KR101850953B1 - Electrochemiluminescent probe for detection of homocysteine - Google Patents

Abstract

The present invention relates to an electrochemiluminescent probe having selectivity for homocysteine which reacts with homocysteine to produce electrochemiluminescence.

Description

[0001] The present invention relates to an electrochemiluminescent probe for detection of homocysteine,

The present invention relates to an electrochemiluminescent probe for detecting homocysteine, and more particularly, to an electrochemiluminescent probe for detecting homocysteine comprising an iridium complex having homocysteine selectivity which reacts with homocysteine to exhibit electrochemiluminescence.

Homocysteine (Hcy) is a naturally occurring sulfur - containing amino acid produced by the demethylation of methionine, an essential amino acid found in both animal and plant proteins. Homocysteine concentrations between 5 and 15 μM in plasma are generally considered normal. Increased concentrations of homocysteine cause cardiovascular disease, Alzheimer's, neural tube defects and osteoporosis. The average homocysteine concentration in the Americans was 8 μM, and it was reported that 20-30% of elderly people aged 60 years or older suffered from an increase in homocysteine concentration (> 13 μM) over the period 1991-2004.

Methods such as high performance liquid chromatography (HPLC), colorimetric analysis, radioimmunoassay, photoluminescence (PL) immunoassay and fluorescent molecule sensor have been proposed as methods for determining homocysteine concentration [(a) Clin. Chem. 48, 941-944; (b) J. Am. Chem. Soc. 126, 438-439; (c) Lipid Res. 39, 925-933; (d) Inorg. Chem. 46, 11075-11108]. Such methods exhibit excellent performance in measuring homocysteine concentration, but these approaches have the disadvantage of requiring spectrometry coupled with a bulky light source.

Electrochemical luminescence refers to a luminescence process through electron transfer between electrochemically generated radicals. Since it does not require a separate light source or bulky equipment, it is easy to miniaturize the equipment and has high sensitivity It can be said that it is optimized for on-site diagnostic detection system. Most of the electrochemiluminescent system based sensors that have been published so far are based on immunoassay, and some of them are actually commercialized and used variously. Immunoassay-based ECL sensors have been limited to specific proteins or some enzymes or some monomers associated with them because they depend on specific biological interactions such as antigen-antibody reaction or DNA hybridization. In addition, most of these biological-based assays require separate additional washing steps to wash off the signal material, since the analysis of a specific substance is based on the presence or absence of the signal by the signal substance. As an alternative to this, studies have been made on fluorescent single-molecule chemical sensors with specific receptors, but fluorescence-based sensors require a molecular excitation process through a separate light source in order to confirm signal changes, There is a limit to the difficulty of price cuts. In addition, since the above-described methods are not easy for an ordinary person, such as an expert, to handle analytical instruments or interpret data, it is difficult for anyone to easily use the system for field diagnosis of a concept that can diagnose diseases at all times .

Therefore, a method for measuring the concentration of homocysteine through a simple and easy analysis method is needed so that anyone can easily and economically diagnose cardiovascular disease early.

The first problem to be solved by the present invention is to provide an electrochemiluminescent probe for detecting homocysteine capable of qualitatively or quantitatively measuring the concentration of homocysteine.

A second object of the present invention is to provide a method for detecting homocysteine using an electrochemiluminescent probe for detecting homocysteine.

A third problem to be solved by the present invention is to provide an electrochemiluminescence sensor including an electrochemiluminescent probe for detecting homocysteine.

Unimolecular chemical sensors that selectively cause signal changes upon binding or reacting with specific analytes by introducing a light receptor into a specific receptor or reactive site have no limitations on the object to be analyzed, The change of the signal causes change, so there is an advantage that no additional analysis process such as washing is required.

Chemotherapy approaches combined with electrochemiluminescence (ECL) have several advantages over traditional analytical techniques. Electrochemistry does not require bulky equipment, additional reagents or isotopes, and allows for simple and economical chemical analysis. Since luminescence can only be generated through an electrochemically induced chemiluminescence process, it is highly susceptible to background fluorescence emission. In addition, chemical methods can selectively recognize and detect the target molecule due to the irreversible chemical reaction between the synthetic probe and the target molecule.

