KR20150041982A - Method for Detecting Theophylline Using Silver Nanoclusters Formation - Google Patents
Method for Detecting Theophylline Using Silver Nanoclusters Formation Download PDFInfo
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Abstract
The present invention relates to a method for detecting or quantifying theophylline using silver nanoclusters. More particularly, the present invention relates to a method for detecting or quantifying theophylline using silver nanoclusters formed by adding silver ions to a reaction product of a double- To a method for detecting or quantifying the theophylline.
The detection or quantification method of theophylline using silver nanocluster formation according to the present invention is an easy and simple detection or quantification method which does not require labeling of expensive fluorescent material, Practical application is easy to analyze the sample.
Description
The present invention relates to a method for detecting or quantifying theophylline using silver nanoclusters. More particularly, the present invention relates to a method for detecting or quantifying theophylline using silver nanoclusters formed by adding silver ions to a reaction product of a double- To a method for detecting or quantifying the theophylline.
Theophylline, a bronchodilator used as a treatment for asthma and chronic obstructive pulmonary disease, has a narrow therapeutic range and has side effects that can cause permanent nerve damage if the blood concentration is too high Ferapontova et al ., Electroanalysis . 21: 1261-1266, 2009). Due to these problems, a detection system for precise quantitative analysis of theophylline drugs is needed.
Most of the theophylline is analyzed by gas and liquid chromatography, ultraviolet spectrophotometry and enzyme immunoassay in a diagnostic laboratory. However, these conventional methods require complicated experimental procedures and skilled techniques and have a disadvantage of long analysis time Have. In addition, these existing methods are difficult to distinguish theophylline from the structurally similar caffeine (Caffeine) and theobromine (Theobromine), which makes it difficult to accurately analyze theophylline and can generate a false positive signal .
As an alternative to this, RNA aptamers that specifically bind to theophylline and cause structural changes have been introduced and various detection systems have been developed based thereon (Jenison et < RTI ID = 0.0 > al ., Science . 263: 1425-1429,1994; Pernites et al ., Biosens . Bioelectron . 26: 2766-2771, 2011; Ferapontova et al ., J. Am . Chem. Soc . 130: 4256-4258, 2008; Ferapontova et al ., Langmuir . 25: 4279-4283, 2009; Stojanovic et al., J. Am . Chem . Soc . 126: 9266-9270, 2004; Rankin et al., Nucleosides Nucleotides Nucl . Acids . 25: 1407-1424, 2006). However, RNA has a problem that it is easily degraded through chemical and enzymatic action, and it is disadvantageous in that it is difficult to manufacture compared to DNA and is expensive.
To overcome these drawbacks, recently Teramae et al . Has developed a theophylline fluorescence detection system by introducing riboflavin, a fluorescent ligand that competitively binds the theophylline to the basic site and exhibits a fluorescence signal change (Sato et < RTI ID = 0.0 > al ., Chem .- Eur . J. 18: 12719-12724, 2012). The system was able to detect theophylline easily without labeling a particular fluorescent substance, but the introduced fluorescent ligand competitively binds to the basic site with theophylline and inhibits the binding of theophylline, thereby affecting the detection sensitivity. Fluorescent ligands also have low fluorescence emission intensities and a fatal disadvantage of poor photostability.
As a result, the present inventors have solved the problems of the above-described prior arts and have made efforts to easily and inexpensively analyze theophylline. As a result, silver nano clusters having fluorescence characteristics, which are made of DNA as a template, A recent report that can be made (Ma et al ., Nanotechnology . 22: 305502, 2011), it has been found for the first time that the formation of silver nanoclusters can be controlled by the presence of theophylline. Based on this finding, it is not necessary to label expensive fluorescent materials, Has developed a simple and easy new type of theophylline detection system using nanoclusters.
It is an object of the present invention to provide a simple and easy new type of theophylline detection or quantification method using silver nanoclusters which do not require labeling of expensive fluorescent materials and have high fluorescence signal and light stability.
In order to accomplish the above object, the present invention provides a method for preparing a biocompatible nucleic acid comprising the steps of: (a) reacting a sample containing a theophylline with a double stranded DNA comprising an abasic site and arranged in the direction of azithromycin; (b) adding silver ions and a reducing agent to the reactant of the double-stranded DNA and the theophylline-containing sample to form silver nanoclusters; And (c) analyzing the formed silver nanoclusters to detect or quantify the theophylline.
