KR101572901B1 - Method for detecting target material and fluorescence sensor based on conjugated polyelectrolyte and aptamer probe using 2-step FRET - Google Patents

Abstract

The present invention relates to a method for detecting a target substance based on a conjugated polyelectrolyte and an extramamer probe using a two-step FRET and a fluorescence sensor, which is capable of detecting a target substance to a level of ~ nM and has a spacer length and a cationic By controlling the concentration of the conjugated polyelectrolyte, it is possible to finely control the fluorescence signal and to improve the dynamic detection range and the detection limit of the target substance.

Description

 FIELD OF THE INVENTION [0001] The present invention relates to a conjugated polyelectrolyte and aptamer probe using 2-step FRET,

TECHNICAL FIELD The present invention relates to a conjugate polymer electrolyte and a method of detecting a target substance based on an electrode and a fluorescence sensor using 2-step FRET. By controlling the spacer length of the electrode tip and the concentration of the cationic conjugated polymer electrolyte, To a detection method and a fluorescence sensor which can finely control the characteristics of the sensor such as limit and detection range.

Recently, conjugated polyelectrolyte (CPE) has been widely applied as a chemical and biosensor due to its unique electrical and optical properties. In addition, the CPE is used as an optical antenna for signal amplification due to inherent light harvesting and molecular wire effects. Therefore, a wide range of CPEs have been designed and studied with targeting moieties to detect chemical and biological molecules such as DNA, RNA, protein, ATP and thrombin utilizing signal amplification characteristics.

An aptamer is an oligonucleotide that selectively binds to a specific target molecule with high affinity and specificity. Some single-stranded aptamers having a guanine (G) -rich base sequence can be obtained from a linear structure via intramolecular hydrogen bonding in the presence of a specific alkali metal ion in the presence of a double G-quadruplex, such as a G-quadruplex It can be deformed into a folded structure. In particular, potassium ions promote such deformation. Extensive research has been conducted on plummeter-based probe systems for detecting metal ions and / or biomolecules. However, when the structural change of the plummeter is applied to a chemical or biosensor, the range of detectable concentration is limited due to a narrow dynamic range. Therefore, many studies focus on useful tuning or broadening of the dynamic range, and in order to develop an efficient sensor, a dynamic detection range that can be adjusted not only for high selectivity and sensitivity but also for wide / range should be considered.

The phenomenon of fluorescence resonance energy transfer (FRET) has been widely used as various sensor schemes. A two-stage and multi-stage FRET-based sensor system with long-range energy transfer, large stokes shift and high sensitivity as well as conventional single-stage FRET with one fluorescent donor and fluorescent receptor has been developed come. However, the application and design of a two- or multi-step FRET-based detection system with high selectivity and sensitivity is still a challenging problem.

Potassium ion (K ⁺ ) is a physiologically important intracellular cation. Decreases in extracellular K ⁺ ions are associated with apoptosis, whereas increases in K ⁺ ions can lead to hyperexcitability. Potassium ions also play an important role in other biological processes such as neurotransmission, such as maintaining muscle strength and enzyme activity. Thus, considerable efforts have been made to create a bioassay for detecting K ⁺ ions. Nevertheless, there are still many problems that still need to be addressed in effective detection in aqueous solutions, high selectivity for other intracellular and extracellular cations (e.g., Na ⁺ ), high detection sensitivity, and adjustable detection range.

The inventors of the present invention have proposed a fluorescence sensor and a method of detecting a target substance using a two-step FRET based on a conjugated polyelectrolyte and an electrothermal probe while searching for an efficient detection method and a detection sensor of a target substance in vivo. The length of the spacer and the concentration of the cationic conjugated polyelectrolyte can be controlled to finely control the fluorescence signal and further to control the detection limit and the detection range.

Accordingly, the present invention provides a method for detecting potassium ion based on a conjugated polyelectrolyte and an extramamer probe using two-step FRET, and a fluorescence sensor capable of controlling the detection limit and detection range.

The present invention provides a method for detecting potassium ions based on a conjugated polyelectrolyte and an extramamer probe using two-step FRET, and a fluorescence sensor capable of controlling a detection limit and a detection range.

The conjugated polyelectrolyte and platemer probe-based fluorescence sensor using the two-step FRET according to the present invention can detect the target substance up to the nM level and can control the spacer length of the platamer probe and the concentration of the cationic conjugated polyelectrolyte The fluorescence signal can be finely adjusted and the detection range and detection limit of the target substance can be improved.