The inventors of the present invention have developed an electrochemiluminescent probe capable of quantitatively and / or qualitatively detecting the amount of homocysteine through the selective reaction of monomolecular based on cyclic iridium complex and homocysteine and electrochemiluminescence Thereby confirming the possibility of early diagnosis of various cardiovascular diseases and the like, thereby completing the present invention.

The present invention provides an electrochemiluminescent probe for detecting homocysteine comprising a compound represented by the following Formula 1:

[Chemical Formula 1]

In Formula 1,

L is a non-luminescent ligand;

A is a fused to 2-to 3-ring heteroaryl containing N and Z < ₁ >; Wherein said condensed heteroaryl is selected from quinoline, benzoisoquinoline, phenanthridine, benzothiazole, benzoxazole and benzoimidazole; The condensed heteroaryl is unsubstituted, or C _{1 - 10} alkyl, C _{3 - 8} cycloalkyl, C _{1 - 10} alkyloxy, nitro, and cyano is substituted with one to three is selected from;

R ₁ , R ₂ and R ₃ may be the same or different and each independently may be an electron donation group, preferably H, hydroxy, cyano, C _1-10 alkyl, C _{3- 12} cycloalkyl, C _5-12 aryl, C _1-10 alkyloxy and C _5-12 aryloxy; The C _{1 - 10} alkyl or said C _{1 - 10} alkyloxy is unsubstituted, C _{5 - 12} aryl which is substituted by; The C _{5 - 12} aryl or C _{5 - 12} aryl-oxy are unsubstituted or C _{1 - 6} alkyl or C _{1 - 6} alkyl being substituted with aryloxy;

Z ₁ is selected from O, S and NR _a ; R _a is H or C _{1 - 10} is alkyl.

In the present invention, A is a structure that contributes to lowering the LUMO energy level of the compound of Chemical Formula 1 without affecting the reaction with homocysteine, and may be any compound capable of releasing electrochemiluminescence even in the reaction with homocysteine. Preferably a 2 to 3 ring condensed heteroaryl compound containing N and Z < ₁ >, and more preferably may be selected from the following structures.

In the above formulas,

R ₄ is hydrogen, C _{1 -} is selected from the ₁₀ alkyloxy, nitro, and cyano, _{- 10} alkyl, C _{3 - 8} cycloalkyl, C ₁

m is an integer of 0 to 3,

Z ₁ is selected from O, S and NR _a ; R _a is H or C _{1 - 10} is alkyl.

When A has the above structure, the LUMO energy level of the compound of formula (1) to be produced is stabilized, has a low LUMO energy level, and can release stably electrochemiluminescence even after reaction with homocysteine. Particularly, when it has a structure of quinoline or phenanthridine, it has been confirmed that it exhibits high electroluminescent emission efficiency in reaction with homocysteine.

In the present invention, R ₁ , R ₂ and R ₃ may be an electron donation group. The substituent affects the ligand field stabilization energy (LFSE) of the iridium complex, and may contribute to an improvement in the rate of reaction with homocysteine. Specifically, the iridium complex in which R ₁ and R ₂ are methoxy exhibited red-shifted emission and a high HOMO level, and the reaction rate was remarkably improved. According to the present invention, R ₁ is methoxy, R ₂ is showed a hydrogen iridium complex is R ₁ and R ₂ are both similar properties and are hydrogen iridium complex, and R ₁ is hydrogen, R ₂ is methoxy The iridium complexes showed faster reaction rate and lower detection limit than the two iridium complexes.

In the present invention, L is not limited as long as it is a ligand used for preparing an iridium complex, but is preferably a non-luminescent ligand. More preferably, L does not have a direct effect on reactivity with homocysteine, And most preferably may be selected from the following structures, but the present invention is not limited thereto.

In the above formulas,

_{R 6, R 7, R 8} , R 9, R 10, R 11 and R ₁₂ are the same or different and are each independently hydrogen, C _{1 - 10} alkyl, halo C _{1 - 6} alkyl and C _{5 - 12} Aryl.

In the present invention, the ligand is specifically exemplified as follows.

In the present invention, the compound of Formula 1 may selectively react with homocysteine to increase the electrochemiluminescence signal. Preferably, the compound of Formula 1 reacts with homocysteine to form a hexagonal ring, May be to increase the electrochemiluminescent signal.

