KR20100113388A - Fluorescein-cu (ii) complex having selectivity for cyanide, preparation method thereof and detection method of cyanide using the same - Google Patents

Abstract

PURPOSE: A fluorescein-copper(ii) composite, a manufacturing method thereof, and a detection method of the cyanide ion using thereof are provided to secure the fluorescence quenching effect of the composition by being combined with a copper ion. CONSTITUTION: A fluorescein-copper(ii) composite marked with chemical formula 1, is included in an optical sensor for the cyanide ion detection. In the chemical formula 1, R1 is hydrogen, halogen, or C1~C4 alkyl. AR is an aryl group including fluorophore inside the structure to form a fused ring with a neighboring phenyl ring.

Description

Fluorescein-Cu (II) complex having cyanide ion selectivity, preparation method thereof and method for detecting cyanide ion using same {Fluorescein-Cu (II) complex having selectivity for cyanide, preparation method etc and detection method of cyanide using the same}

The present invention relates to a fluorescent sensor comprising a fluorescein-copper (II) complex having cyanide ion selectivity and a cyanide ion detection method using the same. More particularly, the fluorescein derivative is combined with copper ions in an aqueous solution. Fluorescence quenching effect, and when cyanide ions are added to the composite, copper ions are released to form cyanide ions and reactants, while fluorescence is re-emitted to effectively detect cyanide ions. The present invention relates to a fluorescence sensor comprising a lecein-copper (II) complex and a cyanide ion detection method using the same.

Due to the important role of biological and environmental processes, the development of selective optical signal systems for negative ions has received significant attention in recent decades. In particular, cyanide ions are harmful anions that cause toxicity in the biological and environmental fields. Cyan ions are very toxic to mammals, causing vomiting, leading to loss of consciousness and eventually death. Due to the strong toxicity of cyanide ions, concentrations in drinking water cannot exceed 22 mM, according to the World Health Organization.

Thus, various colorimetric and fluorescent probes have been actively reported in recent decades. For example, a general approach is to use cyanide ion complexes or to add zinc (II) -porphyrin, Ru (II) -pyridine, boronic acid derivatives and CdSe quantum dots. In addition, a hydrogen bonding reaction can be used. As a third approach for detecting cyanide ions, a nucleophilic group addition method of cyanide ions has been applied that can minimize interference by other anions such as fluorine and acetate. For example, oxazine, pyrylium, squalane, Cyanide ions were detected by adding nucleophilic groups of cyanide ions to trifluoroacetophenone, acryltriazene, acridinium and salicylaldehyde. Finally, a method of using copper-cyanide ion affinity for cyanide ion sensing has recently been reported. However, no examples of "Off-On" type fluorescent probes for cyanide ions working in aqueous solution have been reported.

It is an object of the present invention to provide a fluorescent sensor capable of selectively detecting cyanide ions using a fluorescein derivative capable of selectively detecting copper ions and a cyanide ion detection method using the same.

In order to achieve the above object, the present invention provides a cyanide ion detection fluorescent sensor comprising a fluorescein-copper (II) complex represented by the following formula (1).

[Formula 1]

Where

R ₁ represents hydrogen, halogen or C _1-4 alkyl,

Ar is an aryl group containing a fluorophore in its structure, and forms an fused ring with an adjacent phenyl ring or is substituted with a substitutable carbon atom of the adjacent phenyl ring.

The present invention also provides a method for detecting cyanide ions comprising reacting the fluorescein-copper (II) complex with cyanide ions.

The present invention also provides a microfluidic device comprising the fluorescent sensor of the present invention as a microfluidic device comprising a loading channel, a chaotic mixer comprising a microchannel and an outlet for fluorescence measurement.

In the fluorescein-copper (II) complex of the present invention, the fluorescence of the fluorescein derivative is quenched in 100% aqueous solution, but when cyanide is added, the fluorescein derivative is returned to the fluorescein derivative while forming a copper-cyanide ion reactant. Can be used as an "off-on" type fluorescent sensor.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a fluorescent sensor for detecting cyanide ions comprising a fluorescein-copper (II) complex represented by the following formula (1).

