KR101837827B1 - Biomolecule detecting sensor, method for manufacturing the same and biomolecule detecting method - Google Patents

Biomolecule detecting sensor, method for manufacturing the same and biomolecule detecting method Download PDF

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KR101837827B1
KR101837827B1 KR1020160028630A KR20160028630A KR101837827B1 KR 101837827 B1 KR101837827 B1 KR 101837827B1 KR 1020160028630 A KR1020160028630 A KR 1020160028630A KR 20160028630 A KR20160028630 A KR 20160028630A KR 101837827 B1 KR101837827 B1 KR 101837827B1
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biochemical molecule
biochemical
nanostructure
fluorescence
dimensional fluorescence
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이내응
쉬리바스타바 사잘
이원일
손영민
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성균관대학교산학협력단
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Abstract

A biochemical molecule detection apparatus is disclosed. The biochemical molecule detection apparatus comprises: a substrate; At least one nanostructure fixed to the substrate and grown vertically from the substrate; And an amphoteric complex which is fixed on the surface of the nanostructure and is capable of selectively binding with a biochemical molecule and comprises at least one double helix DNA intercalated with a fluorescent dye for generating fluorescence, As the amount of the biochemical molecule that reacts with the platameric complex increases, the fluorescence can be reduced.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a biomolecule detection apparatus, a biomolecule detection apparatus, and a biomolecule detection method.

The present invention relates to a biochemical molecule detection apparatus capable of quantitatively detecting biochemical molecules using fluorescence from a substrate, and more particularly, to a quantitative biochemical molecule detection apparatus having a three-dimensional structure, a method for producing the same, The present invention relates to a quantification method capable of detecting a biochemical molecule using a biochemical molecule detection apparatus.

Conventional fluorescent extender sensors have been developed as sensors for detecting biochemicals by measuring and analyzing the presence or absence of fluorescence and the change in brightness using a spectrophotometer, but they are limited to measurement in a liquid phase, There is a problem in that there is a limit to the measurement using the detector of FIG. To solve this problem, the use of a signal amplification substrate utilizing a fluorescent amplification nanostructure has been attempted, but it is difficult to realize a sufficient fluorescence amplification function.

For example, in the case of conventional fluorescence magnets using zinc oxide nanorods, the absolute amount of fluorescence intensity due to the probe magnets fixed over the entire three-dimensional structure can not be accurately analyzed during fluorescence measurement using a conventional spectrometer Has a disadvantage. In order to quantify the fluorescence using the three-dimensional structure based on the fluorescence-amplified nanomaterial, a new fluorescence quantification technique capable of quantifying the fluorescence signal originating from the entire three-dimensional structure as well as the fluorescence originating from the top is required. Additional nanomaterial control technology is needed to complement this.

In order to utilize the fluorescence quantitation technology based on the three-dimensional imaging, the zinc oxide nano-rods must be grown to be completely perpendicular on the substrate and the density and the aspect ratio can be controlled in order to improve the accuracy. If the density of the nanorods is excessively high, the binding of the target material may be reduced due to the narrow space. If the density of the nanorods is too low, the amount of the target material to be bonded as a whole may decrease, The introduction of techniques to control the density and aspect ratio is necessary to solve and complement the problems arising in the prior art. In addition, when using fluorescence image-based fluorescence quantitation technology using an optical camera as a detector, the fluorescence signal of a single plane based on a certain point with respect to the longitudinal direction of the zinc oxide nano-rod is different depending on the imaging and analysis 3-D fluorescence quantitation technique is needed to compensate the fluorescence analysis of the nanorod in the longitudinal direction.

It is an object of the present invention to provide a three-dimensional structure capable of increasing the surface area capable of binding with a biochemical molecule by using a three-dimensional structure and detecting low-concentration biochemical molecules by using a three- And a method for producing the same.

Further, it is a further object of the present invention to provide a method for detecting a biochemical molecule using a quantification method using a three-dimensional fluorescence image.

According to an aspect of the present invention, there is provided a biochemical molecule detection apparatus comprising: a substrate; At least one nanostructure fixed to the substrate and grown vertically from the substrate; And an amphoteric complex which is fixed on the surface of the nanostructure and is capable of selectively binding with a biochemical molecule and comprises at least one double helix DNA intercalated with a fluorescent dye for generating fluorescence, As the amount of the biochemical molecule that reacts with the platameric complex increases, the fluorescence can be reduced. Fluorescence intensity before reacting with a biochemical molecule can be checked by comparing fluorescence intensity after reacting with a biochemical molecule.

