KR101022927B1 - Biochip using aptamer and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a biochip using aptamer and a method of manufacturing the same.

In particular, the present invention forms a plurality of protruding electrodes on the lower substrate of the container that can accommodate the aqueous solution containing the target material to increase the contact area of the electrode to the target material to improve the immobilization ability and improve the sensitivity of the aptamer It relates to a biochip used and a method of manufacturing the same.

Further, according to the present invention, the plate-shaped lower substrate; A plurality of protruding electrodes protruding from the lower substrate and made of a conductive material to have conductivity; And an aptamer attached to the plurality of protruding electrodes and comprising an aptamer for detecting a target substance.

Aptamers, protruding electrodes, aptamers, biochips, biosensors

Description

Biochip using aptamer and its manufacturing method {Biochip using aptamer and manufacturing method

The present invention relates to a biochip using aptamer and a method of manufacturing the same.

In particular, the present invention forms a plurality of protruding electrodes on the lower substrate of the container that can accommodate the aqueous solution containing the target material to increase the contact area of the electrode to the target material to improve the immobilization ability and improve the sensitivity of the aptamer It relates to a biochip used and a method of manufacturing the same.

Aptamers are single-stranded DNA or RNA molecules that have high affinity and selectivity for specific chemical or biological molecules by evolutionary methods using oligonucleotide libraries called systemic evolution of ligands by exponential enrichment (SELEX). Oligomers that bind to and are obtained (C. Tuerand L. Gold, Science 249, 505-510, 2005; AD Ellington and JW Szostak, Nature 346, 818-822, 1990; M. Famulok, et. Al. , Acc. Chem. Res. 33, 591-599, 2000; DS Wilson and Szostak, Annu. Rev. Biochem. 68, 611-647, 1999). Aptamers, recognized as replacements for antibodies, can be used as components of biosensors that can recognize molecules in detection and analysis systems, just like antibodies. Oligonucleotide-based aptamers have several advantages over protein-based antibodies (R. Nutiu, et. Al., Pure Appl. Chem. 76, 1547-1561, 2004). First, aptamers are obtained in vitro, and second, various organic and inorganic substances including toxins can be used as target molecules of aptamers. When specific aptamers that specifically bind are identified and obtained, they can be reproduced with low cost and high consistency by automated oligomer synthesis methods. In addition, aptamers are relatively easy to introduce useful functional groups such as fluorescent molecules or photoreactive groups, and the structure of aptamers is reversible due to the denaturation-renaturation process by heat. It can have a long time of activity. Recently, the first FDA-approved aptamer molecules have been developed (E. W. Ng, et. Al., Nat. Rev. Drug Discov. 5, 123-132, 2006). The aptamer is an RNA-based molecule with an anti-vascular endothelial growth factor (VEGF165) and dissociation constant (Kd) value of several tens of picomoles (pM) and is known to be effective in ocular vascular diseases.

As a molecule that plays a role in recognizing a target protein, such as an antibody, the field of aptamer has been developed a lot, but the aptamer itself does not have a property that can easily report a signal change upon binding to the target protein. In order to supplement this property of aptamers and maximize their function as probe molecules, an example of the attachment of a fluorescent substance to one end of an aptamer has been reported (N. Hamaguchi, et. Al., Anal. Biochem. 294, 126). 131, 2001). In this method, in the absence of a target protein, the aptamer forms a secondary structure, which reduces the fluorescence intensity of the fluorescent substance labeled on the aptamer. When binding to the target protein, the secondary structure of the aptamer is changed to cause a fluorescent signal. Use what is increased. This method has a disadvantage that each of the aptamers must be labeled with a fluorescent material, it is difficult to fix on the chip, there is a problem that takes a lot of cost and time required for diagnosis.

Among these problems, the difficulty of immobilization was slightly improved by applying a sol-gel to the chip in a state in which a target material was mixed and gelled.