1 is a diagram showing a process for producing a cationic conjugated polyelectrolyte ( P1 ).
Figure 2 shows the absorption and emission spectra of P1 , 6-FAM and TAMRA in aqueous solution.
FIG. 3 is a graph showing the solubility of Pl by confirming the intensity of the PL spectrum according to the concentration of P1 ([ P1 ] = 2.5 × 10 ^-7 M to 2.5 × 10 ^-5 M).
Fig. 4 is a diagram showing a base sequence of three types of platemaker probes having different spacers and the molecular structure of the phosphors, and a potassium ion detection system by single step FRET in the absence of a conjugated polyelectrolyte.
FIG. 5 is a graph showing PL spectra of (a) platemer 1, (b) platamer 2 and (c) platamer 3 having different spacers depending on the presence or absence of potassium ion in the absence of conjugated polymer electrolyte . Fluorescence signals for [piezometer] = 10 nM and [K ⁺ ] = 50 mM in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl) were confirmed by excitation at 490 nm.
6 is a graph showing (a) PL spectrum and (b) FRET ratio (I ₅₈₅ nm / I ₅₁₇ nm) of plummeter 1 with increasing potassium ion concentration ([K ⁺ ]). The concentration used here was excited at 490 nm with [piezometer] = 10 nM, [K ⁺ ] = 50 mM in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl)
7 is a _{graph showing the relationship} between (a) PL spectrum, (b) FRET ratio (I _{585 nm} / I _{517 nm} ), and (c) PL spectrum of _platemmer 2 with increasing potassium ion concentration [K ⁺ d) FRET ratio (I _{585 nm} / I _{517 nm} ). The concentration used here was excited at 490 nm with [piezometer] = 10 nM, [K ⁺ ] = 50 mM in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl)
8 is a graph showing dynamic detection ranges of potassium ion (K ⁺ ) in (a) compressor 1, (b) compressor 2, and compressor 3. The concentration used here is [platamer] = 10 nM and [K ⁺ ] = 0 to 300 mM in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl)
9 is a diagram showing a potassium ion detection system by two-step FRET.
10 is a graph showing a PL spectrum of platemaker 1 when cationic conjugated polymer ( P1 ) is added depending on the presence or absence of potassium ions. The concentrations used here were excited at 390 nm with [piezometer] = 10 nM and [K ⁺ ] = 20 mM in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl). The figure shows a photograph of a solution according to the presence (right) and absence (left) of potassium ion under UV irradiation at 365 nm.
FIG. 11 is a graph showing the PL spectrum of an extramammer (5'-GGTT GGTG TGGT TGG-TAMRA-3 ') excited at 390 nm and labeled with P1 / TAMRA (a system without intermediate mediator 6-FAM). The concentration used here is [Platamer] = 10 nM and [ Pl ] = 1.0 x 10 ^-7 M to 6.0 x 10 ^-7 M in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl) .
12 is a diagram showing the intermolecular energy transfer between 6-FAM and TAMRA of electrotamper / P1 , which is an electrostatic complex in the absence of potassium ions.
Figure 13 is a concentration ([K ^+]) of the aptamer 1 / P1-based sensor according to an increase (a) 2- step FRET derived PL spectra and (b) the normalized spectrum, and (c) FRET ratio of the potassium ion (I _{585 nm} / I _{425 nm} ). The concentrations used here were excited at 390 nm with [piezometer 1] = 10 nM and [ Pl ] = 4.5 x 10 ^-7 M in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl).
Figure 14 is a concentration ([K ^+]) of the aptamer 2 / P1-based sensor according to an increase (a) 2- step FRET induced PL spectrum (b) the normalized spectrum and (c) FRET ratio of the potassium ion (I _585nm / I _425nm) and a graph showing aptamer 3 / P1-based sensors of the (d) 2- step FRET inducing the PL spectrum, (e) the normalized spectrum and (f) FRET ratio (I _585nm / I _425nm). The concentration used here was the concentration of conjugated polyelectrolyte [Tryptophan] = 10 nM and charge ratio [+]: [-] = 6: 1 in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl) And excited at 390 nm.
Fig. 15 is a graph showing the relationship between the concentration of the potassium ion and the molecular weight of the probe. In order to confirm the binding force between the molecular tympanic probe and the target potassium ion, a conjugated polyelectrolyte is added to the platemaker solution so that most of the aptamers form linear electrostatic complexes. Fig. 5 shows a change in PL spectrum. The concentration used here is [platamer] = 10 nM and [ Pl ] = 4.5 x 10 ^-7 M in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl)
16 is a graph showing the PL spectrum of the platemaker 1 / P1 sensor in the presence of bovine serum albumin (BSA). The concentrations used here were [piezometer] = 10 nM and [ P1 ] = 4.5 × 10 ^-7 M in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl) % BSA concentration was used.
17 is a diagram showing the intermolecular separation between the 6-FAM and the TAMRA (loss of fluidity by a total electrical attraction in the conjugated polyelectrolyte) of the plumbers having potassium ion (K ⁺ ) in the presence of P1 .
18 is a diagram showing the dynamic detection range for potassium ion (K ⁺ ) of platemaker 3 / P1 -based sensor according to the charge ratio ([+] of P1 : [-] of plummeter). The concentrations used here were: [pi] tamer 3] = 10 nM and [ Pl ] = 1.5 x ^10-7 M ([+]: [-] in Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl) -] = 1: 1), 4.5 x ^10-7 M ([+]: [-] = 3: 1), 7.5 x ^10-7 M ^-7 M ([+]: [-] = 6: 1) and excited at 390 nm.
FIG. 19 is a diagram showing the selectivity of potassium ions according to the coexistence of (a) various metal ions and (b) metal ions of potassium ion based sensor of platemaker 1 / P1 . FIG. The concentrations used here were: [Aptamer 1] = 10 nM, [metal ions] = 20 mM, and [ P1 ] = 4.5 x 10 ^{-7 in} 20 mM Tris-HCl buffer (20 mM, pH = 7.4, 100 mM NaCl) M.
Figure 20 is aptamer 2 / a P1-based sensors (a) various metal ions, and (c) metal ion (b) various metal ions, and (d) the metal ions of potassium during ions coexist and aptamer 3 / P1-based sensors And the selectivity of potassium ion according to coexistence with the potassium ion. The concentration used here was 10 mM, [metal ions] = 20 mM and charge ratio [+]: [-] in the Tris-HCl buffer solution (20 mM, pH = 7.4, 100 mM NaCl) ] = 6: 1 was used as the conjugated polyelectrolyte.

The present invention

(CPE / G-quadruple spiral structure) due to the electrostatic attraction between the cationic conjugated polyelectrolyte (CPE) and the G-quadruple helix structure, thereby realizing efficient FRET (fluorescence resonance energy transfer) Method.

The G-quadruple helix structure is formed by the interaction of the target substance and the linear molecular structure of a molecular tympanic probe.

The molecular pressure tamer probes were designed in three ways depending on the presence or absence of spacers and labeled at two ends with two different phosphors (5 'terminal and 3' terminal phosphor), respectively.

The cationic conjugated polyelectrolyte (CPE) is a first fluorescent donor and is subjected to a one-step FRET with a 5'-end fluorescent material (6-FAM) of a molecular tympanum probe as a first fluorescent receptor. Thereafter, the 5'-end fluorescent material is again subjected to a 2-step FRET with a 3'-end fluorescent material (TAMRA) as a second fluorescent donor, which is a final fluorescent acceptor.

Also, by adjusting the spacer length of the molecular pressure tamer probe, the FRET efficiency can be controlled by adjusting the distance between the phosphors at the end of the plamma probe when the conjugated polyelectrolyte is not present (1-step FRET).

(2-step FRET) by adjusting the concentration of the cationic conjugated polyelectrolyte (CPE) to adjust the detection limit and detection range through different signal amplification effects.

The present invention is strongly influenced by the order of introduction of CPE, linear plumamer probe and potassium ion due to strong binding force between platemaker and potassium ion, and can be detected even in the presence of protein, which is a biological substance.

Hereinafter, the present invention will be described in detail.

The method for detecting a target substance using two-step FRET according to the present invention can control the fluorescence efficiency by 1- or 2-step FRET by controlling the spacer length of the molecular pressure thermometer probe and the concentration of the cationic conjugated polyelectrolyte, And a detection range of the detection signal.

As used herein, the term " detection " refers simply to " detection " of whether a target substance is present in a sample and " quantitative analysis "

The "target substance" in the present specification can be detected without limitation as long as it is a substance that simply changes the specific base sequence of the molecular pressure thermometer to form a G-quadruple helical structure by interacting with the molecular pressure thermometer. That is, the target substance refers to chemical and biological species such as DNA, RNA, protein, ATP, and thrombin, and various metal ions capable of forming a G-quadruple helical structure by changing the base sequence of the molecular pressure tamer probe.

The metal ion is preferably potassium ion, but not limited thereto.

The " aptamer " is characterized by being able to bind to the target substance with a very high selectivity and affinity as a nucleic acid ligand. In particular, single-stranded DNA with a very rich sequence of guanine bases can form a secondary structure and form a G-quadruplex structure by hydrogen bonding of internal molecules. The aptamer preferably has a base sequence capable of forming a G-quadruple helix structure and is characterized in that G-quadruple helix structure formation is promoted in the presence of an electrometer-specific target substance. Formation of the G-quadruple helix structure in the presence of potassium ions can be further promoted.

The above-mentioned "molecular-massamer probe" has a platamer that specifically binds to a target substance to be detected. It forms a G-quadruple helical structure from a linear structure by interaction with a target substance, Refers to oligonucleotide probes labeled with two different phosphors (5'-terminal and 3'-terminal fluorophores). The target substance can be detected by confirming that the fluorescence signals of the two phosphors labeled at the ends are changed by morphological deformation of the G-quadruple helical structure.

The present invention can be used without limitation in the case of compressors capable of forming a G-quadruple helical structure in the presence of a target substance. In particular, the molecular tympanum probe for detecting potassium ions of the present invention is characterized by having an alkaline protease sequence GGTT GGTG TGGT TGG (SEQ ID NO: 1) that specifically binds to potassium ions. However, (SEQ ID NO: 2), TGAG GGTG GGGA GGGT GGGG AA (SEQ ID NO: 3), GTGG GTCA TTGT GGGT GGGT GTGG (SEQ ID NO: 4), TTTA AGGG TGTG GGTG TGGG TGTG SEQ ID NO: 5), etc.).