The detection or quantification method of theophylline using silver nanocluster formation according to the present invention is an easy and simple detection or quantification method which does not require labeling of expensive fluorescent material, Practical application is easy to analyze the sample.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing the principle of development of a method for the analysis of theophylline using double-stranded DNA comprising the basic region of the present invention. FIG. In the absence of theophylline, the cytosine arranged opposite to the basic region forms an Ag + -cytosine complex through interaction with silver ions (Ag + ), and a fluorescent substance (NaBH 4 ) Thereby forming a nanocluster. On the other hand, in the presence of theophylline, the abasic site facing the cytosine binds to theophylline and blocks the interaction of silver ions and cytosine. As a result, a silver nanocluster having fluorescence characteristics is not formed even when a reducing agent is added.
FIG. 2 is a graph illustrating the formation of silver nanoclusters according to the presence or absence of the theophylline. In the absence of the theophylline of (a) 3 and (b), the maximum fluorescence signal was generated at 640 nm through the formation of silver nanoclusters from double stranded DNA containing an Abyssin site, while theophylline of (b) , The fluorescence signal was greatly reduced. In (a) 1 and 2, the effect of theophylline was examined using single-stranded DNA, a component of double-stranded DNA, in which case the formation of silver nanoclusters was not affected.
FIG. 3 shows the results of experiments on the formation of silver nanoclusters and the effect of theophylline according to DNA bases arranged opposite to the basic region of double-stranded DNA. When the DNA base aligned with the basic region is adenine, thymine, and guanine, silver nanoclusters with fluorescence properties are not formed, while cytotoxic Only the silver nanoclusters showing a high fluorescence signal at 640 nm were formed. In this case, the fluorescence signal was reduced by theophylline.
FIG. 4 shows the results of experiments on formation of silver nanoclusters according to the order of addition of theophylline. When theophylline was reacted with double-stranded DNA to form silver nanoclusters (1), theophylline effectively inhibited the formation of silver nanoclusters, resulting in a reduced fluorescence signal. On the other hand, when theophylline was added (2) after formation of the silver nanoclusters, a high fluorescence signal similar to that in the absence of theophylline was generated.
FIG. 5 shows the fluorescence signals of silver nanoclusters produced when different concentrations of theophylline were added. The fluorescence signal was linearly decreased in the theophylline concentration range of 0-40M and the detection limit was 1.8M.
Figure 6 shows the results of an experiment for the specificity of the theophylline detection system. We analyzed the effect of structurally similar caffeine and theobromine on the formation of silver nanoclusters using glucose and creatinine in the blood. It was confirmed that the fluorescence signal was decreased only when the theophylline was present unlike the case where the other substance was present.
Figure 7 shows the results of an experiment to see if the developed detection system could be used for the analysis of theophylline present in the blood. As the concentration of artificially added theophylline in the blood increased, the fluorescence signal of the silver nanocluster decreased and showed a linear signal change in the theophylline concentration range of 0-40 M. Also, based on the linear graph obtained in this section, it was confirmed that the concentration of theophylline added in the blood can be accurately analyzed.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
The present invention applies the novel silver nanoparticle-forming technology (Ma et al., Nanotechnology. 22: 305502, 2011) used for SNP detection to the novel detection agent theophylline to show that theophylline can modulate silver nanocluster formation Based on this, we developed a theophylline detection system based on fluorescence signal which solved the problems of existing theophylline detection system.
The present invention relates to a method for detecting theophylline incorporating a conventional riboflavin fluorescent ligand (Sato et al., Chem . -Eur. 18: 12719-12724, 2012), the sensitivity of detection of theophylline was increased by introducing silver nanoclusters which have the same point of measuring fluorescence signal change but high fluorescence signal and excellent light stability.
In one aspect, the present invention provides a method for detecting a fibronectin comprising the steps of: (a) reacting a sample containing theophylline with a double stranded DNA comprising an abasic site, (b) adding silver ions and a reducing agent to the reactant of the double-stranded DNA and the theophylline-containing sample to form silver nanoclusters; And (c) analyzing the formed silver nanoclusters to detect or quantify the theophylline.
In the method of the present invention, the double stranded DNA of step (a) may include a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, wherein the silver ion and / The reducing agent is AgNO 3, Ag (CH 3 CO 2 ) And NaBH 4 , and the analysis of the silver nanoclusters in the step (c) can be characterized by the fluorescence signal.
In the present invention, the theophylline-containing sample may be blood, saliva, or urine.
Abasic moiety "in the present invention means a base-free site in which the base is deleted.