In one embodiment, the double helix DNA comprises a first nucleic acid strand having a first nucleotide sequence capable of selectively binding to the biochemical molecule; And a second nucleic acid strand having a second nucleotide sequence complementary to the first nucleotide sequence, wherein the fluorescent dye may be intercalated between the first nucleic acid strand and the second nucleic acid strand have.

In one embodiment, a gallium nitride or gallium nitride alloy thin film is formed on the surface of the substrate, and the nanostructure may be a zinc oxide nanorod grown on the thin film.

In one embodiment, the surface of the nanostructure may be carboxylated.

A method of fabricating a biochemical molecule detection device according to an embodiment of the present invention includes: growing one or more nanostructures on a substrate vertically; Immobilizing an amphoteric complex comprising at least one double helix DNA capable of selectively binding to a biochemical molecule on the surface of the nanostructure; And intercalating the fluorescent dye into the double helix DNA.

In one embodiment, the step of vertically growing one or more nanostructures on the substrate includes the steps of: forming a thin film of gallium nitride or gallium nitride on the substrate using a zinc oxide precursor solution to which polyethyleneimine molecules are added, And growing a zinc oxide nanorod on the thin film.

The method of detecting a biochemical molecule according to an embodiment of the present invention includes the steps of: reacting the biochemical molecules at different concentrations with the biochemical molecule detection apparatus; Dividing each of the biochemical molecules at different concentrations into a plurality of sections along a longitudinal direction of the nanostructure and obtaining two-dimensional fluorescence images for each of the plurality of sections; Obtaining, for each of the different concentrations of the biochemical molecule, one three-dimensional fluorescence image using the two-dimensional fluorescence images; Quantifying the change in fluorescence intensity of the three-dimensional fluorescence image according to the concentration of the biochemical molecule; And detecting the biochemical molecule using the biochemical molecule detection apparatus based on the quantification result.

In one embodiment, the two-dimensional fluorescence images may be images obtained by visualizing fluorescence generated along a direction perpendicular to the longitudinal direction of the nanostructure.

In one embodiment, the three-dimensional fluorescence image can be obtained by incorporating the two-dimensional fluorescence images.

The present invention as described above has an effect of increasing the concentration range capable of detecting biochemical molecules and detecting a low concentration of biochemical molecules.

The present invention can control the density of the nanostructures fixed on the substrate and the aspect ratio of the nanostructure so that the efficiency with which the biochemical molecules can be bonded can be increased and the nanostructure can be grown on the substrate .

The present invention has an effect of quantifying the concentration of an unknown biochemical molecule even when an unknown biochemical molecule is detected by deriving an analytical function of the fluorescence intensity according to the concentration of the biochemical molecule.

1 is a view for explaining a biochemical molecule detection apparatus according to an embodiment of the present invention.
2 is a view for explaining a state after a biochemical molecule is reacted with a biochemical molecule detection apparatus according to an embodiment of the present invention.
3 is a flowchart illustrating a method of manufacturing a biochemical molecule detection apparatus according to an embodiment of the present invention.
4 is a flowchart illustrating a method of detecting a biochemical molecule according to an embodiment of the present invention.
5 is a view for explaining a method of detecting a biochemical molecule according to an embodiment of the present invention.
FIG. 6 is a graph illustrating an analysis function using a two-dimensional fluorescence image obtained at the top of a nanostructure as the concentration of ATP added to the biochemical molecule detection apparatus according to an embodiment of the present invention is increased.
FIG. 7 is a graph showing an analysis function derived by the method of detecting a biochemical molecule according to an embodiment of the present invention.

While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the invention is not intended to be limited to the particular embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings. Wherein like reference numerals refer to like elements throughout.

FIG. 1 is a view for explaining a biochemical molecule detection apparatus having a three-dimensional structure according to an embodiment of the present invention. FIG. 2 is a schematic diagram of a biochemical molecule detection apparatus having a three- And Fig.

1, a biochemical molecule detection apparatus 100 having a three-dimensional structure according to an embodiment of the present invention includes a substrate 10, at least one nanostructure 20, and double helix DNAs 30 and 40 Platameric < / RTI > For example, the substrate 10 may be a sapphire substrate, and a thin film layer made of a gallium nitride compound is formed on the surface of the substrate 10. The gallium nitride compound may be composed of, for example, gallium nitride (GaN), gallium nitride (aluminum gallium nitride (AlGaN), or the like). Such a thin film layer can serve as a buffer.