The spacer of the molecular pressure tamer probe may be, but is not limited to, the base sequence -TTTT A- or -TTTT ATTT TA-. Adjustment of the spacer length of the molecular pressure tamer probe can be accomplished by introducing one or more spacers into the molecular pressure tamer probe. The FRET efficiency is greatly reduced as the distance between the fluorescent donor and the fluorescent receptor of the molecular pressure tamer probe is greatly decreased. Therefore, the distance between the two phosphors can be controlled by adjusting the spacer length in the molecular pressure tamer probe.

In the present invention, three molecular pressure thermometer probes having different spacer lengths were designed (15 bases (5 '-6-FAM-GGTT GGTG TGGT TGG (SEQ ID NO: 1) -TAMRA-3' TAMRA-3 ') and 30 bases (5'-6-FAM-TTTT ATTT TAGG TTGG TGTG GTTG GTTT TA (SEQ ID NO: 7) -TAMRA- 3 ').

In one embodiment of the present invention, the molecular pressure tamer probe may be labeled with 6-carboxyfluorescein at the 5 'terminus and 6-carboxytetramethylrhodamine at the 3' terminus.

The "phosphor" includes a material capable of emitting light, that is, a material capable of absorbing energy from other phosphors and conjugated polyelectrolytes as a signal coloring group. Typical phosphors include fluorescent dyes, semiconductor nanocrystals, lanthanide chelates and green fluorescent proteins.

Examples of fluorescent dyes include fluorescein, 6-carboxyfluorescein, 6-carboxytetramethylrhodamine, rhodamine, Texas Red, tetramethylrhodamine, carboxydodamine, carboxy (Cyanine), Cy3 (trade name), Cy3.5 (trade name), Cy5 (trade name, trade name) Cy5.5 (trade name), Cy-chrome, picoeritrin, PerCP (peridinin chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy-4 ', 5'- Dimethoxyfluorescene), NED, ROX (5- (and -6) -carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Floor 430, Alexa Floor 488, Alexa Floor (trademark) Alexa Floor (trade name) 532, Alexa Floor 546, Alexa Floor 568, Alexa Floor 594, Alexa Floor 633, Alexa Floor 647, Alexa Floor 660, Alexa Floor ) 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br 2, BODIPY 530/550, 556/568, BodipiTM 564/570, BodipiTM 576/589, BodipiTM 581/591, BodipiTM 630/650, BodipiTM 650/665, P. R6G, Bodipi TMR, Bodipi TR, conjugated cargoes thereof, and combinations thereof. Examples of lanthanide chelates include europium chelates, terbium chelates and samarium chelates.

The " cationic conjugated polyelectrolytes " in the present specification can expect the antenna effect of the polymer in which the fluorescence of the phosphor is amplified by FRET.

The cationic conjugated polyelectrolyte can form a complex by hydrophobic interaction with electrostatic primer probe and electrostatic attraction.

The cationic conjugated polyelectrolyte can be produced by introducing a side chain having a cationic ion group into a hydrophobic conjugated polymer main chain (polyparaphenylene, polyfluorene, polyparaphenylenevinylene, polythiophene, polycarbazole, etc.) Can be used at the same time. Preferably, the cationic conjugated polyelectrolyte of Table 1 can be used as the cationic conjugated polyelectrolyte.

Cationic conjugated polymer electrolyte Poly [((9,9'-bis ( 4- (6- N, N, N -trimethylammoniumhexyl) phenyl) fluorene) -2,7-diyl) - alt - (benzene-1,4-diyl)] dibromide Poly [((9,9'-bis ( 4- (6- N, N, N -trimethylammoniumhexyl) phenyl) fluorene) -2,7-diyl) - co - ((9,9'-bis (3,4 -bis (6- N, N, N -trimethylammoniumhexyl) phenyl) fluorene) -2,7-diyl) - co - (benzene-1,4-diyl)] tribromide Poly [((9,9'-bis ( 3,4-bis (6- N, N, N -trimethylammoniumhexyl) phenyl) fluorene) -2,7-diyl) - alt - (benzene-1,4-diyl) ] tetrabromide Poly [((9,9'-bis ( 3,4-bis (6- N, N, N -trimethylammoniumhexyl) phenyl) fluorene) -2,7-diyl) - co - ((9,9'-bis ( 3,4,5-tris (6- N, N , N -trimethylammoniumhexyl) phenyl) fluorene) -2,7-diyl) - co - (benzene-1,4-diyl)] pentabromide Poly [((9,9'-bis ( 3,4,5-tris (6- N, N, N -trimethylammoniumhexyl) phenyl) fluorene) -2,7-diyl) - alt - (benzene-1,4- diyl)] hexabromide Poly [9,9'-bis (6- N , N, N -trimethylammoniumhexyl) fluorene- alt -phenylene] dibromide Poly [9,9'-bis (6- N, N, N- trimethylammoniumhexyl) fluorene] dibromide Poly [9,9'-bis (6- N, N, N- trimethylammoniumhexyl) fluorene] dibromide Poly [9,9'-bis (2- ( 2- (2-methoxyethoxy) ethoxy) ethyl) fluorene- alt -phenylene] dibromide Poly [9,9'-bis (6- N , N, N -trimethylammoniumhexyl) fluorene- alt -1,4- (2,5-bis (6- N, N, N -trimethylammoniumhexyloxy) phenylene)] tetrabromide Poly [3- (6-trimethylammoniumhexyl) thiophene] bromide Poly [9,9'-bis (6- N, N, N- trimethylammoniumhexyl) fluorene- alt- thiophene] dibromide Poly [2,5-bis (6- N , N, N -trimethylammoniumhexyl) phenylene- alt -phenylene] dibromide

It is preferable that the concentration of the cationic conjugated polyelectrolyte (CPE) is selected by adding a cationic conjugated polyelectrolyte to the molecular pressure thermometer probe solution and selecting the concentration range showing the maximum FRET signal. The charge ratio of the cationic conjugated polyelectrolyte and the anionic molecular compressamer probe is preferably [+]: [-] = 1: 1 to 10: 1. By adjusting the charge ratio between the two materials, the antenna effect of the conjugated polymer can be adjusted and consequently the FRET efficiency can be controlled.

The term " fluorescence resonance energy transfer (FRET) "phenomenon in this specification refers to a phenomenon in which energy is transferred by resonance when different kinds of phosphors are close to each other. At this time, there must be a portion overlapping between the fluorescence spectrum of the fluorescent material used as the fluorescent donor and the absorption spectrum of the fluorescent material used as the fluorescent receptor.

The two-step FRET can be confirmed in an electrostatic complex (CPE / G-quadruple spiral structure) by interaction of a cationic conjugated polyelectrolyte (CPE) with a G-quadruple helix structure. When the cationic conjugated polyelectrolyte (CPE, first fluorescence donor) is energized and excited, the fluorescence signal is transmitted through the two-step FRET to transmit the fluorescence signal. That is, primary FRET occurs from the cationic conjugated polymer to the fluorescent substance (6-FAM) at the 5'-end of the molecular extramamer probe to transfer and amplify energy, and the fluorescent substance at the 5'- (TAMRA) at the 3 'end, which is the final fluorescent receptor.

