O ₃ ) and the nanoparticle to be coated is gold (Au), the nanohybrid particles to which the biomolecules are immobilized may be added to the γ-iron oxide (γ-Fe ₂ O ₃ ). It still maintains its magnetic properties.

The collected particles are dried. The drying may be performed at room temperature.

9 is a graph measuring absorbance according to the concentration of glutathione (hereinafter, referred to as 'GSH'). 9 is produced by reacting dithionitrobenzene (hereinafter referred to as 'DTNB') to a biomolecule-nanohybrid particle having GSH immobilized (or hybridized) to a nanohybrid particle (Au / γ-Fe ₂ O ₃ ). The absorbance of 2-nitro-5-thiobenzoic acid was measured in the UV-visible region.

Referring to FIG. 9, GSH reacts with DTNB to produce yellow by-products. As the concentration of GSH was quantified with respect to the absorbance by measuring the degree of absorption of DTNB while varying the concentration of GSH, it can be seen that the absorbance increases as the concentration of GSH increases.

Table 1 is a table showing the amount of GSH immobilized GSH to the nanohybrid particles (Au / γ-Fe ₂ O ₃ ).

Au (mg) Au / γ-Fe ₂ O ₃ (mg) Absorbed GSH (mM) GSH / (Au / γ-Fe ₂ O ₃ ) Matrix (mM / g) GSH / Au (mM / g) 2.3 12.3 18.22 1.48 7.92 4.6 14.6 46.20 3.16 10.04 6.9 16.9 78.74 4.66 11.41 10.6 20.6 138.61 6.73 12.05

Hereinafter, a method of detecting biomolecules using nanohybrid particles will be described.

First, nanohybrid particles including core particles reacting to magnetic force and nanoparticles coated or adsorbed around the core particles are prepared. Next, biomolecules are reacted with the nanohybrid particles to hybridize (or immobilize) the biomolecules. Then, magnetic force is used to extract or analyze the nanohybrid particles hybridized with the biomolecules. The extracted hybrid nanoparticles are analyzed by using an analyzer. The analyzer is capable of measuring particle size and observing particle structure, a spectrometer for measuring absorbance, an X-ray for observing X-ray rotation patterns, and fluxgate. Sensors and the like. After extracting the nanohybrid particles hybridized with the biomolecules, the biomolecules may be injected into an antigen or an antibody that reacts with the biomolecules, and may be analyzed by observing an antigen-antibody reaction.

According to a preferred embodiment of the present invention, the nanoparticles are coated (or adsorbed) on iron (Fe) oxide (γ-Fe ₂ O ₃ or Fe ₃ O ₄ ) to make nanohybrid particles, and the nanohybrid particles By targeting the target biomolecules to measure the magnetic properties of the magnetic material, the biomolecule analysis process can be simplified and the analysis can be completed in a short time. Biomolecules may be separated and purified (or extracted) by immobilizing biomolecules on the nanohybrid particles, or nanohybrid particles immobilized on target biomolecules may be used as biochips. For example, a real antigen is immobilized on a glass plate on which Au is deposited, and hybridization of the biocomposite particle and the antigen hybridized with the target biomolecule to the nanohybrid particle is carried out by a magnetic force microscope. It can be measured. In addition to external measuring devices such as the magnetic force microscope, various magnetoresistances, alternating current (AC), direct current (DC) or a fluxgate sensor (a biomolecule detection device using magnetic force) designed as shown in FIG. It can be measured, which shows that the nanohybrid particles according to the preferred embodiment of the present invention are practical as biochips.

10A to 10D are diagrams schematically showing an apparatus for detecting biomolecules. FIG. 10A is a diagram schematically illustrating a layout of an analysis device for detecting biomolecules, and FIG. 10B is an enlarged view of a region 'A' of FIG. 10A, and FIG. 10C is a portion of FIG. 10B. FIG. 10D is a cross-sectional view of the II ′ of FIG. 10B. Hereinafter, a fluxgate sensor (analytical device for detecting biomolecules) designed as shown in FIGS. 10A to 10D will be described.