The one or more nanostructures 20 can be grown vertically from the substrate 10. For example, the nanostructure may be a zinc oxide nanorod. The surface of the nanostructure may be carboxylated for binding to double helix DNA (30). When zinc oxide nanorods are grown on a thin film made of gallium nitride (GaN) or gallium nitride (aluminum gallium nitride (AlGaN), etc.), zinc oxide nanorods can be grown perfectly vertically.

The platameric complex is intercalated with a fluorescent dye that generates fluorescence, and the platameric complex is immobilized on the surface of the nanostructure 20 and can selectively bind to the biochemical molecule. Fluorescent dye may be released when reacting with biochemical molecules. For example, double-stranded DNAs (30, 40) can be placed on the nanostructure surface by bonding one end of the double-stranded DNA (30, 40) with the surface of the carboxylated nanostructure.

The double helix DNAs 30 and 40 include a first nucleic acid strand 30 that can function as an aptamer, that is, a first nucleotide sequence region capable of selectively binding with a biochemical molecule, And a second nucleic acid strand 40 having a second nucleotide sequence region complementary to the first nucleotide sequence region and forming a complementary strand with the first nucleotide sequence region. The first nucleic acid strand 30 and the second nucleic acid strand 40 may form double helix DNAs 30 and 40 through hybridization. In addition, the fluorescent dye 50 may be intercalated between the first nucleic acid strand 30 and the second nucleic acid strand 40. Fluorescence may appear in the double stranded DNAs 30 and 40 through the fluorescent dye 50. For example, Sybr green I may be used as the fluorescent dye (50).

Referring to FIG. 2, when the double helix DNAs 30 and 40 react with biochemical molecules, the double helix is released and the fluorescent dye 50 can be released. When the fluorescent dye solution 50 is released, the intensity of fluorescence generated from the platemater complex including the double helix DNAs 30 and 40 is reduced. It is detected whether the biochemical molecule has reacted . For example, ATP molecules may be used as the biochemical molecule, but the present invention is not limited thereto.

3 is a flowchart illustrating a method of manufacturing a biochemical molecule detection apparatus according to an embodiment of the present invention.

Referring to FIG. 3, a method for fabricating a biochemical molecule detection apparatus having a three-dimensional structure according to an embodiment of the present invention includes vertically growing one or more nanostructures on a substrate (S110) (S120) of fixing the platemater complex comprising one or more double helix DNAs capable of selectively binding (S120) and intercalating the fluorescent dye into the double helix DNA (S130).

One or more nanostructures are vertically grown on the substrate (S110). A zinc oxide nano rod is vertically grown on a sapphire substrate having a thin film layer formed of a gallium nitride compound (gallium nitride (GaN) or aluminum gallium nitride (AlGaN)). For example, a zinc oxide nanorod can be grown on the thin film completely using a zinc oxide precursor solution to which a polyethyleneimine molecule is added on the substrate on which a thin film of gallium nitride or gallium nitride alloy is formed. The density at which the nanostructures are arranged on the substrate and the aspect ratio of the nanostructures during the growth of the at least one nanostructure can be controlled by the addition of polyethyleneimine and the addition of the polyethyleneimine allows the nanostructures to grow from the substrate in a highly dense, Growth. When the nanostructure is grown at a high density, the surface area capable of reacting with the biochemical molecule can be improved, so that the detection performance of the biochemical molecule can be improved.

After the surface of the vertically grown zinc oxide nanorods is carboxylated, an amphoteric complex containing at least one double helix DNA capable of selectively binding with a biochemical molecule is immobilized on the surface of the nanostructure (S120). The double helix DNA comprises a first nucleic acid strand having a first nucleotide sequence capable of selectively binding to a biochemical molecule and a second nucleotide strand complementary to the first nucleotide sequence and forming a complementary strand with the first nucleotide sequence And hybridizing the second nucleic acid strand having the second nucleotide sequence region with the second nucleic acid strand.

Next, the fluorescent dye is intercalated into the double helix DNA (S130). Through this process, a biochemical molecule detection apparatus having a three-dimensional structure can be manufactured.