[Formula 1]

Where

R ₁ represents hydrogen, halogen or C _1-4 alkyl,

Ar is an aryl group containing a fluorophore in its structure, and forms an fused ring with an adjacent phenyl ring or is substituted with a substitutable carbon atom of the adjacent phenyl ring.

The compound of Formula 1 is, for example,

,

,

을 나타내고,Ar forms a fused ring with a phenyl ring

,

,

Indicates

을 나타내며,When Ar is substituted with a substitutable carbon atom of a phenyl ring

,

Wherein X and Y each independently represent an oxygen or sulfur atom,

을 나타내며, R ₂ , R ₃ , R ₅ , R ₆ and R ₇ are each independently hydrogen, halogen, hydroxy, C _1-4 alkyl, or

,

R ₄ represents hydrogen, hydroxy, C _1-4 alkyl, C _1-4 alkoxy, -COOR ₁₁ , -CON (R ₁₂ ) (R ₁₃ ) or -N (R ₁₄ ) (R ₁₅ ),

Wherein R ₁₁ to R ₁₇ each independently include a compound representing hydrogen or C _1-4 alkyl.

More specifically, the compound of Formula 1 includes any one of the compounds of Formulas 1a to 1d.

[Formula 1a]

[Chemical Formula 1b]

[Formula 1c]

≪ RTI ID = 0.0 &

Compound of Formula 1a is for example

X and Y each independently represent an oxygen atom,

R ₁ and R ₃ each independently represent hydrogen or halogen,

을 나타내고, R ₂ is

Indicates

R ₄ represents hydrogen, hydroxy, C _1-4 alkyl, C _1-4 alkoxy, -COOR ₁₁ , -CON (R ₁₂ ) (R ₁₃ ) or -N (R ₁₄ ) (R ₁₅ ),

Wherein R ₁₁ to R ₁₇ each independently include a compound representing hydrogen or C _1-4 alkyl.

For example, the compound of Formula 1a may include a compound of Formula 1a-1.

[Formula 1a-1]

In addition, the compound of Formula 1b is, for example

X and Y each independently represent an oxygen atom,

R ₁ and R ₅ each independently include a compound representing hydrogen, halogen, or C _1-4 alkyl.

For example, the compound of Formula 1b includes a compound of Formula 1b-1.

[Formula 1b-1]

In addition, the compound of Formula 1c is, for example

X and Y each independently represent an oxygen atom,

R ₁ represents hydrogen or halogen,

을 나타내고, R ₆ is

Indicates

R ₇ includes compounds representing hydrogen or hydroxy.

For example, the compound of Formula 1c may include a compound of Formula 1c-1.

[Formula 1c-1]

In addition, the compound of Formula 1d is for example

X and Y each independently represent an oxygen atom,

R ₁ represents hydrogen or halogen,

R ₈ and R ₉ each independently represent hydrogen, halogen or C _1-4 alkyl,

R ₁₀ includes compounds representing hydrogen or hydroxy.

For example, the compound of Formula 1d may include a compound of Formula 1d-1.

[Formula 1d-1]

R ₈ and R ₉ represent hydrogen, fluoro, chloro, or methyl.

The fluorescein-copper (II) complex of the present invention can be used as a fluorescent probe of type “Off-On” for cyanide ions in 100% aqueous solution.

According to the mechanism of Scheme 1, the fluorescein derivative of Formula 2 exhibits green fluorescence in a 100% aqueous solution of pH 6.5 to 8.5, and fluorescein-copper quenching when bound with copper ions ( II) A complex is formed, and when cyanide ions are added to the complex, copper ions are released to react with cyanide ions to produce green fluorescence while producing very stable Cu (CN) ₂ .

Scheme 1

The cyanide ion is not particularly limited but may be derived from a cyanide alkali metal salt such as sodium cyanide.

The compound of formula 1 of the present invention may also be prepared by reacting a copper salt with a compound represented by the following formula (2).

[Formula 2]

Where

R ₁ represents hydrogen, halogen or C _1-4 alkyl,

Ar is an aryl group containing a fluorophore in its structure, and forms an fused ring with an adjacent phenyl ring or is substituted with a substitutable carbon atom of the adjacent phenyl ring.

The compound of Formula 2 may be prepared, for example, according to the method described in Korean Patent No. 690,199 as a fluorescein derivative, but is not particularly limited thereto.