However, compared to attaching an existing aptamer on a substrate and diagnosing a fluorescence signal, the amount of aptamer that can be fixed to the chip was increased slightly.

Accordingly, the present invention uses a method for fixing the aptamer to the protruding electrode to solve the above problems by increasing the degree of attachment of the aptamer to improve the immobilization ability of the biochip using the aptamer and its manufacturing method To provide.

In addition, the present invention is to solve the above problems by using a protruding electrode to increase the attachment degree of the aptamer by increasing the amount of target material to be combined to improve the sensitivity as a sensor using a biochip with aptamer It is to provide a method for producing the same.

The present invention for achieving the above object, the plate-shaped lower substrate; A plurality of protruding electrodes protruding from the lower substrate and made of a conductive material to have conductivity; And an aptamer attached to the plurality of protruding electrodes to detect a target substance.

In addition, the present invention is characterized in that the conductive material is made of a conductive material, and further formed on the lower substrate, further comprising a lower electrode for providing an electrical connection between the protruding electrode and the external analysis system.

In addition, the present invention is characterized in that the lower substrate is formed to a certain thickness, and further comprises a sol-gel composition for allowing the target material to be easily attached to the aptamer.

In addition, the target material of the present invention is characterized in that it can bind with the aptamer as a nucleic acid, protein or polysaccharide.

In addition, the present invention (A) forming a lower electrode on the lower substrate; (b) forming a protruding electrode to protrude from the lower electrode of the lower substrate; (c) forming an outer wall on the lower substrate to complete an accommodation container; And (d) attaching an aptamer to the protruding electrode.

In addition, the present invention is characterized in that it further comprises the step of removing the outer wall formed on the lower substrate.

In addition, the step (a) of the present invention comprises the steps of forming a polymer on the lower substrate; Forming a lower electrode pattern on the polymer; And forming a conductive material on the lower electrode pattern formed on the polymer to complete the lower electrode.

In addition, the step (d) of the present invention to form a linker by coating the protruding electrode; And a step of attaching the aptamer to the linker by pouring a gel-sol composition including an aptamer into the container.

According to the present invention as described above, as the electrode is formed on the lower substrate to protrude, the available area of attachment of the aptamer is widened, so that the degree of attachment of the aptamer is increased, thereby improving immobilization ability.

In addition, according to the present invention, as the degree of adhesion of the aptamer is increased by using the protruding electrode protruding from the lower substrate, the amount of the target material to be bonded is increased, thereby improving sensitivity.

Hereinafter, a biochip using an aptamer and a method of manufacturing the same according to an embodiment of the present invention will be described in detail with reference to FIG. 1.

1 is a cross-sectional view of a biochip using an aptamer according to a preferred embodiment of the present invention.

Referring to FIG. 1, a biochip using an aptamer according to a preferred embodiment of the present invention is formed vertically on an accommodation container 10, a lower substrate 10a of the accommodation container 10, and is made of a metal conductive material. A plurality of conductive protruding electrodes 12 and the lower substrate 10a of the receiving container 10 and formed of a conductive material are used to make electrical connections between the protruding electrodes 12 and an external analyzer (not shown). The lower electrode 12a to be provided, the aptamer 14 attached to the protruding electrode 12 to detect the target material, and formed at a predetermined thickness from the lower substrate 10a and include a conductive material or It is provided with a sol-gel composition 16 made of a porous material to enable the flow of the target material.

In such a configuration, the receiving container 10 includes a lower substrate 10a and an outer wall 10b formed perpendicular to the lower substrate 10a, and may accommodate the sol-gel composition 16 therein.

Here, it is preferable that the lower substrate 10a of the receiving container 10 is a glass, metal or plastic substrate whose surface is modified with amine, lysine, aldehyde, carboxylic acid, ester, maleimide or carbohydrate in a flat plate shape. In particular, a glass substrate whose surface is modified with an amine is preferable, but a glass substrate having no surface modification is also possible.