FIG. 4 is a flowchart for explaining a method of detecting a biochemical molecule according to an embodiment of the present invention, and FIG. 5 is a view for explaining a method of detecting a biochemical molecule according to an embodiment of the present invention.

4 and 5, a method for detecting a biochemical molecule according to an embodiment of the present invention includes the steps of reacting (S210) the biochemical molecules at different concentrations to the biochemical molecule detection device (S210) (S220) for each biochemical molecule of concentration, dividing the biomolecule molecule into a plurality of sections along the longitudinal direction of the nanostructure and obtaining two-dimensional fluorescence images for each of the sections divided into the plurality of sections, Obtaining a three-dimensional fluorescence image using the two-dimensional fluorescence images for each of the biochemical molecules of the three-dimensional fluorescence image (S230), quantifying the fluorescence intensity change of the three-dimensional fluorescence image according to the concentration of the biochemical molecule S240) and detecting the biochemical molecule using the biochemical molecule detection device (S250) based on the quantification result .

The biochemical molecules of different concentrations are respectively reacted with the platemater complex of the biochemical molecule detection apparatus (S210). In the case of double-stranded DNA and biochemical molecules in a biochemical molecule detection device, the fluorescent dye can be separated, and the intensity of fluorescence generated from the platameric complex containing the double-stranded DNA can be reduced.

Next, for each of the biochemical molecules having different concentrations, the biomolecule is divided into a plurality of sections along the longitudinal direction of the nanostructure, and two-dimensional fluorescence images are acquired for each of the sections divided into the plurality of sections (S220) . The two-dimensional fluorescence images may be images that visualize fluorescence occurring along a direction perpendicular to the longitudinal direction of the nanostructure with respect to each point. Such a two-dimensional fluorescence image can be obtained through a CCD camera or a CID camera. For example, the two-dimensional fluorescence images may be images obtained by visualizing fluorescence generated from the upper portion of the nanostructure along a direction perpendicular to the longitudinal direction of the nanostructure. Two-dimensional images can be obtained for each concentration of biochemical molecules.

Next, for each of the biochemical molecules having different concentrations, one three-dimensional fluorescence image is obtained using the two-dimensional fluorescence images (S230). One three-dimensional fluorescence image can be obtained by incorporating two-dimensional fluorescence images for each of the different concentrations of the biochemical molecule. As an example, a three-dimensional fluorescence image can be obtained by stacking and integrating two-dimensional fluorescence images sequentially for each point. Three-dimensional fluorescence images can be obtained through digital imaging techniques.

Next, the fluorescence intensity change of the three-dimensional fluorescence image according to the concentration of the biochemical molecule is quantified (S240). For example, the fluorescence intensity of a three-dimensional fluorescence image can be quantified by deriving an analytical function of the fluorescence intensity of the three-dimensional fluorescence image according to the concentration of the introduced biochemical molecule.

Fluorescence intensities for three-dimensional fluorescence images can be measured by fluorescence intensity normalized through a program, and fluorescence intensities for three-dimensional fluorescence images are for concentrations of introduced biochemical molecules. Therefore, it is possible to measure the intensity of fluorescence with respect to the concentration of the biochemical molecule, and measure the fluorescence intensities for the three-dimensional fluorescence images with different concentrations of the biochemical molecules, Can be derived. The analytical function can be represented as a linear graph of the intensity of three-dimensional fluorescence images according to each concentration.

Finally, based on the quantification result, the biochemical molecule detection apparatus detects the biochemical molecule (S240).

When the analytical function is derived, a biochemical molecule having an unknown concentration is put into a biochemical molecule detection apparatus, and fluorescence intensity of the three-dimensional fluorescence image is measured. The concentration corresponding to the fluorescence intensity measured in the analytical function is measured The biochemical molecule can be detected by determining the unknown concentration of the biochemical molecule.

FIG. 6 is a graph illustrating an analysis function using a two-dimensional fluorescence image obtained at the top of a nanostructure as the concentration of ATP added to the biochemical molecule detection apparatus according to an embodiment of the present invention is increased.

The concentration of ATP was increased from 1 pM to 100 uM in a biochemical molecule detection apparatus with a 10 - fold increase in concentration. Two - dimensional images according to each concentration were obtained from a biochemical molecular detector having a three - dimensional structure. That is, a camera was disposed toward the upper surface of a nanostructure of a biochemical molecule detection apparatus having a three-dimensional structure, and two-dimensional images corresponding to respective concentrations were obtained through a camera.