More specifically, the compound of Formula 2 may be prepared according to the following Scheme 2,

1) Dialkyl iminodiacetate fluorescein by reacting 2 ', 7'-dichlorofluorescein, paraformaldehyde and dialkyl iminodiacetate (dialkyliminodiacetate fluorescein),

2) The compound of formula 2 may be obtained by hydrolyzing the dialkyl iminodiacetate fluorescein obtained in step 1) with a base.

In step 1), 2 ', 7'-dichlorofluorescein is reacted with iminium ions of a product of paraformaldehyde and diethyl iminodiacetate, and is aminomethylated dialkyl through a Mannich reaction. Prepare a compound of dialkyliminodiacetate fluorescein.

In step 2), the dialkyl iminodiacetate fluorescein (dialkyliminodiacetate fluorescein) is hydrolyzed with KOH to prepare a compound of Formula 2.

Scheme 2

The compound of formula 2 prepared in the above step can be reacted with the copper salt in a 100% aqueous solution of pH 6.5 to 8.5 to obtain a fluorescein-copper (II) complex.

The copper salt is not particularly limited, but perchlorate, chloride, or acetate salts of copper can be used.

The present invention also relates to a method for detecting cyanide ions comprising reacting the fluorescein-copper (II) complex with cyanide ions.

Fluorescein-copper (II) complex of formula 1 according to the present invention shows a selective chelate amplification fluorescence effect at concentration-dependent excitation wavelength of 505 nm with CN ⁻ in 100% aqueous solution of pH 6.5 to 8.5 Fluorescein-copper (II) complexes can be used as fluorescent probes to detect cyanide ions.

In addition, cyanide ion detection may be measured through colorimetric change. More specifically, when copper ions are added to the fluorescein derivative, phenolate is formed, and light yellow turns to bright pink, and when cyanide ions are added to the solution, pink turns to yellow again.

In addition, cyanide ions can be detected by detecting a chemotactic response by co-cultivating a organism having motility and chemical sensitivity in a medium containing a fluorescein-copper (II) complex and a cyanide ion.

With life having a sensitivity to the mobility and chemical may use a ciliate (Ciliate), paramecium (Paramecium), stenter (Stentor), flagellate (flagellate), nematode (Nematode), or bacteria and the like. More specifically, nematodes such as Caenorhabditis elegans may be used.

The chemotaxis reaction can be confirmed by real-time observation with a stereoscopic mirror, but is not particularly limited thereto.

The invention also relates to a microfluidic device comprising a loading channel, a chaotic mixer comprising microchannels and an outlet for fluorescence measurement,

It relates to a microfluidic device comprising the fluorescent sensor of the present invention.

The microfluidic device includes microchannels manufactured using microfluidic technology, more specifically, soft lithography. The microfluidic device includes glass, silica, polycarbonate, poly (Methyl methacrylate) (Poly (methylmethacrylate)), or poly (dimethylsiloxane) (Poly (dimethylsiloxane), PDMS) may be used, but is not particularly limited thereto.

The wall of the microchannel in the chaos mixer may be formed in a herring-bone form to induce chaotic advection and to enhance mixing of laminar flows.

In addition, the microchannel may include the fluorescent sensor of the present invention capable of detecting cyanide ions.

The overall size of the microfluidic device of the present invention may be manufactured to about 1.5 to 2.5cm in width, 0.5 to 1cm in length, and may be used by attaching one or two or more such devices to one glass plate.

Hereinafter, the present invention will be described in more detail through examples according to the present invention and comparative examples not according to the present invention, but the scope of the present invention is not limited to the examples given below.

Example 1 Preparation of Fluorescein Derivatives of Formula 2

Diethyl iminoacetate (1.52 mL, 8.68 mmol) and paraformaldehyde (0.224 g, 7.47 mmol) were placed in 20 mL of CH ₃ CN and refluxed for 30 minutes. 2 ', 7'-dichlorofluorescein (1.00 g, 2.49 mmol) in 30 mL of CH ₃ CN / H ₂ O (1: 1) was added to the solution and the reaction mixture was refluxed for 24 h. CH ₃ CN was removed and the product and residue were dissolved in 30 mL of boiling ethanol. After cooling to room temperature, 5 mL of ether was added to the solution. The precipitate produced at 4 ° C. was filtered to give diethyl iminodiacetate fluorescein (1.21 g, 61%).