The outer wall 10b of the container 10 is preferably made of Teflon, glass, plastic, or the like, and may be formed of the same material as the lower substrate 10a of the container 10 or made of a different material. Alternatively, various types of height variations are possible.

Next, the protruding electrode 12 is formed of at least one material of gold, silver, copper, platinum, palladium, aluminum, copper, tungsten, titanium, chromium, iron, carbon, and carbon nanotubes. In addition, the protruding electrode 12 may be composed of a single material or fragments of two or more different materials, and is preferably oriented so as to be substantially perpendicular to the lower substrate 10a.

This protruding electrode 12 may have any suitable geometric shape. For example, the protruding electrode 12 may have a rod shape or a horn shape, and the cross section may have a circular or polygonal shape. The distal end of each protruding electrode 12 may also include one or more rounded triangles and pointed tips.

In addition, the protruding electrode 12 may have, for example, an aspect ratio of 1 or more and 20 or less. Here, the aspect ratio is defined as the ratio of the height of the protruding electrode 12 to the maximum width of each protruding electrode 12. Here, the reason why the protruding electrode 12 should be 1 or more is because the area is increased by 50% or more, so that the desired sensitivity and an increase in immobilization capacity can be sufficiently obtained. Because this is difficult. Preferably, the height of each protruding electrode 12 is preferably constant, but may be different from each other.

The lower electrode 12a is formed of at least one of gold, silver, copper, platinum, palladium, aluminum, copper, tungsten, titanium, chromium, iron, carbon, and carbon nanotubes.

The lower electrode 12a may be formed in a flat plate shape on the lower substrate 10a or may be formed in a flat plate shape inside the lower substrate 10a.

The lower electrodes 12a may be formed to be separated from each other with respect to each of the protruding electrodes 12, or may be formed so that all of the protruding electrodes 12 are connected to each other. The protruding electrodes belonging to the group by forming a group in rows or columns It can be formed so as to be connected only to (12).

Meanwhile, the aptamer 14 may be formed perpendicularly or inclined to the surface of the protruding electrode 12.

In addition, the aptamer 14 used herein refers to long or short oligonucleotides having high binding power to various targets, and the aptamer 14 is a nucleic acid optimized to specifically bind to a given ligand. . Such aptamers 14 are obtained by SELEX techniques that separate and amplify nucleic acids with specific enzyme functions or nucleic acids with ligand functions that bind to specific receptors from randomly synthesized nucleic acid sequence libraries. Because the aptamer sequence is folded into a unique tertiary structure and is produced to bind only a given ligand with high selectivity and binding power, the tertiary structure of the aptamer-ligand complex is optimized for specific ligand recognition only. The reason why the aptamer 14 can specifically recognize the ligand is that a large part of the ligand is surrounded by the nucleic acid, and the aptamer-ligand binding is the repulsive force, structural complementarity or hydrogen bond according to the ligand type. Or by chemical bonds.

The aptamer 14 can detect a protein or a microorganism, and there is no limitation in the kind thereof. The aptamer 14 is preferably stabilized with fluorine in the middle of the aptamer 14 for stabilization.

Next, since the sol-gel composition 16 includes a conductive material or has a porosity, and can add a biomaterial in a sol or gel state, the sol-gel composition 16 can highly integrate protein or low molecular weight material, thereby making a biochip manufactured. High efficiency screening (HTS) is possible using. In addition, since the sol-gel composition 16 is capable of accumulating enzymes used for measuring protein activity, the biochips thus prepared can be used in a method for measuring enzyme activity. The activity measuring enzymes include enzymes used for disease analysis, toxicity analysis, environmental analysis, food bacteria analysis.