When the target substance is not present, the cationic conjugated polyelectrolyte (CPE) and the molecular pressure thermometer probe form an electrostatic complex of a linear structure. Due to the linear structure of the molecular extreme probe, the distances between the two phosphors at both ends are distanced, 2-step FRET occurs.

The CPE / G-quadruple helix structure is formed by 1) forming a linear electrostatic complex (CPE / linear structure plamer probe) by interaction of a cationic conjugated polyelectrolyte (CPE) and a linear molecular structure Followed by addition of potassium ions, or 2) formation of a G-quadruple helix structure by interaction of potassium ion with a molecular-massamer probe, which is a linear structure, followed by addition of CPE. This means that the present sensor system is not significantly influenced by the introduction order of CPE, linear plumer probe, and potassium ion.

In the presence of bovine serum albumin (BSA), the turn on / off of PL spectrum according to presence or absence of potassium ion was confirmed, and it is possible to detect it in actual biological environment.

In addition,

(CPE / G-quadruple spiral structure) by interaction of a cationic conjugated polyelectrolyte (CPE) with a G-quadruple spiral structure, wherein the G-quadruple spiral structure comprises a molecular mass (5'-terminal and 3'-terminal fluorescent material), and the cationic conjugated polymer electrolyte (hereinafter referred to as " fluorescent material " CPE) is a first fluorescent donor, and a 1-step FRET is made with a 5'-terminal fluorescent substance of a molecular tympanic probe as a first fluorescent receptor, and the 5'-terminal fluorescent substance is again a second fluorescent donating substance, Stage FRET is carried out by adjusting the spacer length of the molecular tympanic probe and the concentration of the cationic conjugated polyelectrolyte (CPE), whereby the fluorescence signal, Provides a fluorescent sensor using the two-step FRET, it characterized in that the adjustment range.

The "target material" can be detected without limitation as long as it is a substance that simply changes the specific base sequence of the molecular pressure tamer to form a G-quadruple helical structure by interacting with the molecular pressure tamer. That is, the target substance refers to chemical and biological species such as DNA, RNA, protein, ATP, and thrombin, and various metal ions capable of forming a G-quadruple helical structure by changing the base sequence of the molecular pressure tamer probe.

The metal ion is preferably potassium ion, but not limited thereto.

Therefore,

A molecular extramamer probe having spacers at both ends and two different phosphors (5 'terminal and 3' terminal phosphor) is used to form a G-quadruple helical structure in the presence of the potassium ion to be detected, . The FRET phenomenon can be controlled by the spacer length of the molecular pressure tamer probe. The cationic conjugated polyelectrolyte (CPE) forms a G-quadruple helical structure and an electrostatic complex (CPE / G-quadruple helical structure) upon introduction of a cationic conjugated polyelectrolyte (CPE) A 1-step FRET is generated by the 5'-terminal fluorophore of the molecular pressure tamer probe. Subsequently, the 5'-end fluorescent material is subjected to a two-step FRET with a 3'-end fluorescent material which is a final fluorescent acceptor as a second fluorescent donor. The concentration of the cationic conjugated polyelectrolyte (CPE) can be controlled to adjust the fluorescence efficiency, and consequently, the detection limit and detection range of the sensor can be adjusted.

Hereinafter, the present invention will be described in detail with reference to Production Examples and Examples. However, the following Preparation Examples and Examples are illustrative of the present invention, and the content of the present invention is not limited by the following Production Examples and Examples.

Manufacturing example  1. Samples and measurement methods

1-1. Preparation of samples and molecular-pressure tamer probes

All chemicals of the present invention were purchased from Aldrich Chemical Co. and used as is unless otherwise noted. K ⁺ 15 bases in order to detect ions (aptamer 1: 5'-6-FAM- GGTT GGTG TGGT TGG-TAMRA-3 '), 20 bases (aptamer 2: 5'-6-FAM- TTTT AGGT TGGT < / RTI > GTGG TTGG-TAMRA-3 ') and 30 bases (Adtamer 3: 5'-6-FAM-TTTT ATTT TAGG TTGG TGTG GTTG GTTT TA- Aptamer was purchased from Sigma-Genosys.

1-2. Preparation of cationic conjugated polyelectrolyte (CPE)

(1) Manufacturing process

The solubility of the cationic conjugated polyelectrolyte (CPE) in water and its interaction with biomolecules are very important for biological applications. In addition, the optical properties of CPEs are highly dependent on their solubility in aqueous media. On the other hand, hydrophobic interactions also play an important role in the interaction between CPE and biomolecules.

Thus, a fluorene-based cationic conjugated polymer electrolyte ( P1 ) was designed and synthesized as an optical platform for chemical and biosensor applications. In order to improve solubility in water, ionic tetraalkylammonium bromide and ethylene oxide were included in the side chain. The phenyl ring was also included in the main chain of the polymer to improve the hydrophobic interaction with the oligonucleotide salt. The production process of the cationic conjugated polyelectrolyte ( P1 ) is shown in Fig.

As shown in Fig. _{1, (PPh 3) 4 Pd} (0) under the catalyst, toluene / water for 36 hours at 85 ° C (2: 1 volume ratio) of 2,7-bis (4,4,5,5-in 2-yl) -9,9-bis (6'-bromohexyl) fluorene (2,7-bis (4,4,5,5-tetramethyl -1,3,2-dioxaborolan-2-yl) -9,9-bis (6'-bromohexyl) fluorene) (1) and 2,7-dibromo-9,9- (2,7-dibromo-9,9-bis (3,4-bis (2- (2-methoxyethoxy) ethoxy) phenyl) fluorene) (3), Suzuki coupling reaction was carried out to synthesize polyfluorene-based cationic P1 , and the reaction was continuously carried out by using trimethylamine dissolved in methanol and quaternization A water - soluble conjugated polymer was synthesized by introducing an ion group at the terminal of the alkyl chain. The number-average (M _n ) and weight-average molecular weights (M _w ) of neutral P1 were found to be 25,000 g mol ^-1 and 31,000 g mol ^-1 (PDI = 1.26).

(2) Production of monomers

1) Synthesis of 2,7-bis (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) -9,9-bis (6'-bromohexyl) fluorene (One)

Compound (1) is reacted with a known method [J. Am. Chem. Soc. 2006, 128, 1188-1196. Yield: 0.95 g (42%).

¹ H NMR (300 MHz, CDCl ₃ ,?): 7.82 (d, 2H), 7.73 (m, 4H), 3.34 s, 24H), 1.17 (q, 4H), 1.04 (m, 4H), 0.55 (m, 4H).

¹³ C NMR (75 MHz, CDCl ₃ , 隆): 150.27, 144.09, 133.99, 128.93, 119.67, 83.97, 55.24, 40.12, 34.19, 32.84, 29.15, 27.93, 23.55.

_{Anal.calcd for C 36 H 52 B 2} Br 2 O 4: C, 59.21; H, 7.18; B, 2.96; Br, 21.88. Found: C, 59.31; H, 7.21.

2) 2,7-dibromo-9,9-bis (3,4-hydroxylphenyl) fluorene (2)

Pyrocatechol (1.5 g, 13.6 mmol), 2,7-dibromofluorenone (2.0 g, 5.92 mmol) and 3-mercaptopropionic acid (1 mL ) Was slowly added concentrated sulfuric acid (0.5 mL). The reaction mixture was stirred at 80 < 0 > C for 2 hours and then toluene was added. The precipitate was washed and filtered several times with hot water and dried to obtain the product. Yield: 86% (2.76 g).