Referring to FIG. 6, as the concentration of ATP increases, the fluorescence intensity decreases. This is because the first nucleic acid strand and the second nucleic acid strand of the double-stranded DNA are released by ATP and the intercalated cyto- green I is separated from the first and second nucleic acid strands, This is because no fluorescence is generated from Sybr green I). Although the analytical function has a linear graph shape, it can be confirmed that the fluorescence intensity measured at each concentration has a large error from the intensity value of the analytical function.

FIG. 7 is a graph showing an analysis function derived by the method of detecting a biochemical molecule according to an embodiment of the present invention.

Referring to FIG. 7, an analysis function for fluorescence intensities of three-dimensional fluorescence images measured by injecting ATP molecules while increasing the concentration by 10 times from 1 pM to 100 uM can be confirmed. As the concentration increases, the fluorescence intensities tend to decrease. As shown in the graph of FIG. 6, as the concentration of ATP increases, the fluorescence intensity decreases, but a more accurate linearization graph is obtained than the graph of FIG. That is, by using three-dimensional fluorescence images, a more accurate analysis function can be derived.

Also, as compared with FIG. 6, it can be confirmed that the intensity of fluorescence increases as a whole. This means that the fluorescence intensity quantified from the three-dimensional fluorescence image is further increased, and the detection of ATP molecules at low concentration is also possible because the fluorescence intensity is increased. This is because it is difficult to confirm whether ATP molecules are detected when the fluorescence intensity is small.

Therefore, by using the biochemical molecule detection method using the biochemical molecule detection apparatus according to the embodiment of the present invention, it is possible to detect low concentration biochemical molecules (that is, the range of concentration capable of detecting biochemical molecules increases) The concentration of the molecule can be quantified.

While the invention has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. Accordingly, the true scope of the present invention should be determined by the following claims.

100: Biochemical molecule detecting apparatus having a three-dimensional structure 10:
20: nanostructure 30: first nucleic acid strand
40: second nucleic acid strand 50: fluorescent dye

Claims (9)

Board;
At least one nanostructure fixed to the substrate and grown vertically from the substrate; And
A platameric complex fixed on a surface of the nanostructure and capable of selectively binding with a biochemical molecule and comprising at least one double helix DNA intercalated with a fluorescent dye for generating fluorescence,
As the amount of the biochemical molecule that reacts with the platelet complex increases, the fluorescence decreases
By including one or more platemeric complexes on the surface of the nanostructures grown perpendicular to the substrate,
Acquiring two-dimensional fluorescence images for each of the plurality of sections divided by the plurality of sections along the longitudinal direction of the nanostructure, acquiring one three-dimensional fluorescence image using the two-dimensional fluorescence images Lt; RTI ID =
Biochemical molecule detection device.
The method according to claim 1,
The double-
A first nucleic acid strand having a first nucleotide sequence capable of selectively binding to said biochemical molecule; And
And a second nucleic acid strand having a second nucleotide sequence complementary to the first nucleotide sequence,
Wherein said fluorescent dye is intercalated between said first nucleic acid strand and said second nucleic acid strand.
The method according to claim 1,
Wherein a thin film of gallium nitride or gallium nitride is formed on the surface of the substrate,
Wherein the nanostructure is a zinc oxide nanorod grown on the thin film.
The method according to claim 1,
Wherein the surface of said nanostructure is carboxylated.
delete delete Reacting the biochemical molecules at different concentrations with the biochemical molecule detection device of any one of claims 1 to 4, respectively;
Dividing each of the biochemical molecules at different concentrations into a plurality of sections along a longitudinal direction of the nanostructure and obtaining two-dimensional fluorescence images for each of the plurality of sections;
Obtaining, for each of the different concentrations of the biochemical molecule, one three-dimensional fluorescence image using the two-dimensional fluorescence images;
Quantifying the change in fluorescence intensity of the three-dimensional fluorescence image according to the concentration of the biochemical molecule; And
And detecting the biochemical molecule using the biochemical molecule detection apparatus based on the quantification result.
8. The method of claim 7,
Wherein the two-dimensional fluorescence images are images obtained by visualizing fluorescence generated along a direction perpendicular to the longitudinal direction of the nanostructure.
8. The method of claim 7,
Wherein the three-dimensional fluorescence image is obtained by integrating the two-dimensional fluorescence images.
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