Such sol-gel compositions 16 include, for example, bismuth titanate ferroelectrics containing samarium (BSmT; for example, compositions having the formula Bi _3.35 Sm _0.65 Ti ₃ O ₁₂ ), tetramethylorthosilicate (TMOS) , Tetraethylorthosilicate (TEOS), methyltrimethoxysilane (MTMOS), ethyltriethoxysilane (ETrEOS), trimethoxysilane (TMS), methyltrimethoxysilicate (MTMS) and 3-aminopropyltrimeth Any one or more silicate monomers selected from the group consisting of oxysilicates (3-ATMS), polyglyceryl silicates (PGS), diglyceryl silanes (DGS), 3-glycidoxy oxy trimethoxy silane (GPTMOS), (N -Triethoxysilylpropyl) -O-polyethylene oxide urethane (PEOU), glycerol and one or more additives selected from the group consisting of polyethylene glycol (PEG) having a molecular weight of 100 to 10,000 are mixed by mixing in various proportions.

2A to 2E are views illustrating a method of manufacturing a biochip using an aptamer according to an embodiment of the present invention.

Referring to FIG. 2A, a biochip using an aptamer according to an embodiment of the present invention forms a lower electrode 12a formed of a conductive material on a lower substrate 10a such as an insulating substrate.

Here, the lower substrate 10a is preferably a glass, metal or plastic substrate whose surface is modified with amines, lysine, aldehydes, carboxylic acids, esters, maleimides or carbohydrates. Glass substrates are preferred, but glass substrates with unmodified surfaces are also possible.

The lower electrode 12a is formed of at least one of gold, silver, copper, platinum, palladium, aluminum, copper, tungsten, titanium, chromium, iron, carbon, and carbon nanotubes.

For example, the lower electrode 12a may coat a polymer layer on the lower substrate 10a and imprint the polymer layer coated on the lower substrate 10a into a mold corresponding to the shape of the lower electrode 12a. After imprinting, the lower electrode 12a is manufactured by depositing on the imprinted polymer layer.

Thereafter, as illustrated in FIG. 2B, a plurality of protruding electrodes 12 made of a conductive material are formed on the lower substrate 10a thus formed.

For example, the protruding electrode 12 may coat the polymer layer on the lower substrate 10a on the lower substrate 10a, and the polymer layer coated on the lower substrate 10a may correspond to the shape of the lower electrode 12a. After imprinting into a mold, the lower electrode 12a is manufactured by depositing on the imprinted polymer layer.

Next, referring to FIG. 2C, the outer wall 10b capable of accommodating the sol-gel composition is formed in the state in which the protruding electrode 12 made of the conductive material is formed on the lower substrate 10a.

Then, a sol-gel composition including an aptamer 14 is poured into the container 10 on the surface of the protruding electrode 12 formed on the lower substrate 10a as shown in FIG. 2D, and as shown in FIG. 2E. Curing 14 is formed on the protruding electrode 12 and a sol-gel composition is formed around the protruding electrode 12 on which the aptamer 14 is immobilized.

As the immobilization method, a piezoelectric method such as a direct contact method, an ink jet method, or an electrical method can be used.

In the immobilization method, the direct contact method is to couple the aptamer 14 to the protruding electrode 12 by direct chemisorption or physisorption. For this purpose, the surface of the protruding electrode 12 is first functionalized with a relatively short linker molecule. . For example, the surface may be amine-functionalized by coating. This amine-functionalized linker immobilizes the aptamer 14 on the surface.

Although not shown later, the outer wall 10b of the container may be removed to manufacture a biochip without the outer wall 10b (of course, the outer wall 10b may not be removed).

Next, a biomolecule detection system such as nucleic acid, protein or polysaccharide by the biochip according to the present invention will be described in more detail. In particular, it will be described by way of example DNA that is not easily denatured in the biological molecule, and easy to analyze. The purpose of such an analytical detection system is, for example, by contacting a DNA sample (target DNA) to be analyzed with respect to a biochip in which the aptamer 14 is arranged, and hybridizing as shown in FIGS. 3A to 3C. It is intended to accurately measure the hybridization result. As a result, the distribution of DNA with a particular sequence in the sample to be analyzed is measured.