More preferably, the compound represented by Formula 1 according to the present invention is an iridium complex, which reacts with homocysteine to form a ring with each other. Specifically, as shown in Reaction Scheme 1 below, the formyl group of Formula 1 reacts with homocysteine to form a 1,3-thiazinane ring. At this time, strong electrochemiluminescence (ECL) do.

The electrochemiluminescence may be generated because the LUMO energy level of the reaction product of the compound of formula (I) and homocysteine is lower than the HOMO energy level of the electrochemiluminescent compound.

An iridium complex according to the prior art, for example, an iridium complex (probe 2) based on two 4- (2-pyridyl) benzaldehyde and two 4- (2-pyridyl) benzaldehyde And an acetylacetonate-based iridium complex (probe 3) exhibit a decrease in electrochemiluminescence (ECL) when reacting with homocysteine because of a high LUMO energy level. Therefore, the iridium complex can not be used as an electrochemiluminescent probe.

On the other hand, when viewed from an electrochemical point of view, the LUMO energy level becomes unstable and the reduction potential becomes larger negative value when the formulator having strong electron-attracting property disappears due to the reaction. At this time, if the reduction potential has a large negative value, the electron transfer process to the LUMO energy level of the electrochemiluminescent hole reactant becomes difficult, and it is difficult to form the excited state of the molecule.

However, since the compound of Formula 1 according to the present invention is designed so that the LUMO energy level is remarkably low, even when the LUMO energy level is increased due to the reaction with homocysteine, the compound does not interfere with the electrochemiluminescent electron transfer process Feature.

[Reaction Scheme 1]

In the above Reaction Scheme 1, 1 is the compound of Formula 1 according to the present invention, and 2 is the iridium complex (probe 2) according to the prior art.

In the present invention, the electrochemiluminescent coactivator may be tripropylamine or 2- (dibutylamino) ethanol, but is not limited thereto.

Meanwhile, the present invention provides a method for detecting homocysteine in vivo using the electrochemiluminescent probe.

The living body may be a living body of a human being or a living thing which is not a human being.

The present invention also provides an electrochemical luminescent sensor including an electrochemiluminescent probe.

The electrochemiluminescence sensor can be used to detect the level of homocysteine in the ex vivo detection sample.

According to the present invention, the electrochemiluminescence sensor including the electrochemiluminescent probe may exhibit a change in electrochemiluminescence in a blue-shifted region due to a reaction with homocysteine.

The present invention also relates to a process for producing 4- (isoquinolin-1-yl) benzaldehyde by reacting 4-formylphenylboronic acid with 1-haloisoquinoline; Reacting 4- (isoquinolin-1-yl) benzaldehyde with iridium chloride hydrate to produce a metal-linked iridium (III) chloro-linked dimer; And reacting the metal cyclized iridium (III) chloride-linked dimer, sodium carbonate and acetylacetone to prepare a compound represented by the formula (1). to provide.

The electrochemiluminescent probe according to the present invention is a combination of an electrochemiluminescence-based analysis system and a monomolecular chemical sensor, and has high selectivity and sensitivity to homocysteine. Specifically, the probe of the present invention exhibits strong electrochemiluminescence by electrical triggering when homocysteine is present in an aqueous solution. The electrochemiluminescent probe according to the present invention has excellent selectivity for homocysteine and exhibits remarkable electrochemiluminescence distinct from various other amino acids, so that it is possible to quantitatively and easily analyze the amount of homocysteine.

It is well known that the amount of homocysteine present in the body is directly related to cardiovascular disease. When the electrochemiluminescence probe according to the present invention is used, it is possible to obtain a homocysteine Since the quantity can be quantified, it is industrially useful because it can be applied as equipment for field diagnosis.