¹ H NMR (CDCl ₃ ) δ 8.06 (d, 1H, J = 6.8 Hz), 7.69 (quintet, 2H, J = 7.4 Hz), 7.20 (d, 1H, J = 6.2 Hz), 6.69 (s, 2H) , 4.30-4.53 (d, 4H, J = 14 Hz), 4.21 (q, 8H, J = 7.1 Hz), 3.54 (s, 8H), 1.28 (t, 12H, J = 7.1 Hz);

¹³ C NMR (CDCl ₃ ) δ 170.6, 169.0, 155.9, 151.6, 148.6, 135.6, 130.6, 128.3, 127.3, 125.8, 124.3, 117.9, 110.9, 109.9, 83.0, 61.6, 54.5, 49.1, 31.2, 14.4;

HRMS (FAB) m / z = 825.1805 (M + H + Na) ⁺ , calc. _{for C 38 H 40 C l2 N} 2O13 Na = 825.1805.

Next, diethyl iminodiacetate fluorescein (500 mg, 0.62 mmol) prepared above was dissolved in 25 mL of distilled water, and potassium hydroxide (KOH) was added thereto. The reaction mixture was refluxed for 12 hours. After cooling the reaction mixture to room temperature, 1M hydrochloric acid was added to adjust the pH to 2-3. The precipitated solid was filtered off and washed with cold distilled water (1 mL). After drying at 90 degreeC using a vacuum pump, imino diacetic acid fluorescein (402 mg, 90% yield) was obtained.

Melting point (m.p.): 230 ° C., dec .;

¹ H-NMR (DMSO d 6) 8.03 (d, 1H, J = 7.3 Hz), 7.80 (m, 2H), 7.37 (d, 1H, J = 7.3 Hz), 6.63 (s, 2H), 4.30 (s , 4H), 3.55 (s, 8H);

¹³ C-NMR (D ₂ O) 181.4, 173.1, 171.6, 170.1, 155.6, 133.7, 133.4, 132.2, 130.5, 130.2, 130.1, 129.9, 127.4, 111.9, 105.2, 56.7, 50.1;

MS (FAB) m / z = 691.09 (M + H) ⁺ , calc. for C ₃₀ H ₂₅ C _l2 N 2 _O13 = 691.07

Experimental Example 1 Detection of Cyanide Ions Using Fluorescein Derivatives of Chemical Formula 2

In order to examine the anion binding properties of a derivative of fluorescein of the formula (2) prepared in Example ^{1, CN -, SCN -,} H 2 PO 4 -, HSO 4 -, NO 3 -, CH 3 CO 2 -, F ^-, Cl ^-, Br ^- and I ^- in dissolving the sodium salt of a secondary distilled water to prepare a stock solution (1mM). In addition, a host stock solution (0.01 mM) was prepared using secondary distilled water.

The test solution was added 4-40 μl of a fluorescein derivative stock solution of Formula 2 into a test tube, and then added 1 mM copper perchlorate salts solution and each anion stock solution prepared with secondary distilled water, and 0.02 M The solution was diluted to 4 mL with HEPES buffer (pH 7.4). The final concentration of the probe was 6 μM, 1 equivalent of copper ions and 100 equivalents of each anion were added.

All fluorescence measurements were taken at excitation wavelength (505 nm). The slit width of the excitation and emission was set to either 1.5 nm or 3 nm.

As shown in FIG. 1, when copper ions were added, fluorescent quenching appeared, and when CN ions were added, fluorescence was recombined with copper. Except for CN ions, the other anions did not change fluorescence.

<Experiment 2> Fluorescence titration test for cyanide ions by concentration

CN ions were added in 5, 6, 10, 13, 16, 20, 30, 32, 38, 40, 50, 70, 100, 200 equivalents to observe the fluorescence change, and the results are shown in FIG.

In addition, the fluorescence change was observed for the concentration of CN ions 1 to 10 μM, and the results are shown in FIG. 3. In this case, the excitation wavelength is 505 nm, the emission wavelength is 522 nm, and the slit: 3 nm / 3 nm).