3a to 3c are electrical measurements of the change in the amount of charge according to the presence or absence of aptamer 14 and the target DNA 19 on the protruding electrode 12 using a biochip using an aptamer according to an embodiment of the present invention 2 is a diagram illustrating a biomolecule detection process using a biomolecule detection system capable of measuring the binding of the target DNA 19.

This will be described in detail as an example of voltage measurement. (For reference, current measurement and all electrochemical measurements such as changes in voltage or current due to voltage or current change are possible.) First, as shown in FIG. When the target DNA 19 is not coupled to the aptamer 14 attached to 12), a constant voltage of Vin is applied to the reference electrode 18 and the output Vout of the lower electrode 12a is measured. The potential difference between Vin and the lower electrode 10a becomes Vd.

Next, as shown in FIG. 3B, the target DNA sample is poured into the container 10, and the target DNA sample (target DNA) is brought into contact with the aptamer 14 attached to the protruding electrode 12. As shown in FIG. 3C, the target DNA 19 is hybridized with the aptamer 14 by a structural specific bond or a chemical bond.

When Vin is turned on after the target DNA 19 is hybridized with the aptamer 14 by hydrogen bonding, the voltage applied to the lower electrode 12a becomes the voltage Vd 'according to the target DNA binding. The voltage value changes in proportion to the extent coupled to the aptamer 14.

Here, the reason why the voltage value is changed when the target DNA is attached to the aptamer 14 attached to the protruding electrode 12 is due to the change in capacitance or impedance between the reference electrode 18 and the protruding electrode 12. This results in a change in output voltage.

In this way, the degree of change in the voltage value of the reference electrode 18 and the lower electrode 14 can be read and analyzed for the degree of binding of the aptamer 14 and the target DNA 19 in the biochip structure according to the change amount.

1 is a cross-sectional view of a biochip using aptamers according to a preferred embodiment of the present invention.

2a to 2e is a view showing a method of manufacturing a biochip using aptamers according to an embodiment of the present invention.

3A to 3C are diagrams illustrating a biomolecule detection process using a biomolecule detection system using a biochip using an aptamer according to an embodiment of the present invention.

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

10: accommodating container 10a: lower substrate

10b: outer wall 12: protruding electrode

12a: lower electrode 14: aptamer

16 sol-gel composition 18 reference electrode

19: target substance

Claims (8)

Plate-shaped lower substrate; A plurality of protruding electrodes protruding from the lower substrate and made of a conductive material to have conductivity; An aptamer attached to the plurality of protruding electrodes for detecting a target substance; And A biochip using an aptamer formed on the lower substrate to a predetermined thickness and comprising a sol-gel composition for easily attaching the target material to the aptamer. The method according to claim 1, A biochip using an aptamer made of a conductive material, having conductivity, and formed on the lower substrate, and further including a lower electrode providing electrical connection between the protruding electrode and an external analysis system. delete The method according to claim 1 or 2, The target material is a biochip using an aptamer, characterized in that the binding to the aptamer as a nucleic acid, protein or polysaccharide. (a) forming a lower electrode on the lower substrate; (b) forming a protruding electrode to protrude from the lower electrode of the lower substrate; (c) forming an outer wall on the lower substrate to complete an accommodation container; And (d) attaching an aptamer to the protruding electrode, Step (d) Coating the protruding electrode to form a linker; And A method of manufacturing a biochip using an aptamer, comprising: attaching an aptamer to the linker by pouring a gel-sol composition including an aptamer into the container. The method according to claim 5, (e) a method for manufacturing a biochip using an aptamer, further comprising removing an outer wall formed on the lower substrate. The method according to claim 5, Step (a) is Forming a polymer on the lower substrate; Forming a lower electrode pattern on the polymer; And And forming a conductive material on the lower electrode pattern formed on the polymer to complete the lower electrode. delete

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