In another aspect of the present invention, there is provided a silver nanocluster comprising (a) silver nanoparticles formed by reacting a double-stranded DNA comprising an abasic region with an azobatic- Adding a material capable of regulating formation of the cluster; And (b) analyzing a silver nanocluster formed by a substance capable of regulating formation of the added silver nanocluster to detect or quantify a substance capable of regulating the formation of the silver nanocluster. And to a method of detecting or quantifying a substance capable of regulating the formation of a substance.
In the present invention, the DNA platamer can be applied to the detection and quantification of a substance capable of regulating the formation of silver nanoclusters by introducing double stranded DNA in which abasic-cytosine is arranged facing each other. Small molecules, and metal ions. Specifically, thrombin and PDGF, ATP, cocaine, and Hg 2 + can be included in the nanoclusters.
The abdomen refers to a small single-stranded oligonucleotide capable of specifically recognizing a target substance with high affinity.
[Example]
Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the technical scope of the present invention is not limited to these embodiments.
Example One: Abasic Containing a site Double strand DNA making
In this example, the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 5 was prepared so as to form a double-stranded DNA in which the ASB-Cytosine was arranged facing each other.
SEQ ID NO: 1 5'-TGGTGG X GGCAGC-3 ': Abasic-site-containing DNA
SEQ ID NO: 2 5'-GCTGCC C CCACCA-3 ': Complementary DNA (C)
SEQ ID NO: 3 5'-GCTGCC A CCACCA-3 ': Complementary DNA (A)
SEQ ID NO: 4 5'-GCTGCC T CCACCA-3 ': Complementary DNA (T)
SEQ ID NO: 5 5'-GCTGCC G CCACCA-3 ': Complementary DNA (G)
The basic site to which theophylline is bound is indicated by X , and the aligned DNA bases are labeled C , A , T and G , respectively.
Example 2: Formation of silver nanoclusters
In this example, the formation of silver nanoclusters was analyzed using double stranded DNA comprising the aspartic region prepared in Example 1.
As a result, silver ions (AgNO 3 ) and a reducing agent (NaBH 4 ) were added to the double stranded DNA to form an Ag + -cytosine complex (FIG. 1), and silver nanoclusters were formed to generate a maximum fluorescence signal at 640 nm (Fig. 2B).
Example 3: Detection of theophylline using silver nanoclusters
In this example, the possibility of detection and quantitative analysis of theophylline using silver nanocluster formation was confirmed.
In the presence of theophylline, it was found that theophylline binds only to the basic site of double-stranded DNA and inhibits the formation of silver nanoclusters, resulting in a significant decrease in fluorescence signal (FIG. 2).
In addition, fluorescence signals of silver nanoclusters produced when different concentrations of theophylline were added were measured. As a result, it was confirmed that as the concentration of theophylline increased, formation of silver nanoclusts was inhibited and a decreased fluorescence signal was generated ).
Example 4: Detection conditions of theophylline using silver nanoclusters
In this example, detection conditions of theophylline using silver nanocluster formation were analyzed.
4-1: Double strand DNA ≪ / RTI > Single strand DNA Theophylline's influence on
The effect of theophylline was analyzed using single-stranded DNA, a component of double-stranded DNA containing abasic sites, confirming that the formation of silver nanoclusters was not affected in this case.
As a result, it was confirmed that theophylline binds only to the abasic region of double-stranded DNA and inhibits the formation of silver nanoclusters (Fig. 2a).
4-2: DNA Formation of silver nanoclusters and effect of theophylline on base
We analyzed the formation of silver nanoclusters and the effect of theophylline on DNA bases aligned with the basic region of double - stranded DNA.
As a result, silver nanoclusters with fluorescence properties were not formed when the DNA base aligned with the basic region was adenine, thymine, and guanine, whereas cytosine It was confirmed that a silver nanocluster showing a fluorescence signal at 640 nm was formed only when arranged, and it was confirmed that the fluorescence signal was reduced by theophylline only in this case (FIG. 3).
4-3: Formation of silver nanoclusters by changing the order of addition of theophylline
The order of addition of theophylline to the double stranded DNA prepared in Example 1 was changed to analyze the formation of silver nanoclusters.
When theophylline was reacted with double-stranded DNA to form silver nanoclusters (Fig. 4, 1), it was confirmed that theophylline effectively inhibited the formation of silver nanoclusters, resulting in a reduced fluorescence signal.
It was confirmed that a high fluorescence signal similar to that in the case where theophylline was added after the formation of the nanocluster (2 in FIG. 4) was observed.