2 and 3 illustrate the change in fluorescence according to the concentration of CN ions when 1 equivalent or 0.5 equivalents of copper ions are added, respectively, and it can be seen that cyanide ions having a level of μM can be detected in 100% aqueous solution. Could. In addition, it was found that the increase in the copper concentration did not affect the fluorescence change.

In addition, when the pH dependent reaction experiment was carried out in the range of pH 6.5-8.5 showed similar results to the above (not shown)

In addition, cyanide ions can also be measured through colorimetric change. When copper ions are added, the bright yellow turns bright pink, and when cyanide ions are added to the solution, the pink turns yellow again.

Production Example 1 Fabrication of Microfluidic Devices

In situ analysis was performed using a microfluidic device for chemical analysis of the fluorescein-copper (II) complex of the present invention on cyanide ions.

The microfluidic device was fabricated with PDMS (polydimethylsiloxane) using soft lithography according to the method described in Korean Patent No. 819,452. The microfluidic device consists of three main components: two loading channels, a chaotic mixer and an outlet for fluorescence measurement. For each loading channel, the inlet port is connected to a syringe pump (PHD2000 Infusion, Harvard Apparatus, Holliston, MA, USA) that controls the flow rate of the loaded sample. The microchannels in the chaos mixer have herring-bone-shaped obstacles installed on the wall, causing chaotic advection and improving the mixing of laminar flows. The outflow channel is formed with a flat wall to facilitate fluorescence measurement.

Specifically, after the SU-8 2100 photosensitive resin was coated on the silicon wafer, UV was exposed to fabricate a silicon master having a microchannel having a height of 250 μm. This silicone master was referred to as tridecafluoro-1,1,2,2, tetrahydroxytyl-1-1-trichlorosilane [(tridecafluoro-1,1,2,2-tetrahydroctyl) -1-1-trichlorosilane After surface treatment], the PDMS polymer and the curing agent were mixed to remove the bubbles and poured into a silicone master, followed by thermosetting at 80 ° C. for 1 hour. After curing, the cured polymer was removed, and holes were formed in the inlet and outlet portions to be attached to the glass plate after the oxygen plasma treatment. After firmly fixing to the glass plate, the microtube was connected to the inlet and the outlet part. The channel is 250 μm wide and 100 μm high and the overall size of the microfluidic device is 9.60 mm long and 2.48 mm wide. In addition, the loading channel and the outflow channel are finished with holes having a diameter of 600 µm.

Experimental Example 3 Cyanide Ion Detection Using a Microfluidic Device

In order to test the cyanide ion detection capability of the sensor using the microfluidic device manufactured in Preparation Example 1, the concentration of cyanide ions is set to 0.1, 1, 10 and 100 equivalents (1.24 μM to 1.24 mM), and the copper salt is 6.24 μM, the sensor was added at a concentration of 12.48 μM, and the flow rate was measured at 2 μl / min.

Fluorescence images were obtained under a fluorescence stereo microscope equipped with a Peltier-cooled CCD camera (SPOT INSIGH ™ Diagnostic instruments, Sterling Heights, MI, USA), and a Java-based image processing program, IMAGE-J (NIH, Bethesda, USA) Further analysis.

4 is a measurement of the fluorescence intensity according to the concentration of cyanide ions, (A) is added only the sensor and the copper salt, (B) to (E) is 0.1, 1, 10 of the concentration of the sensor, copper salt and cyanide ions And 100 equivalents.

As shown in FIG. 4, in the case of (A), the fluorescence was gradually quenched while passing through the microchannel in the chaotic mixer, and in the case of (B) to (E), the fluorescence was strongly dependent on the cyanide ion concentration.

Experimental Example 4 Detection of Cyanide Ions Using Nematodes

The cyanide ion detection ability of the fluorescein-copper (II) complex of the present invention was confirmed in vivo . For this purpose, C. elegans , a bacterial feeding nematode, was used. Nematodes live in the gaps between soil particles and have been considered ideal organisms for testing the toxicity of water-soluble environments such as urban and industrial wastewater.