FIG. 1 is a diagram thermodynamically explaining the homocysteine (Hcy) detection of a probe according to an embodiment (a) and a comparative example (b) of the present invention obtained by Density functional theory (DFT) calculation.
FIG. 2 is a view for thermodynamically explaining the Hcy detection of the probe 1, the probe 4, and the probe 5 manufactured according to the embodiment of the present invention obtained by the DFT calculation.
Figure 3 shows the presence (hue) / absence (black) of hcy (5 mM) probe 1 (a), probe 4 (b) and probe 5 (c) a confirming a phosphorescence emission spectrum measurement result under _{(H 2 O / MeCN (1} :.. 1 v / v, pH 7.4, 10 mM HEPES) λex = 500 nm (a)
4 is a graph showing the results of evaluating electrochemiluminescence characteristics using a probe according to an embodiment of the present invention.
5 is a graph illustrating the electrochemiluminescence characteristics of the probe according to the concentration of homocysteine according to an embodiment of the present invention.
FIG. 6 shows the results of evaluating the homocysteine selectivity of a probe according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It is to be understood by those skilled in the art that these embodiments are for further explanation of the present invention and that the scope of the present invention is not limited thereto.

Reagents and devices

All materials were purchased from Sigma-Aldrich or TCI and used without any further purification. A deuterated solvent for NMR was purchased from CIL. NMR spectra were measured using a Bruker Advance DPX-300. ESI-MS data were measured using a QUATTRO LC Triple Quadrupole Tandem Mass Spectrometer and expressed in m / z units. MALDI-TOF was measured using a Voyager-DE STR Biospectrometry Workstation. For analytical thin layer chromatography, a Kieselgel 60 F254 plate was purchased from Merck. Column chromatography was performed using Merck silica gel 60 and Merck aluminum oxide 60.

Electrochemical and electrochemiluminescence measurement

Electrochemical studies were performed using CH Instruments 650B. CV and DPV were measured for individual solutions to investigate electrochemical oxidation / reduction characteristics. The ECL spectra were obtained by CCD camera, and the ECL intensity was obtained by PMT module. The ECL signal was measured by directly connecting a 10-mL ECL cell to the CCD or PMT module. All ECL data were collected simultaneously with CV experiments. The ECL solution was generally used in an aqueous solution containing 50% or 99.9% water, using 10 mM TPA (tripropylamine) as the coreactant. The reason TPA is used as co-reactant is because TPA is the most widely used and electrochemical characteristics are best studied. ECL measurements were carried out at room temperature without further treatment. The electrochemical experiments used Ag / Ag + reference electrode in organic solvent and Ag / AgCl reference electrode in aqueous solution. Ferrocene was used as an internal standard, and the electrochemical properties of other materials were compared using this. The Pt working electrode was cleaned by poling the pad with 0.05 M alumina and sonication in water and ethanol for 5 minutes. One solution was used for only one experiment and all data was discarded. The reported ECL values were highly reliable with mean values obtained from at least three or more repeated experiments.

Example 1. Synthesis of Compound 1 (Probe 1)

Example 1.1. Synthesis of 4- (isoquinolin-1-yl) benzaldehyde (5)

Acid (1.15 g, 7 mmol), 1- chloro-isoquinoline (1.05 g, 7 mmol) 4-formyl-phenyl beam in THF (40 mL) and _{H 2 O (40 mL),} Pd (PPh 3) 4 (404 mg, 0.35 mmol) and potassium carbonate (1.93 g, 14 mmol) was added thereto and refluxed for 24 hours. The reaction mixture was then cooled to room temperature and extracted with dichloromethane (DCM). The organic layer was washed with brine, dried over Na ₂ SO _4. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1: 5) to give a white solid (1.37 g, 84% yield).

^{300 MHz 1 H NMR (chloroform-} d): δ 10.15 (s, 1 H), 8.65 (d, 1H, J = 6.3 Hz), 8.02-8.08 (m, 3H), 7.93 (d, 1H, J = 10.1 1H, J = 7.8 Hz), 7.72 (d, 2H, J = 7.1 Hz), 7.89 (d, 2H, J = 10.1 Hz) .

Example 1.2. Synthesis of Compound 6

(5 g) of Example 1 (1 g, 4.29 mmol) and iridium chloride hydrate (576 mg, 1.93 mmol) were added to 2-ethoxyethanol (30 mL) and H ₂ O . After cooling at room temperature, cold water (~ 50 mL) was poured into the reaction mixture. The resulting dark brown precipitate was filtered to give a crude metal-cyclized iridium (III) chloro-bridged dimer 6.