From the above results, it was confirmed that the detection of theophylline should be performed in the order of forming silver nanoclusters after the reaction of the double-stranded DNA and theophylline.
Example 5: Specificity of theophylline detection system
In this embodiment, the specificity of the newly developed theophylline detection system is analyzed.
We analyzed the effect of structurally similar caffeine and theobromine on the formation of silver nanoclusters using glucose and creatinine in the blood.
As a result, it was confirmed that the fluorescence signal decreased only when the theophylline known to bind to the aligned basic site, which is opposite to the cytosine of the double-stranded DNA, was present, unlike the case where other substances were present (FIG. In addition, the presence of theophylline was visually confirmed easily by confirming the fluorescence signal using a UV lamp.
Example 6: Application of theophylline detection system to biological samples
In the present example, it was confirmed whether the developed detection system could be used for the analysis of theophylline present in the blood.
As the concentration of theophylline added artificially in the blood increased, the fluorescence signal of the silver nanocluster decreased (FIG. 7a), and a linear signal change was observed in the theophylline concentration range of 0-40 M (FIG. 7b). In addition, based on the linear graph obtained from this section, the concentration of theophylline added in the blood could be analyzed accurately.
These results confirm that the newly developed theophylline detection system can be applied to the analysis of actual clinical samples.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
<110> Korea Advanced Institute of Science and Technology <120> Method for Detecting Theophylline Using Silver Nanoclusters Formation <130> P13-B232 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Abasic-site-containing DNA <400> 1 tggtggggca gc 12 <210> 2 <211> 13 <212> DNA <213> Artificial Sequence <220> Complementary DNA (C) <400> 2 gctgccccca cca 13 <210> 3 <211> 13 <212> DNA <213> Artificial Sequence <220> Complementary DNA (A) <400> 3 gctgccacca cca 13 <210> 4 <211> 13 <212> DNA <213> Artificial Sequence <220> Complementary DNA (T) <400> 4 gctgcctcca cca 13 <210> 5 <211> 13 <212> DNA <213> Artificial Sequence <220> Complementary DNA (G) <400> 5 gctgccgcca cca 13
Claims (7)
(a) reacting a sample containing theophylline with a double-stranded DNA comprising an abasic site and arranged in the direction of azithromycin;
(b) adding silver ions and a reducing agent to the reactant of the double-stranded DNA and the theophylline-containing sample to form silver nanoclusters; And
(c) analyzing the formed silver nanoclusters to detect or quantify the theophylline in the theophylline-containing sample.
(a) a silver nanocluster is formed by reacting a double-stranded DNA having an abasic region with an arrangement of azobatic-cytosine and a silver ion, wherein silver nanoclusters are formed, Adding a material; And
(b) analyzing silver nanocrystals formed by the substance capable of regulating the formation of the added silver nanoclusters to detect or quantify a substance capable of regulating the formation of the silver nanoclusters.
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Cited By (3)
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| CN105834445A (en) * | 2016-03-23 | 2016-08-10 | 湖南科技大学 | Synthesizing method for silver nanoparticles high in scattering strength and method for detecting sulfur ions |
| CN106404726A (en) * | 2016-05-26 | 2017-02-15 | 吉林大学 | Fluorescent probe based on double-stranded DNA protection and application of same to preparation of drug used for detecting Plasmodium falciparum lactate dehydrogenase |
| CN115121803A (en) * | 2021-03-11 | 2022-09-30 | 上海交通大学医学院附属仁济医院 | Method for synthesizing polymeric nanoclusters based on DNA framework structure |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105834445A (en) * | 2016-03-23 | 2016-08-10 | 湖南科技大学 | Synthesizing method for silver nanoparticles high in scattering strength and method for detecting sulfur ions |
| CN105834445B (en) * | 2016-03-23 | 2017-08-25 | 湖南科技大学 | A kind of Nano silver grain synthesis of strong scattering intensity and the method for detection sulphion |
| CN106404726A (en) * | 2016-05-26 | 2017-02-15 | 吉林大学 | Fluorescent probe based on double-stranded DNA protection and application of same to preparation of drug used for detecting Plasmodium falciparum lactate dehydrogenase |
| CN115121803A (en) * | 2021-03-11 | 2022-09-30 | 上海交通大学医学院附属仁济医院 | Method for synthesizing polymeric nanoclusters based on DNA framework structure |
| CN115121803B (en) * | 2021-03-11 | 2024-04-30 | 上海交通大学医学院附属仁济医院 | Synthetic method of polymeric nanoclusters based on DNA framework structure |
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