10A to 10D, a substrate 210 such as glass, a polymer, or the like is prepared, and the substrate 210 may be selectively etched to contain biomolecules or biocomposite particles 220 (hereinafter, 'bio Molecular support cells'). In this case, the biomolecule carrying cells 220 are implemented to form a matrix form regularly arranged in the horizontal and vertical directions. That is, one biomolecule carrying cell 220 is formed to be spaced apart from the other biomolecule carrying cell 220 at the same interval, and the biomolecule carrying cells 220 thus formed are regularly arranged in the horizontal and vertical directions. Form.

After depositing a metal film on the entire surface of the substrate 210 on which the biomolecule supporting cell 220 is formed, the magnetic force change detection lines 230b arranged in a horizontal or vertical direction are selectively formed by etching. The metal film may be a metal film such as gold (Au) or aluminum film (Al). When copper (Cu) is used as the metal film, the magnetic change detection wiring 230b may be formed using a damascene process. That is, after forming a region pattern (for example, a trench) in which the magnetic change detection wiring 230b is to be formed, the copper film may be deposited and chemically mechanically polished to form the magnetic change detection wiring 230b.

In the above-described embodiment, the case where the magnetic change sensing wiring 230b is formed after the biomolecule supporting cell 220 is formed has been described as an example, but the biomolecule supporting cell 220 after the magnetic change sensing wiring 230b is formed. Of course, it can also be formed.

The magnetic force change detection wiring 230b may be implemented in an n-angle (n≥4) or a circle shape surrounding each of the biomolecule carrying cells 220, and a section between each of the biomolecule carrying cells 220 may be implemented in a straight line form. . At this time, the number of turns of the magnetic change detection wiring 230a is set to 1 or more.

After depositing a metal film on the back surface of the substrate 210 on which the biomolecule carrying cell 220 is formed, the magnetic force generating wire 230a is formed to be selectively etched so as to be arranged in a direction perpendicular to the magnetic change detection wiring 230b. The metal film may be a metal film such as gold (Au) or aluminum film (Al). When copper (Cu) is used as the metal film, the magnetic field generating wiring 230a may be formed using a damascene process. That is, after forming a region pattern (for example, a trench) in which the magnetic field generating line 230a is to be formed, the copper layer may be deposited and chemically mechanically polished to form the magnetic field generating line 230a.

The center of the track formed by the magnetic force generation wiring 230a is formed to correspond to the center of the biomolecule carrying cell 220. The magnetic force generating wiring 230a may be implemented in an n-square (n≥4) or circle shape corresponding to each biomolecule carrying cell 220, and a section other than the part corresponding to each biomolecule carrying cell 220 is straight. It can be implemented in the form. At this time, the number of turns of the magnetic force generating wiring 230a is set to 1 or more.

Each region where the magnetic force change detecting wiring 230b and the magnetic field generating wiring 230a intersect corresponds to each of the biomolecule carrying cells 220, and the crossing regions are regularly arranged to form a matrix as a whole.

In the above-described embodiment, the case where the magnetic change detection wiring 230b is formed before the magnetic force generating wiring 230a has been described as an example. However, the magnetic force change detecting wiring 230b may be formed after the magnetic force generating wiring 230a is formed. Of course you can.

Each magnetic force generating wiring 230a is configured to be electrically connected to the electric classification means 240a. In addition, each magnetic force change detection wiring 230b is also configured to be electrically connected to the electrical classification detecting means 240b. The electric classification means 240a is connected to the electricity generating means 260, and the electric classification detecting means 240b is connected to the magnetic force change detecting means 250.

As such, the biomolecule carrying cell 220 to be detected may be selected by applying an electrical signal to each of the magnetic field generating lines 230a and the magnetic force change detecting lines 230b arranged in a matrix form.