^{1 H NMR (300 MHz, CDCl} 3, δ): 8.90 (d, 4H), 7.87 (d, 2H), 7.56 (d, 2H), 7.45 (s, 2H), 6.64 (d, 2H), 6.52 ( s, 2 H), 6.35 (d, 2 H).

¹³ C NMR (75 MHz, CDCl ₃ , 隆): 153.87, 144.94, 137.58, 135.07, 130.56, 128.92, 128.22, 125.33, 122.71, 120.98, 118.54, 115.25, 64.15.

3) 2,7-dibromo-9,9-bis (3,4-bis (2- (2-methoxyethoxy)

To a mixture of compound (2) (1.0 g, 1.9 mmol), K ₂ CO ₃ (3.0 g, 21.7 mmol) and KI (0.15 g, 0.9 mmol) was added 1- bromo -2- (2-methoxyethoxy) Ethane (2.0 g, 10.9 mmol). The reaction mixture was refluxed for 2 days and cooled to room temperature. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. After the residue was extracted with methylene chloride, the extract was washed several times with hot water and dried with magnesium sulfate. The residue was purified by column chromatography to obtain the title compound (3). Yield: 63% (1.1 g)

^{1 H NMR (400 MHz, CDCl} 3, δ): 7.45 (d, 2H, J = 8.0 Hz), 7.36 (m, 4H), 6.67 (m, 4H), 6.53 (d, 2H, J = 8.4 Hz) 4H, J = 5.2 Hz), 3.94 (t, 4H, J = 5.2 Hz), 4.04 (t, (m, 8 H), 3.44 (m, 8 H), 3.27 (s, 12 H).

¹³ C-NMR (100 MHz, CDCl ₃ ,?): 153.01, 148.30, 148.11, 137.60, 137.10, 130.67, 128.91, 121.48, 121.44, 120.76, 115.17, 113.81, 71.73, 71.67, 70.46, 69.46, 69.39, 68.73, 68.48, 64.52, 58.76.

Anal.calcd for C ₄₅ H ₅₆ Br ₂ O ₁₂ : C, 56.97; H, 5.95; Br, 16.84. Found: C, 57.11; H, 5.99.

(3) Polymerization and quaternization

Monomer (1) (0.164 g, 0.220 mmol), the monomer (3) (0.209 g, 0.220 mmol) and 15mg of (PPh _{3) 4} aqueous potassium carbonate 2M in a mixture of Pd (0) (4 mL, N 2 furged ) And a phase transfer catalyst, Aliquat ^® 336 (8 mL, N ₂ furged) were added and mixed. The reaction mixture was diluted with 85 After heating at < RTI ID = 0.0 > 0 C < / RTI > an excess of bromobenzene was added as an end-capper and further reacted for 12 hours. The reaction mixture was cooled to room temperature and precipitated in methanol (200 mL) with vigorous stirring. This was filtered to obtain polymer fibers and washed with a Soxhlet apparatus and acetone for 2 days to remove oligomers and catalyst residues. The neutral polymer was obtained by drying under reduced pressure. Yield: 66% (186 mg). M _n = 25,000 g mol ^-1 (M _w / M _n = 1.26).

The quaternization of the neutral precursor was performed using trimethylamine. Condensed trimethylamine (3-5 mL) was added dropwise at -78 <0> C in a solution of neutral precursor (100 mg) in 10 mL of THF. The mixture, which was mixed at low temperature, was kept at room temperature overnight. Methanol was added to re-dissolve the precipitate and 2 mL of trimethylamine was added at -78 &lt; 0 &gt; C. The resulting mixture was further stirred at room temperature for 24 hours. After removing the solvent under reduced pressure, acetone was added to precipitate the charged polymer ( P1 ). The precipitated P1 was then collected and vacuum dried. Yield: 85% (93 mg).

^{1 H NMR (300 MHz, DMSO} -d 6, δ): 8.36 (s, 2H), 7.80-7.62 (br, 6H), 7.22 (br, 6H), 6.92 (br, 4H), 4.03 (br, 6H ), 3.69 (br, 8H), 3.41 (br, 10H), 3.02 (s, 18H), 2.11 (br, .

1-3. How to measure

^{The 1} H and ¹³ C-NMR spectra were measured with a JEOL (JNM-AL300) FT NMR system operating at 300 MHz and 75 MHz, respectively. The number- and weight-average molecular weights of the polymers were determined using the Agilent GPC 1200 series (Gel Permeation Chromatography (GPC) instrument).

The UV / Vis absorption spectrum was measured with a Jasco (V-630) and the photoluminescence (PL) spectrum was measured using a Jasco (FP-6500) spectrophotometer with a Xenon lamp as the excitation source. The quantum yield of PL of the polymer was measured on the basis of a fluorescein solution prepared at pH = 11.

All PL experiments were performed in 20 mM Tris-HCl buffer (pH = 7.4) containing 100 mM NaCl as the salt. Three concentrates of potassium tympanic acid (10 ^-6 M) were prepared as deionized water. The 20 uL pressure buffer concentrate was diluted in 2 mL buffer and heat treated with K ⁺ ions at 60 ° C for 30 min. The heat-treated solution was cooled to room temperature for 1 hour and the PL spectrum was measured by exciting 6-FAM at 490 nm (1-step FRET).

A cationic conjugated polyelectrolyte ( P1 ) was then added to the solution and the polymer was excited at 390 nm to measure the PL spectrum again (2-step FRET). For the selectivity evaluation, the same protocol was repeated in the presence of NaCl, CaCl ₂ , LiCl, MgCl ₂ , NH ₄ Cl, CuCl ₂ , AgCl, ZnCl ₂ and AlCl ₃ (20 mM each) instead of KCl.

Experimental Example 1. Characterization of cationic conjugated polyelectrolyte

1-1. Optical Characterization of Conjugated Polymer Electrolyte (P1)

UV-Vis absorption and fluorescence (PL) spectra of P1 in aqueous solution are shown in Fig.

As shown in Fig. 2, the maximum values of the UV and PL spectra of P1 were observed at? _Abs = 391 nm and? _PL = 425 nm. The molar absorption coefficient and the PL quantum efficiency measurements of P1 are ^{respectively, 5.2 × 10 -4 M -1 cm} -1 and ~ 58%. Spectral overlap between the absorption and emission spectra of P1 (FRET fluorescent donor) and 6-FAM (intermediate FRET fluorescent receptor) and 6-FAM (intermediate FRET fluorescent donor) and TAMRA (FRET fluorescent receptor) was observed.

1-2. Evaluation of solubility of conjugated polyelectrolyte (P1) in water

To determine the solubility of P1 in water, a stock solution of P1 was prepared in dimethylsulfoxide (DMSO) and diluted with water to give a sample of 2.5 × 10 ^-7 M to 2.5 × 10 ^-5 M Prepared. To remove the organic solvent effect on solubility, DMSO was maintained at 0.12 volume (vol.)% In water. The PL spectrum according to the concentration of P1 ([ P1 ] = 2.5 x 10 ^-7 M to 2.5 x 10 ^-5 M) is shown in Fig.

As shown in Figure 3, the solubility of P1 in water was estimated to be 5 [mu] M.