Example 1.3. Synthesis of Compound 1

A chloro-bridged dimer (6, 330 mg, 0.245 mmol), sodium carbonate (260 mg, 0.245 mmol) of Crude metal cyclized iridium (III) of Example 2 was added to 2-ethoxyethanol mg, 2.45 mmol) and acetylacetone (0.252 mL, 2.45 mmol) were added, and the mixture was refluxed for 8 hours. Then cooled to room temperature and the reaction mixture was extracted with DCM. After the organic layer was washed with water, and dried over Na ₂ SO _4. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / methanol = 20: 1) to give a dark brown solid. (229 mg, 61% yield).

^{300 MHz 1 H NMR (DMSO-} d 6): δ 9.59 (s, 2H), 9.06 (d, 2H, J = 8.9 Hz), 8.46 (m, 4H), 8.24 (d, 2H, J = 7.1 Hz) , 7.98 (m, 6H), 7.40 (d, 2H, J = 8.3 Hz), 6.70 (s, 2H), 5.28 (s, 1H), 1.70 (s, 6H).

75 MHz ¹³ C NMR (CDCl ₃ ): δ 193.268, 185.179, 152.895, 151.009, 140.470, 137.315, 135.466, 134.930, 131.164, 129.581, 128.464, 127.605, 126.898, 126.500, 121.723, 121.592, 28.690.

HRMS (FAB ⁺ , m-NBA): m / z measurement 756.1602 (calculated C ₃₇ H ₂₇ N ₂ IrO ₄ [M] ⁺ 756.1600).

Example 2. Preparation of probe 4

Was synthesized in the same manner as in Example 1, and purified by silica gel column chromatography (EA / Hex = 1: 1) to prepare a black solid probe 4 (yield 31.6%).

300 MHz < ¹ > H NMR (chloroform-d): [delta] 10.21 (s, 2H); 8.94-8.97 (m, 2H); 8.50 (d, 2H, J = 6.3 Hz); 7.99-8.02 (m, 2H); 7.92 (s, 2H); 7.78-7.81 (m, 4H); 7.61 (d, 2H, J = 6.39 Hz); 6.80 (m, 2H); 5.19 (s, 1 H); 3.95 (s, 6H), 1.76 (s, 6H).

Example 3. Preparation of probe 5

Was synthesized in the same manner as in Example 1 and purified by silica gel column chromatography (EA / Hex = 1: 1) to obtain a black solid probe 4 (yield of 22.7%).

^{300 MHz 1 H NMR (chloroform-} d): δ 9.46 (s, 2H); 8.44 (d, 2H, J = 6.23 Hz); 8.34 (d, 2H, J = 8.24 Hz); 7.94 (t, 2H, J = 7.97 Hz, 7.78 Hz); 7.60-7.65 (m, 4H); 6.96 (s, 2H); 6.27 (s, 2 H); 5.23 (s, 1 H); 3.85 (s, 6H), 1.75 (s, 6H).

Example 4. Preparation of probe 6

Probe 6 was prepared in the same manner as in Example 1.

Example 5. Preparation of probe 7

Probe 7 was prepared in the same manner as in Example 1.

Example 6. Preparation of probe 8

Probe 8 was prepared in the same manner as in Example 1.

Comparative Example 1. Synthesis of probe 2

Comparative Example 1.1. Synthesis of Compound 7

4-formylphenylboronic acid (1.15 g, 7 mmol), 2-bromopyridine (0.667 mL, 7 mmol), Pd (PPh 3) were added to 40 mL of THF and 40 mL of N ₂ -bubbed H ₂ O ₃ ) ₄ (404 mg, 0.35 mmol) and potassium carbonate (1.93 mg, 14 mmol) were added thereto and refluxed for 24 hours. After cooling to room temperature, the reaction mixture was extracted with DCM. After the organic layer was washed with water, and dried over Na ₂ SO _4. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1: 5) to give a white solid (836 mg, 76% yield).

Comparative Example 1.2. Synthesis of Compound 8

Compound 7 (1 g, 6.44 mmol) and iridium chloride hydrate (865 mg, 2.90 mmol) were added to 2-ethoxyethanol (45 ml) and H ₂ O (15 mL) and refluxed for 12 hours. After cooling to room temperature, the reaction mixture was poured into cold water (~ 100 mL). The resulting yellow precipitate was filtered to yield a crude metal cyclized Ir (III) chloro-linked dimer (8).