Using the fluxgate sensor fabricated as described above, the biomolecule carrying cell contains the actual antigens in the biomolecule carrying cell 220 and the target biomolecules dispersed in the solvent are hybridized to the nanohybrid particles. By administering to the antigen contained in (220), hybridization with the antigen can be induced to immobilize the desired target biomolecule using an antigen-antibody reaction. The magnetic force is measured through the magnetic force generating line 230a and the magnetic force change detecting line 230b, and the electric classifying means 240a, the electric generating means 260, the electric class detecting means 240b, and the magnetic change detecting means 250 are measured. The location of the cell is determined through. Through this, when various kinds of antigens are put into each biomolecule carrying cell 220, the kinds of biomolecules can be grasped according to the respective antigen and antibody reactions.

In addition, a method of detecting a specific biomolecule in a solution in which several biomolecules are mixed is as follows. First, when a biocomposite particle in which a known biomolecule is hybridized to a nanohybrid particle is mixed in a solution in which several biomolecules including a specific biomolecule are mixed, a specific biomolecule and a known biomolecule hybridize to a nanohybrid particle. It is immobilized by the reaction of both ends between the biological composite particles. If this is immobilized after flowing into the biomolecule carrying cell 220 containing the known antigen, a system in which a specific biomolecule is immobilized can be obtained. As described above, only a specific biomolecule can be detected in a solution in which several biomolecules are mixed using an analysis device for detecting biomolecules using magnetic force.

The biomolecule detection system according to the present invention can be applied to quantitative analysis by measuring magnetization using hybridization between target biomolecules and probe biomolecules, and can be applied to biomolecule detection using antigen-antibody reactions.

Biomolecule detection according to a preferred embodiment of the present invention does not require a process of extracting and amplifying hybridized DNA, is a non-contact method, and does not need to add a separate marker, thus making the chip simple and having high sensitivity. .

As mentioned above, although preferred embodiment of this invention was described in detail, this invention is not limited to the said embodiment, A various deformation | transformation by a person of ordinary skill in the art within the scope of the technical idea of this invention is carried out. This is possible.

Claims (31)