Specifically, C. elegans wild type strain N2 was obtained from Caenorhabditis Genetics Center (University of Minnesota, USA). Twelve nematodes were necrode growth medium (NGM) agar plates (0.3% NaCl, 0.25% peptone, 2.5% agar, 5 mg / mL cholesterol, 1 mM CaCl ₂ , 1 mM MgSO ₄ ) cultured at 25 ° C. under Escherichia coli OP50. , 25 mM KH ₂ PO ₄ ) were co-cultured according to the alkali-bleaching method. In order to evaluate the effect of copper and cyanide ions on the fluorescence of a fluorescence sensor (fluorescein derivative of Formula 2) under C. elegans , the fluorescein derivative of Formula 2, Cu (ClO ₄ ) ₂ and NaCN An NGM agar plate was prepared. Specifically, before inoculating the plate nematode, 250 μl of a 12.48 μM fluorescein derivative of Formula 2 dissolved in NGM buffer was dropped on an NGM agar plate and dried at 25 ° C. After exposure to the sensor for 3 hours, nematodes were washed three times with NGM buffer (0.3% NaCl, 5 mg / mL cholesterol, 1 mM CaCl ₂ , 1 mM MgSO ₄ , 25 mM KH ₂ PO ₄ ). Half nematodes were collected and observed under a microscope, and the other half were inoculated into NGM plates containing 6.24 μM Cu (ClO ₄ ) ₂ and incubated at 25 ° C. for 3 hours. After washing with NGM buffer, half nematodes were observed under the microscope, and the other half were inoculated into NGM plates containing 12.48 μM NaCN and incubated at 25 ° C. for 3 hours. After incubation, nematodes were washed three times with NGM buffer and then placed on glass slides with Faramount ™ aqueous mounting medium (Dako, Glostrup, Denmark).

Fluorescence photographs of nematodes were magnified 200 times using a confocal laser scanning microscope (LSM510, Carl Zeiss).

FIG. 5 is a fluorescence photograph of a little nematode cultured in a medium (A) and (B) without addition of the fluorescein derivative of Formula 2, and FIG. 6 is a copper ion and / or cyanide ion and Fluorescence image of a nematode exposed to a lecein derivative, (A) is a result obtained by inoculating copper ions in a pre-cultivation of a nematode in a medium containing a fluorescein derivative of formula (2), (B) is a sequential As a result obtained by inoculating a fluorescein derivative of Formula 2 and a nematode previously exposed to copper ions in a medium containing cyanide ions (12.48 μM), (C) except that the concentration of cyanide ions is 124.8 μM ( This is the result obtained through the same experiment as B).

As shown in FIG. 5, nematodes not previously incubated with the fluorescein derivative of Formula 2 did not exhibit any fluorescence, whereas nematodes pre-exposed to the fluorescein derivative of Formula 2 exhibited strong fluorescence.

As shown in FIG. 6, when the nematode was inoculated on an agar plate containing copper ions, fluorescence was quenched (FIG. 6A), indicating that the fluorescein derivative of Formula 2 inside the nematode reacted with copper ions to lose fluorescence. Able to know. In addition, when the nematodes were incubated with cyanide ions, fluorescence appeared again, indicating that the fluorescein derivative of Formula 2 was very suitable for the in vivo image of cyanide ions (FIGS. 6B and C).

In conclusion, the present invention provides a simple method for detecting cyanide ions in a 100% aqueous solution. The fluorescein derivative represented by Formula 2, a fluorescent probe, exhibited a fluorescence quenching effect upon addition of copper ions. The addition of cyanide ions led to an increase in fluorescence of the "Off-On" type. When the sensor system is applied to a microfluidic device, the fluorescein derivative-copper (II) complex of Formula 2 exhibited green fluorescence upon addition of cyanide ions, and finally the in vivo image can be obtained by using a nematode. there was.

FIG. 1 illustrates the change in fluorescence by adding copper ions and various anions to the fluorescein derivative of Formula 2. FIG.

Figure 2 shows the fluorescence spectra of the concentration of the cyanide ion (5 to 200 equivalents) to the fluorescein-copper (II) complex (copper ion 1 equivalent) of the present invention in pH 7.4 HEPES buffer solution.

Figure 3 shows the fluorescence intensity according to the concentration of cyanide ion (1 to 10μM) addition to the fluorescein-copper (II) complex (copper ion 0.5 equivalent) of the present invention in pH 7.4 HEPES buffer.