Comparative Example 1.3. Synthesis of probe 2

Compound 7 (360 mg, 2 mmol), compound 8 (214 mg, 0.2 mmol) and silver trifluoromethanesulfonate (109 mg, 0.4 mmol) in diglyme (10 mL) . The reaction mixture was heated to 110 < 0 > C with continued stirring for 24 hours. The resulting dark orange-red solution was cooled to room temperature and water was added. The present tan solution was filtered and the residue was dissolved in DCM. The solution was purified by silica gel column chromatography to give a red solid (20% yield).

_{^{300 MHz 1 H NMR (CDCl 3}} ): δ 9.71 (s, 1H), 7.99 (d, 1H, J = 4.4 Hz), 7.92 (d, 2H, J = 4.4 Hz), 7.77 (d, 1H, J = J = 8.7 Hz), 7.69 (m, 6H), 7.51 (d, 1H, J = 8.4 Hz), 7.48 , 2H, J = 6.7Hz), 6.96 (m, 7H), 6.79 (d, 1H, J = 7.4Hz).

75 MHz ¹³ C NMR (CDCl ₃ ): δ 194.603, 166.658, 166.593, 165.331, 161.738, 160.245, 159.804, 149.961, 147.505, 147.070, 146.960, 145.799, 143.679, 143.524, 141.489, 137.166, 137.027, 136.469, 136.270, 136.191 , 130.925,130.202. 129.979, 128.840, 124.119, 124.014, 123.310, 122.053, 121.975, 120.330, 120.102, 120.065, 118.966, 118.920.

HRMS (FAB +, m-NBA): m / z observed 683.1538 (calculated for C ₃₇ H ₂₇ N ₂ IrO ₄ [M] ⁺ 683.1548).

Comparative Example 2. Preparation of probe 3

(Piq) ₂ Ir (acac) was synthesized in a manner similar to that of Example 1.

Manufacturing Example 1. Design of ECL probe

(Piq) ₂ Ir (acac) of Comparative Example 1, which is a strong light emitting iridium complex, was selected as a model material for probe 1, probe 1, and reactant (1-Hcy) of homocysteine.

Probes 1, 4, and 5 were designed using density functional theory (DFT) calculations. The calculated DFT is shown in Figures 1 and 2 and Table 1 below. The oxidation potential was measured using a cyclic voltammetric current at a scan rate of 0.1 V / s in an acetonitrile solution containing 0.1 M tetra-n-butylammonium perchlorate as a support electrolyte, and the value was 1 mM ferrocene (Fc / Fc +), and reference was made to the SCE. The energy gap (DELTA E) was calculated from the cross-sectional wavelength between the absorption spectrum and the emission spectrum.

division E ^{0 '} _ox
(V vs SCE)
HOMO
LUMO ΔE
(HOMO-LUMO) Probe 1 0.78 -5.57 -3.39 2.18 Probe 4 0.54 -5.33 -3.42 1.19 Probe 5 0.70 -5.50 -3.34 2.16

As can be seen in Table 1, R ₁ and R ₂ in the compound of Formula 1 were involved in the HOMO energy level but not in the LUMO energy level. In probe 4, the HOMO-LUMO energy gap difference was greatly reduced compared to probes 1 and 5, photoluminescence (PL) decreased, and electrochemiluminescence was emitted in the red shifted region.

Probe 1 was oxidized at about 0.74 V and showed strong orange-red ECL emission near 615 nm under the condition of TPA. In addition, the strength was about twice that of Ru (bpy) 32 +, which is the most widely used ECL material. Probe 1 is a form in which a formyl group is additionally introduced at the 4-position of the phenyl ring of the main ligand as compared with (piq) ₂ Ir (acac) of the comparative example, and the strong electron- Unlike the comparative example, the light-emitting layer exhibited almost no light, and exhibited luminescence characteristics in a region of a very red-shifted region of about 655 nm. However, when Hcy was added, the original strong PL was recovered at around 613 nm, which was very similar to that of (piq) ₂ Ir (acac). The reason for the strong light in the blue-shifted region is related to the increased HOMO-LUMO energy difference. That is, the LUMO level is rapidly increased due to the disappearance of the former, and the energy difference also increases.