Example 1. Detection of potassium ion by FRET

1-1. Potassium Ion Detection System by 1-Step FRET

The molecular structure of the three abdominal probes with different spacer lengths and the potassium ion detection system by FRET are shown in Fig.

4, the base 15 (GGTT GGTG TGGT TGG) guanine (G) with - K ⁺ rich-specific aptamers K ^+, so the presence of the ion to form a K ⁺ G- quadruple helix recognition unit (recognizing unit). The 5'- and 3'-ends of the K ⁺ -specific platemer probes with three spacers were labeled with 6-FAM and TAMRA, respectively.

In the absence of K ⁺ ions, the aptamer remains predominantly in linear form and a weak TAMRA fluorescent signal is expected due to inefficient FRET from 6-FAM to TAMRA. However, in the presence of K ⁺ ions, the aptamer changes from a linear shape to a folded G-quadruple helical structure, bringing the distance between the two phosphors close to each other. When 6-FAM is excited, efficient FRET from 6-FAM to TAMRA occurs and strong TAMRA fluorescence occurs. The change in the shape of the tympanic membrane depending on the presence of K ⁺ ion induces a change in FRET signal, thus enabling potassium detection with high sensitivity and selectivity.

One -2. Detection of potassium ions by FRET according to spacer length

FIG. 5 shows PL spectra obtained by the first-step FRET of (a) platamer 1, (b) platamer 2 and (c) platemer 3 according to the presence or absence of potassium ion. [Uttamer] = 10 nM and [K ⁺ ] = 50 mM in Tris-HCl buffer (20 mM, pH = 7.4, NaCl 100 mM) was used and the solution for the formation of the G- Heat treatment was performed at 60 캜 for 30 minutes and then cooled to room temperature for 1 hour. PL spectra were measured by excitation of 6-FAM at 490 nm.

As shown in Fig. 5, when there is no potassium ion, the aptamer exists in a linear form. It was confirmed that TAMRA signal of pressure tester 1 was higher than that of pressure tester 2 and 3 among the three types of pressure tampers. This can be explained by the intermolecular spacing between the FRET fluorescent donor (6-FAM) and the fluorescent receptor (TAMRA). Compressor 1 has 15 bases between the two dyes, while Compressor 2 and 3 have 20 and 30 bases, respectively. The FRET ratio (k _FRET ) is very sensitive to the intermolecular distance and is inversely proportional to r _DA ⁶ according to equation (1).

식 (1)

Equation (1)

Where Q _D is the PL quantum yield of the fluorescent donor in the absence of a FRET fluorescent acceptor and k ² is related to the relative orientation of the transition dipole moments of the fluorescent donor and the fluorescent acceptor. The integral part is related to the overlap between the fluorescence spectra of the fluorescent donor and the absorption spectrum of the fluorescent receptor, τ _D is the lifetime of the fluorescent donor in the absence of the fluorescent acceptor, r _DA is the intermolecular distance between the fluorescent donor and the fluorescent acceptor ( DA distance), and n is the refractive index of the solvent.

When the K ⁺ ion is added, the FRET signal is enhanced for all plumbers, and a clear turn-on and -off state is observed by 6-FAM excited at 490 nm depending on the presence of K ⁺ ions. Among the three types of compressors, compressor 1 had the highest on / off ratio. This is because, as in the absence of potassium ions, the spacers that form the G-quadruple helical structure by potassium ions and do not bind to potassium ions maintain the distances between the two phosphors in comparison with those of the piezoelectric elements 2 and 3 can see. Compteramines 2 and 3 have one spacer group (TTTT A) and two spacer groups (TTTT ATTT TA and TTTT A) at both ends, respectively, so that these two dyes have a longer intermolecular distance than Comptomer 1 and K After forming the G-quadruple helix structure with ⁺ ions, they were still separated. The FRET ratio (I _{585 nm} / I _{517 nm} ) of platamer 1 was 1.72, which was higher than that of platamers 2 and 3, 0.77 and 0.35, respectively.

One -3. Detection of potassium ions by FRET with increasing potassium ion concentration

(A) PL spectrum and (b) FRET ratio (I _{585 nm} / I _{517 nm} ) of _platemmer 1 with increasing potassium ion concentration ([K ⁺ ]) (B) the FRET ratio (I _{585 nm} / I _{517 nm} ) and (c) the PL spectrum of _platemaker 3 and (d) the FRET ratio (I _{585 nm} / I _{517 nm} ) are shown in FIG.

Figure 6 (a), Figure 7 (a) and the fluorescent signal of the TAMRA-induced FRET with increasing [K ^+] As shown in Fig. 7 (c) was gradually increased fourfold with this G- K ⁺ Which means that the formation of the helical structure increases.

As shown in Figures 6 (b), 7 (b) and 7 (d), the detection limit (LOD) calculated from the calibration curves was 22.5 [mu] M, 0.82 mM and 3.3 mM (3s / slope, where s is the standard deviation of the fluorescence signal from each of the five measured samples).

The dynamic detection range of platemer 1, platemaker 2 and platemer 3 with increasing potassium ion concentration ([K ⁺ ]) is shown in Fig.

As shown in Fig. 8, the upper limit of the dynamic detection range of the three types of platemaker systems was measured to be 140 mM (in the case of platemer 3, effective potassium ion detection may be difficult due to the low FRET ratio).

Example 2. Detection of potassium ion by 2-step FRET

2-1. Potassium ion detection system by 2-step FRET

A potassium ion detection system by two-step FRET with platemaster and CPE as signal platform is shown in Fig.

As shown in FIG. 9, when the K ⁺ ion is present, the G-base abundant plastomer sequence induces a morphological change from a linear shape to a folded G-quadruple structure to enable potassium ion detection. The addition of cationic CPE results in the formation of an electrostatic complex of CPE / G-quadruple helices and an efficient two-step FRET by exciting CPE. Energy can be transferred from CPE to 6-FAM to TAMRA, resulting in a strong TAMRA fluorescence signal.

In the absence of K ⁺ ions, aptamers remain largely linear after forming electrostatic complexes with CPE. Thus, due to the large distance between the two phosphors, the two-step FRET is relatively inefficient and only weak TAMRA fluorescence occurs when CPE is excited. Through this simple method, TAMRA fluorescence signals induced by two-step FRET show clear turn-on and -off depending on the presence of K ⁺ ions. In addition, the dynamic detection range and sensor characteristics such as LOD were successfully modulated from mM to nM by adding P1 as a focusing optical antenna.

2-2. K ⁺ Evaluation of optical characteristics according to existence of ions

FIG. 10 shows a two-step FRET spectrum of platemer 1 depending on the presence or absence of K ⁺ ions after addition of P1 . Here, in the case of the presence and absence of K ⁺ ions it was all added to the solution of the aptamer excess P1 heat treatment. The final P1 concentration was adjusted to [ P1 ] = ~ 4.5 × 10 ^-7 M ([+]: [-] = 6: 1 for the charge ratio = P1 based on polymer repeat units) and the aptamer was electrostatically and hydrophobically And interacted with P1 to form a complex.

Figure 10 is in the P1 P1 by exciting at 390nm with 6-FAM and 6-FAM fluorescence energy transfer is possible and the fluorescent signal of the TAMRA induced by a 2-step FRET to TAMRA was generated as shown in.