Figure 4 shows a fluorescence picture obtained by the addition of cyanide ions by concentration to the fluorescein-copper (II) complex of the present invention in the microfluidic device of the present invention.

Figure 5 shows a fluorescence picture of a little nematode cultured in a medium containing or not the fluorescein derivative of formula (2).

Figure 6 shows a fluorescence picture of a nymph nematode exposed to the fluorescein derivative of Formula 2 and copper ions and / or cyanide ions.

Claims (13)

Fluorescence sensor for detecting cyanide ions comprising a fluorescein-copper (II) complex of Formula 1: [Formula 1]

Where R ₁ represents hydrogen, halogen or C _1-4 alkyl, Ar is an aryl group containing a fluorophore in its structure, and forms an fused ring with an adjacent phenyl ring or is substituted with a substitutable carbon atom of the adjacent phenyl ring. The method of claim 1, Ar forms a fused ring with a phenyl ring

,

,

Indicates When Ar is substituted with a substitutable carbon atom of a phenyl ring

, Wherein X and Y each independently represent an oxygen or sulfur atom, R ₂ , R ₃ , R ₅ , R ₆ and R ₇ are each independently hydrogen, halogen, hydroxy, C _1-4 alkyl, or

, R ₄ represents hydrogen, hydroxy, C _1-4 alkyl, C _1-4 alkoxy, -COOR ₁₁ , -CON (R ₁₂ ) (R ₁₃ ) or -N (R ₁₄ ) (R ₁₅ ), Wherein R ₁₁ to R ₁₇ each independently represent hydrogen or C _1-4 alkyl; The method of claim 2, Fluorescence sensor for detecting cyanide ions, wherein the fluorescein-copper (II) complex of Formula 1 is any one of the compounds of Formulas 1a to 1d: [Formula 1a]

[Chemical Formula 1b]

[Formula 1c]

&Lt; RTI ID = 0.0 &

The method of claim 3, wherein Compound of Formula 1a X and Y each independently represent an oxygen atom, R ₁ and R ₃ each independently represent hydrogen or halogen, R ₂ is

Indicates R ₄ represents hydrogen, hydroxy, C _1-4 alkyl, C _1-4 alkoxy, -COOR ₁₁ , -CON (R ₁₂ ) (R ₁₃ ) or -N (R ₁₄ ) (R ₁₅ ), Wherein R ₁₁ to R ₁₇ each independently represent hydrogen or C _1-4 alkyl; The method of claim 3, wherein Compound of Formula 1b X and Y each independently represent an oxygen atom, R ₁ and R ₅ each independently represent a hydrogen, halogen, or C _1-4 alkyl cyanide ion detection fluorescent sensor. The method of claim 3, wherein Compound of Formula 1c X and Y each independently represent an oxygen atom, R ₁ represents hydrogen or halogen, R ₆ is

Indicates R ₇ is a fluorescent sensor for detecting cyanide ions representing hydrogen or hydroxy. The method of claim 3, wherein Compound of Formula 1d is X and Y each independently represent an oxygen atom, R ₁ represents hydrogen or halogen, R ₈ and R ₉ each independently represent hydrogen, halogen or C _1-4 alkyl, R ₁₀ is a fluorescent sensor for detecting cyanide ions that represents hydrogen or hydroxy. A method for detecting cyanide ions comprising reacting the fluorescein-copper (II) complex of claim 1 with cyanide ions. The method of claim 8, Reaction is a method for detecting cyanide ions carried out in an aqueous solution of pH 6.5 to 8.5. The method of claim 8, Cyanide ion detection is a method for detecting cyanide ions by co-cultivating a organism having motility and sensitivity to chemicals in a medium containing a fluorescein-copper (II) complex and a cyanide ion. The method of claim 8, Organism having sensitivity to movement, and the chemical is ciliate (Ciliate), paramecium (Paramecium), stenter (Stentor), flagellate (flagellate), nematode (Nematode) and any one of cyanide selected bacteria from the group consisting of Method of detecting ions. A microfluidic device comprising a loading channel, a chaotic mixer including a microchannel, and an outlet for fluorescence measurement, Microfluidic device comprising the fluorescent sensor of claim 1. The method of claim 12, The microchannel wall is a microfluidic device in the form of a herring-bone.

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