Fig. 3 shows the phosphorescence emission spectra of probes 1, 4 and 5. Fig. It was confirmed that probe 4 according to the present invention showed no phosphorescence under the absence of hcy, probe 1 showed weak release at 670 nm, and probe 5 showed weaker release than probe 1. It was confirmed that the effect of the electron-hole excitation was further increased when the electron-hole excitation was located at R ₂ due to the introduction of the electron-hole excitation.

Test Example 1. Verification of ECL characteristics of probe 1

The ECL measurement was carried out in an aqueous solution containing 50 μM of probe 1 and 10 mM of TPA. Probe 1 itself showed little ECL signal during the cyclic voltammetry (CV) process, but ECL gradually increased with the addition of Hcy. The results for increased ECL emission are shown in Figure 4A.

No luminescence was observed when there was no Hcy, but after adding 5 mM of Hcy, two maximum ECL peaks were observed near 1.3 V and 1.6 V. In addition, when 10 mM of Hcy was added, the peak at 1.3 V disappears and only the peak near 1.6 V is observed to be observed. The binding titration curve was obtained from the ECL maximum peak value when various concentrations of Hcy were added, which is shown in FIG. 4b. It was confirmed that the ECL signal was linearly increased in the Hcy concentration range of 0-10 mM while the concentration of the probe 1 was maintained at 50 μM. The measured detection limit value is 41 μM. On the other hand, under the same experimental conditions, probe 4 showed no ECL signal in the absence of hcy, but showed intense ECL intensity at about 1.2 V as Hcy was added. The measured detection limit was 0.44 μM, which was very good.

Referring to FIG. 4A, it can be confirmed that two emission peaks are observed as a result of ECL titration. Two peaks at 1.3 V and 1.6 V gradually increased until the first 5 mM of Hcy was added. When Hcy was further added, the peak at 1.3 V gradually decreased and the peak at 1.6 V continued to increase. 4C shows the change in the intensity of the ECL peak at 1.3 V and 1.6 V. FIG. Two peaks at 1.3 V and 1.6 V gradually increased until 100 equivalents of Hcy had been added. Thereafter, the peak at 1.6 V increased and saturates when 200 equivalents of Hcy were added. Of the peak was gradually decreased. Since probe 1 has two formers, it can be expected that the 1: 2 final binding will be produced as the amount of Hcy increases gradually in the first stage when Hcy and probe 1 are combined at a ratio of 1: 1. Thus, the peak at 1.3 V is for a 1: 1 bond and the peak at 1.6 V is predicted for a 1: 2 final bond. The titration curves were plotted through the ratio of the two different ECL signals, and a linearly increasing pattern was obtained in the Hcy concentration range of 6-17 mM, which is shown in FIG. 4d.

The amount of Hcy present in the blood is generally between 5 and 15 μM, so that detection of Hcy of about 5 μM can be used to diagnose the potential risk factors of actual patients. The LOD value (41 [mu] M) of the probe 1 according to the present invention has a higher value than the detection limit value required for diagnosing Hcy or more. In order to prove that the detection limit for Hcy can be lowered by using the probe 1 according to the present invention, an ECL cell capable of measuring an amount of 25 μL was further prepared. The ECL cell was designed to be in direct contact with the PMT module with a distance of about 1 mm, and it was expected that it would have a high ECL sensitivity.

Using a new system, 0.1 μM of probe 1 in 99.9% aqueous solution was added to Hcy at various concentrations and ECL was measured. The linear increase curve was obtained for Hcy concentration in the range of 0-40 μM, Respectively. The concentration range includes almost the range required for actual diagnosis and also shows almost the same level when compared with the previously reported PL-based Hcy probes. The LOD value obtained from this experiment was 7.9 μM.

Test Example 2. Selectivity Test

In order to measure the selectivity of the probe of the present invention against Hcy, competitive binding experiments with other amino acids were conducted. First, 10 μM of other amino acids or glutathione was added to 50 μM of probe 1, followed by addition of 5 mM of Hcy. As shown in FIG. 6A, when 200 equivalents of amino acid were added, there was almost no change in ECL, but when 100 equivalents of Hcy was added, a noticeable increase in ECL was observed. That is, when 100 equivalents of Hcy were added even when 200 equivalents of amino acid was added, the ECL intensity was about 54-97% as compared with when the same amount of Hcy was added when there was no amino acid. PL competition experiments also showed similar results, demonstrating that Hcy selectively recognizes well in the presence of other amino acids without appreciable interference.