The PL spectrum measured by excitation at 390 nm of the platamer (5'-GGTT GGTG TGGT TGG-TAMRA-3 ') labeled with P1 / TAMRA to confirm FRET between P1 and TAMRA is shown in FIG. 11, Fig. 12 shows the intermolecular energy transfer between 6-FAM and TAMRA of the electrostatic complex / platamer / P1 in the absence of ions.

As shown in FIG. 11, direct energy transfer from P1 to TAMRA is also possible without an intermediate mediator, 6-FAM, possibly due to a small spectral overlap between the fluorescence spectrum of P1 and the absorption spectrum of TAMRA. Therefore, where the P1 -6-FAM-TAMRA clothing enclosure in the absence of potassium ions at 390 nm can be confirmed by fluorescence of weak TAMRA. However, the difference in TAMRA fluorescence signal due to FRET depending on the presence or absence of potassium ion is large, and TAMRA fluorescence signal seems to be increased by two-step FRET through P1 and 6-FAM.

In addition, as shown in Figure 12, weak TAMRA release upon the absence of potassium ions may occur from intermolecular FRET between 6-FAM and TAMRA between neighboring molecules.

2 -3. K ⁺ Evaluation of optical characteristics according to ion concentration

The aptamer 1 / P1-based sensor according to an increase in the concentration ([K ^+]) of the potassium ion (a) 2- step FRET induced PL spectra and (b) the normalized spectrum, and (c) FRET ratio (I _585nm / I showed in Figure 13 to _425nm), aptamer 2 / P1 of the sensor-based (a) 2- step FRET derived PL spectrum, (b) the normalized spectrum and (c) FRET ratio (I _585nm / I _425nm) and pressure (D) two-step FRET-induced PL spectra, (e) normalized spectra and (f) FRET ratios (I _{585 nm} / I _{425 nm} ) of the _Timer 3 / P1 based sensor are shown in FIG.

As shown in FIGS. 13 and 14, the 2-stage FRET signal exhibits linear dependence on [K ⁺ ] in the range of [K ⁺ ] = 0 to 50 nM. In addition, fluorescence signals of TAMRA, which were greatly increased compared with those in the absence of conjugated polyelectrolyte due to the light - condensing or optical amplification effect of CPE, were observed for all three pressure chambers. The TAMRA fluorescence signals through the two-step FRET for the platemer 1 / P1 , 2 / P1 , and 3 / P1 sensor systems were ~ 9 and ~ 7, respectively, when compared to the signals observed directly by excitation of the TAMRA phosphor at 566 nm And ~ 7 times.

2-4. K ⁺ Evaluation of binding force between ion and molecular tympanic probe

In order to confirm the binding force between the platemaker probe and the potassium ion, unlike the above detection method in which the platemer solution and the potassium ion are heat-treated together and the conjugated polymer electrolyte is added to confirm the PL spectrum, a conjugated polymer electrolyte And most of the aptamers were allowed to form an electrostatic complex of a linear structure, followed by heat treatment with potassium ions.

Fig. 15 shows the change in PL spectra by adding a conjugated polyelectrolyte to the platamer solution so that most of the aptamer forms an electrostatic complex of a linear structure and then heat-treated with potassium ions.

As shown in FIG. 15, even though the aptamer forms an electrostatic complex with the conjugated polymer electrolyte, the addition of potassium ion forms a G-quadruple helical structure and an increase in fluorescence signal due to FRET can be observed.

2-5. Evaluation of Potassium Ion Detection in a Biological Environment

In order to confirm the operation of the system in a biological environment, potassium ion detection was attempted in a buffer solution containing bovine serum albumin (BSA), a kind of protein. BSA was added as a white powder to the buffer solution by 0.5, 1.0, 2.0 and 5.0 wt%. The PL spectrum of platemaker 1 / P1 sensor in the presence of bovine serum albumin (BSA) is shown in FIG.

As shown in FIG. 16, it was confirmed that the PL spectrum was turned on / off clearly even in the presence of BSA, which means that the present sensor can be operated in a real biological environment. However, it can be seen that the intensity of TAMRA fluorescence by FRET decreases in the presence of BSA (especially in excess 5.0 wt% BSA solution). This is because the BSA with an isoelectric point of 4.8 in the buffer solution having pH = 7.4 exhibits a negative charge and thus electrostatically interacts with the conjugated polyelectrolyte and affects the P1 / platemater complex.

Example  3. Dynamic detection range and detection limit ( LOD ) evaluation

3 -One. P1 Evaluation according to existence of

The excitons generated in the optical unit of the CPE migrate through the backbone of the pi-conjugated structure and are amplified at low energy locations to amplify the PL signal.

13 and the TAMRA fluorescence signal induced by FRET, as shown in 14 is a [K ^+] = ~ were saturated at 50 nM detection limit of 0.3 nM, respectively for a single aptamer 1, 2 and 3, the addition of P1, 3.6 nM and 3.9 nM, respectively. Since the cationic conjugated polyelectrolyte (CPE) acts as an optical antenna, the detection limit (LOD) is significantly improved when compared to the 1-step FRET without CPE. In the absence of CPE ( P1 ), this sensor method can detect [K ⁺ ] ions in the mM range, but by adding CPE ([+]: [-] = 6: 1) . In the absence of CPE, distinct signal differences were observed due to different intermolecular distances between 6-FAM and TAMRA for the three types of compressors. Compared with platameras 2 and 3, platemer 1 showed the lowest LOD value and strongest TAMRA fluorescence signal of 22.5 μM due to the shorter intermolecular distance between 6 FAM and TAMRA. Interestingly, with the addition of CPE, platameras 1-3 all showed similar FRET signals, detection limits (LOD), and dynamic detection range. The intermolecular distance between the expected 6-FAM and TAMRA of the platamer with potassium ion (K ⁺ ) according to the presence or absence of P1 is shown in Fig.

As shown in FIG. 17, when P1 is added to the P1 / platemer / potassium ion complex due to the electrostatic attraction, the spacers of platameras 2 and 3 are also trapped in the polymer matrix by the electrostatic attraction, I think. Thus, since the distance between two phosphors decreases for all three types of plumbers, platamers 1-3 exhibit similar sensor characteristics after forming an electrostatic complex with P1 .

3 -2. Evaluation of the concentration of P1 (charge ratio)

In the absence of P1 , K ⁺ ions were detected with an LOD of 22.5 μM to 3.3 mM in the mM concentration range. In the presence of P1 (charge ratio, [+]: [-] = 6: 1), high sensitivity detection of K ⁺ ions with an LOD of ~ 3 nM in the nM range was realized. The enhanced detection range and LOD due to the P1 addition were further extended by controlling the signal amplification effect through P1 concentration control. To investigate the change of the detection range with respect to the concentration of P1 , conjugated polymer electrolytes having different charge ratios of [+]: [-] = 1: 1 to 6: 1 were added to the platemaker solution.

The dynamic detection range of the plummeter 3 / P1 -based sensor according to the charge ratio ([+] of P1 : [-] of plummeter) is shown in FIG. ([+]: [-] = 1: 1), [4.5] in the presence of [Tryptophan 3] = 10 nM and [ Pl ] = 1.5 x ^10-7 M in Tris-HCl buffer (20 mM, pH = 7.4, NaCl 100 mM) ^{× 10 -7 M ([+]} : [-] = 3: 1), 7.5 × 10 -7 M ([+]: [-] = 5: 1) and ^{9.0 × 10 -7 M ([+} ]: [-] = 6: 1).