Preparing core particles reacting to magnetic force and nanoparticles to be coated or adsorbed around the core particles; Measuring zeta potential with respect to pH between the core particles and the nanoparticles; Irradiating a pH section in which zeta potentials have opposite signs with respect to the core particles and the nanoparticles; Titrating the pH of the solution in which the core particles and the nanoparticles are mixed such that the zeta potentials of the core particles and the nanoparticles have opposite pH signs; Mixing the core particles and the nanoparticles in the solution to form nanohybrid particles in which the nanoparticles are coated or adsorbed around the core particles; Collecting the nanohybrid particles using a magnet; And Method of manufacturing nanohybrid particles for biomolecule detection comprising the step of drying the collected nanohybrid particles to obtain nanohybrid particle powder. The method of claim 1, further comprising dissolving the biomolecule in deionized water; Hybridizing the biomolecules to the nanohybrid particles by mixing the nanohybrid particles with deionized water in which the biomolecules are dissolved; And The method of manufacturing a nano-hybrid particles for biomolecule detection further comprising the step of collecting the bio-composite particles hybridized with the biomolecule and the nanohybrid particles using a magnet. The method of claim 1, wherein the core particles are γ-Fe ₂ O ₃ or Fe ₃ O ₄ , which is an iron oxide-based material. The method of claim 1, wherein the nanoparticles are gold (Au), silver (Ag), platinum (Pt), palladium (Pd), copper (Cu), nickel (Ni), zinc (Zn) or silicon oxide (SiO). ₂ ) a method for producing nanohybrid particles for detecting biomolecules. The method of claim 1, wherein the nanoparticles are gold (Au), the biomolecule is glutathione (glutathione), lectin (lectin), heavy metal ion (heavy metal ion), potassium ion (potassium ion), protein A (protein A) ), A method for producing nanohybrid particles for detecting biomolecules, characterized in that the HIV virus (HIV virus) or DNA. The method of claim 1, wherein the nanoparticles are gold (Au), the biomolecule comprises a thiol group (thiol group) having a mercapto group (-SH), the mercapto group of the biomolecule (- SH) is a method for producing nano-hybrid particles for biomolecule detection, characterized in that the gold (Au) coated around the core particles and reacts according to the following reaction formula. Scheme RSH + Au⇔RSAu + H ₂ (R is a hydrocarbon group). The method of claim 1, wherein the nanoparticles are silver (Ag) and the biomolecule is a urine protein. The method of claim 1, wherein the nanoparticles are silicon oxide (SiO ₂ ), and the biomolecule is lysozyme or bovine serum albumin. . The method of claim 1, wherein the nanoparticles are silicon oxide (SiO ₂ ), the biomolecule is a material containing a hydrocarbon group, Si-OH which is a surface group of the silicon oxide (SiO ₂ ) is the following reaction formula with the biomolecule Method of producing a nano-hybrid particles for the detection of biomolecules, characterized in that the reaction. Scheme SiOH + R⇔SiOR '(R and R' are the first and second hydrocarbon groups) Core particles responsive to magnetic force; And It comprises nanohybrid particles comprising nanoparticles coated or adsorbed around the core particles, The nanohybrid particles react with biomolecules to form biomolecules in which biomolecules are hybridized, and the biomolecules are nanomolecule detection systems using nanohybrid particles having a characteristic of reacting with a magnetic body. The biomolecule detection system according to claim 10, further comprising biomolecules that react with the nanoparticles and hybridize to the nanoparticles. The biomolecule detection system according to claim 10, wherein the core particles are γ-Fe ₂ O ₃ or Fe ₃ O ₄ , which are iron oxide materials. The method of claim 10, wherein the nanoparticles are gold (Au), silver (Ag), platinum (Pt), palladium (Pd), copper (Cu), nickel (Ni), zinc (Zn) or silicon oxide (SiO). ₂ ) a biomolecule detection system using nanohybrid particles, characterized in that. The method of claim 10, wherein the nanoparticles are gold (Au), and the biomolecule is glutathione (glutathione), lectin (lectin), protein A (protein A), HIV virus (HIV virus) or DNA, characterized in that Biomolecule detection system using nanohybrid particles. The method of claim 10, wherein the nanoparticles are gold (Au), the biomolecule comprises a thiol group (thiol group) having a mercapto group (-SH), the mercapto group of the biomolecule (- SH) is a biomolecule detection system using nanohybrid particles, characterized in that reacted with gold (Au) coated around the core particles according to the following reaction formula. Scheme RSH + Au⇔RSAu + H ₂ (R is a hydrocarbon group) The biomolecule detection system using nanohybrid particles according to claim 10, wherein the nanoparticle is silver (Ag) and the biomolecule is a urine protein. The biomolecule detection system according to claim 10, wherein the nanoparticle is silicon oxide (SiO ₂ ) and the biomolecule is lysozyme or bovine serum albumin. The method of claim 10, wherein the nanoparticles are silicon oxide (SiO ₂ ), the biomolecule is a material containing a hydrocarbon group, Si-OH is a surface group of the silicon oxide (SiO ₂ ) is the biomolecule and the following reaction formula Biomolecule detection system using nanohybrid particles, characterized in that the reaction according to. Scheme SiOH + R⇔SiOR '(R and R' are the first and second hydrocarbon groups) Preparing nanohybrid particles comprising core particles reacting to magnetic force and nanoparticles coated or adsorbed around the core particles; Reacting and hybridizing a biomolecule to the nanohybrid particles; Extracting the nanohybrid particles mixed with the biomolecules using a magnetic force; And A method for detecting biomolecules using nanohybrid particles comprising analyzing the extracted nanomolecule hybridized nanohybrid particles using an analytical device using magnetic force. The method of claim 19, further comprising extracting the nanohybrid particles mixed with the biomolecules, and then injecting them into an antigen or an antibody that reacts with the biomolecules to observe and analyze an antigen-antibody reaction. Biomolecule detection method using a nano-hybrid particles. 20. The method of claim 19, wherein the core particles are γ-Fe ₂ O ₃ or Fe ₃ O ₄ which is an iron oxide-based material. The method of claim 19, wherein the nanoparticles are gold (Au), silver (Ag), platinum (Pt), palladium (Pd), copper (Cu), nickel (Ni), zinc (Zn) or silicon oxide (SiO). ₂ ) biomolecule detection method using a nano-hybrid particles, characterized in that. The method of claim 19, wherein the nanoparticles are gold (Au), the biomolecule is glutathione (glutathione), lectin (lectin), protein A (protein A), HIV virus (HIV virus) or DNA, characterized in that Biomolecule detection method using nanohybrid particles. The method of claim 19, wherein the nanoparticles are gold (Au), the biomolecule comprises a thiol group (thiol group) having a mercapto group (-SH), the mercapto group of the biomolecule (- SH) is a method for detecting biomolecules using nanohybrid particles, characterized in that the reaction with gold (Au) coated around the core particles according to the following reaction formula. Scheme RSH + Au⇔RSAu + H ₂ (R is a hydrocarbon group). 20. The method of claim 19, wherein the nanoparticles are silver (Ag), and the biomolecule is a urine protein. 20. The method of claim 19, wherein the nanoparticles are silicon oxide (SiO ₂ ), and the biomolecule is lysozyme or bovine serum albumin. The method according to claim 19, wherein the nanoparticles are silicon oxide (SiO ₂ ), the biomolecule is a material containing a hydrocarbon group, and Si-OH, which is a surface group of the silicon oxide (SiO ₂ ), is represented by the following reaction formula: Biomolecule detection method using nanohybrid particles, characterized in that the reaction according to. Scheme SiOH + R⇔SiOR '(R and R' are the first and second hydrocarbon groups) Board; It is provided on the front surface of the substrate so that the biomolecules hybridized biomolecules or biomolecules contained in the nanohybrid particles including the core particles and nanoparticles coated or adsorbed around the core particles reacting to the magnetic force, And a plurality of biomolecule carrying cells arranged in a matrix in the longitudinal direction. A magnetic force change sensing wiring arranged in a horizontal or vertical direction on the front surface of the substrate; And It includes a magnetic field generating wiring arranged in the direction perpendicular to the magnetic change detection wiring on the back of the substrate, And an area where the magnetic force change detecting wiring and the magnetic field generating wiring intersect is provided to correspond to the biomolecule carrying cell, respectively. 29. The method of claim 28, wherein the magnetic force change detection wiring is provided in an n-square (n≥4) or circle shape surrounding each biomolecule carrying cell, and the number of turns thereof is at least one, and each biomolecule supporting is carried. An interval between cells is a biomolecule detection assay device using nano-hybrid particles, characterized in that provided in a straight form. 29. The magnetic force generating wire of claim 28, wherein a central portion of the orbit corresponds to a central portion of the biomolecule carrying cell, and a portion corresponding to each biomolecule carrying cell is provided in an n-square (n≥4) or circle shape. The orbit is at least one turn, and the section other than the portion corresponding to each biomolecule carrying cell is provided in a straight line form, the analysis device for detecting biomolecules using nanohybrid particles. The magnetic force generating wiring of claim 28, wherein the magnetic force generating wiring is electrically connected to the electric classification means and the electricity generating means so as to detect a magnetic force change in the biomolecule carrying cell, which is an area where the magnetic force change sensing wiring and the magnetic field generating wiring intersect. And the magnetic change detection wiring is connected to an electric classification detection means and a magnetic change detection means.

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