As shown in FIG. 18, as the concentration of P1 increases, the detection range is increased from mM concentration of K ⁺ ([+]: [-] = 1: 1 to 3: 1) and nM ([+]: [-] = 6: 1). The maximum TAMRA fluorescence signal induced by FRET appeared when a conjugated polyelectrolyte ([+]: [-] = 6: 1) with a charge ratio of 1: 6 was added.

Example  4. Potassium-ion  Selectivity evaluation

Piptamer 1 / P1 , The selectivity of potassium ions according to the presence of various metal ions and coexisting metal ions in platemaker 2 / P1 and platemaker 3 / P1 based sensors is shown in Figs. 19 and 20. PL spectra were measured in the presence of 20 mM NaCl, CaCl ₂ , LiCl, MgCl ₂ , NH ₄ Cl, CuCl ₂ , AgCl, ZnCl ₂ , and AlCl ₃ in the same manner as used for K ⁺ detection.

As shown in FIGS. 19 and 20, the FRET-induced TAMRA signal (FRET ratio (I _{585 nm} / I _{425 nm} ) = ~ 8) was strongly measured only in the presence of K ⁺ In the presence of Ca ²⁺ and Mg ²⁺ ions, a weak TAMRA fluorescence signal (I _{585 nm} / I _{425 nm} = 0.2-0.3) was observed, indicating that Ca ²⁺ and Mg ²⁺ ions In addition, since the G-quadruple helix structure can be stabilized, the bonding strength with platameras 1 to 3 is much weaker than that with K ⁺ ions.

To investigate the effect of other ions on the detection of potassium ions, PL spectra of K ⁺ ions and other ions (20 mM) were measured. As in the previous experiment, a high FRET ratio of I _{585 nm} / I _{425 nm} = ~ 8 was measured, indicating that it is capable of specifically binding and detecting potassium ions despite interference from other ions. The strong binding affinity of platamer and K ⁺ ions was hardly affected by the presence of other metal ions.

The results clearly show that a two-step FRET-based sensor system selectively senses K ⁺ ions for various metal ions. More importantly, by simply altering the specific nucleotide sequence of the platamer, this sensor approach can detect a wide range of target species that can form an elliptical and G-quadruple helical structure.

<110> Pusan National University Industry-University Cooperation Foundation <120> Method for detecting target material and fluorescence sensor          based on conjugated polyelectrolyte and aptamer probe using          2-step FRET <130> P20130183EN-00 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> aptamer probe <400> 1 ggttggtgtg gttgg 15 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> aptamer probe <400> 2 gggttagggt tagggttagg g 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> aptamer probe <400> 3 tgagggtggg gagggtgggg aa 22 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> aptamer probe <400> 4 gtgggtcatt gtgggtgggt gtgg 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> aptamer probe <400> 5 tttaagggtg tgggtgtggg tgtg 24 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aptamer probe <400> 6 ttttaggttg gtgtggttgg 20 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> aptamer probe <400> 7 ttttatttta ggttggtgtg gttggtttta 30

Claims (15)

Detection of target substance using two-step fluorescence resonance energy transfer (FRET) including electrostatic complex (CPE / G-quadruple spiral structure) by interaction of cationic conjugated polyelectrolyte (CPE) with G-quadruple helix structure In the method,
The G-quadruple helical structure is formed by the interaction of the target material and the plamer probe, which is a linear structure,
The plumer probe has a spacer and is labeled at two ends with two different phosphors (5'-terminal and 3'-terminal fluorophores), respectively,
The cationic conjugated polyelectrolyte (CPE) is a first fluorescent donor, and a 1-step FRET is performed with a 5'-end fluorescent material of an aptamer probe which is a first fluorescent receptor, and the 5'-end fluorescent material is again a second fluorescent donor 2-step FRET is achieved with the 3'-end fluorescent material, which is the final fluorescent receptor,
A method for detecting a target substance using a two-step FRET capable of controlling the detection limit and detection range by controlling the signal amplification effect of the polymer by controlling the concentration of the cationic conjugated polyelectrolyte (CPE) in the plamer probe and the solution . The method of claim 1, wherein the target material is selected from the group consisting of metal ions, DNA, RNA, protein, ATP, and thrombin capable of forming a G-quadruple helical structure by altering the base sequence of the platamer probe Wherein the target substance is detected by a two-step FRET. 3. The method of claim 2, wherein the metal ion is a potassium ion. The method of detecting a target substance using 2-step FRET according to claim 1, wherein the base sequence of the platemaker probe is any one of potassium ion specific base sequences selected from the group consisting of SEQ ID NOS: 1 to 5. The method according to claim 1, wherein the spacer of the plummeter probe is any one selected from the group consisting of a base sequence -TTTT A- or -TTTT ATTT TA-. 2. The method of claim 1, wherein the adjustment of the distance between the two phosphors of the platemaker probe is performed by introducing one or more spacers into the platemaker probe. The method of claim 1, wherein the fluorescent material is any one selected from the group consisting of fluorescent dyes, semiconductor nanocrystals, lanthanide chelates, and green fluorescent proteins. The method according to claim 7, wherein the fluorescent dye is any one selected from 6-FAM or TAMRA. The method according to claim 1, wherein the cationic conjugated polyelectrolyte is any one selected from the group of those listed in Table 1 below.
[Table 1]

2. The method of claim 1, wherein the concentration of the cationic conjugated polyelectrolyte (CPE) is controlled by controlling the charge ratio of the cationic conjugated polyelectrolyte and the anionic adductamer probe to between 1: 1 and 10: A method for detecting a target substance using two-step FRET. The method of claim 1, wherein the cationic conjugated polyelectrolyte (CPE) and the linearly structured plumamer probe form an electrostatic complex when the target material is absent, and the linear structure of the plamther probe results in an inefficient two-step FRET Wherein the target substance is detected by a two-step FRET. The CPE / G-quadruple helical structure according to claim 1, wherein the CPE / G-quadruple helix structure comprises: 1) an electrostatic composite (CPE / linear structure plamer probe) by interaction of a cationic conjugated polyelectrolyte (CPE) Or 2) adding CPE after forming a G-quadruple spiral structure by interaction of potassium ion with a platamer probe which is a linear structure, or 2) adding 2- A method for detecting a target substance using a step FRET. (CPE / G-quadruple spiral structure) by interaction of a cationic conjugated polyelectrolyte (CPE) with a G-quadruple helix structure, wherein the G- quadruple spiral structure comprises a target material and a linear structure, Wherein the platemaker probe has a spacer and is labeled at two ends with two different phosphors (5'-terminal and 3'-terminal fluorophores), respectively, and the cationic conjugated polyelectrolyte (CPE) Is a first fluorescent donor, and a 1-step FRET is formed by a 5'-terminal fluorescent substance of an Aptamer probe as a first fluorescent receptor, and the 5'-terminal fluorescent substance is again a second fluorescent donor, Step FRET is performed. By controlling the concentration of the cationic conjugated polyelectrolyte (CPE) in the platemaker probe and the solution, the detection limit and detection Wherein the range is adjusted by using a two-step FRET. 14. The method of claim 13, wherein the target material is selected from the group consisting of metal ions, DNA, RNA, protein, ATP, and thrombin capable of forming a G-quadruple helical structure by altering the base sequence of the plamma probe Wherein the fluorescence sensor is a fluorescence sensor using two-step FRET. 15. The fluorescence sensor of claim 14, wherein the metal ion is a potassium ion.

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