On the other hand, in the case of cysteine (Cys), the effect on interference was confirmed because it can act as an interference due to the structural similarity with Hcy. As shown in Figure 6b, weak ECL signals were observed in the presence of 10 mM Cys. However, it was measured that ECcy intensity of Hcy was 3.2 times stronger than that of Cys. This proves that the probe of the present invention is excellent in selectivity for homocysteine.

Next, the selectivity to probe 4 was measured. Various amino acids 10 mM of the probe 4 10 μM (Ala, Gly, His, Lys, Phe, Ser), glutathione and negative ions (Br-, I-, OAc-, N 3 -, NO 3 2 -, PO 4 3 - And SO ₃ ² -), respectively. As shown in FIGS. 6C and 6D, probe 4 showed only a very weak signal change at levels not significantly different from cysteine, glutathione and sulfide, and was measured to exhibit strong ECL intensity only for Hcy.

Test Example 3. Performance comparison with other iridium-based probes

In order to confirm the superiority of the probe according to the present invention, the performance of a compound represented by the formula (1) and a probe structurally similar to the probe was compared and evaluated.

Studies that detected Hcy through PL experiments have been previously reported by other researchers. Specifically, the Huang Group has developed two acetic acid-based iridium complexes (pba) ₂ Ir (acac) (probe 3), two 4- (2-pyridyl) benzaldehyde, , And the Schmittel group reported using a similar approach to develop a PL sensor for Hcy, but little change in ECL was observed.

From the electrochemical point of view, the destabilization of the LUMO distribution of the probe by the reaction of the bubbler causes the reduction potential to shift to a more negative value. Therefore, it is expected that the reduction potential will be shifted to a negative value in comparison with 3 in case of 3-Hcy generated when the probe 3 according to the prior art reacts with Hcy. However, in the ECL process, when the reduction potential is excessively negative, the electron transfer from the TPA radical to the LUMO level of the bonding material becomes difficult, so that the excited state is hardly formed and the ECL emission phenomenon does not occur well . The iridium complex with 2-phenylpyridine as the primary ligand is known to have a slightly more negative reduction potential than the TPA radical, and thus the increase of the LUMO level of 3-Hcy blocks the electron transfer, It makes it hard.

The probe (probe 1) comprising the compound of formula (1) according to the present invention is designed as an efficient Hcy ECL probe in consideration of the above problems. That is, the present inventors tried to stabilize the LUMO level by replacing the pyridine ring with isoquinoline having a stronger electron-withdrawing property, thereby developing a probe that increases luminescence in the presence of Hcy in PL as well as in ECL.

Density functional theory (DFT) calculations predict that the LUMO level of 1-Hcy will be lower than the HOMO level of TPA radicals and that this will effectively produce ECL after probe 1 has reacted with Hcy You can expect. On the other hand, it is predicted that the probe 3 rapidly increases the LUMO level after the reaction with Hcy and becomes higher than the HOMO level of the TPA radical. In this case, the electron transfer in the TPA radical does not occur well, It was expected not to happen.

To actually demonstrate these predictions experimentally, two iridium complexes 2 (probe 2) and 3 (probe 3) were synthesized. Probe 3 is the material previously disclosed in Huang Group, and Probe 2 is expected to show almost the same characteristics as Probe 3 as an analogue to 3. When Hcy was added to probe 2, the PL rapidly increased as in probe 3. However, as expected, the ECL intensity was slightly decreased with the addition of Hcy to probe 2. That is, when 10 mM Hcy was added to 50 μM of probe 2 and a voltage of 0-1.5 V was applied, 2-Hcy showed weaker ECL intensity than Probe 2 showing weak ECL near 1.2 V . The ECL properties of probe 3 and 3-Hcy also showed similar tendencies to 2, 2-Hcy. However, it was observed that the probe 1 according to the present invention shows a similar pattern when Hcy is added to both PL and ECL, which is greatly increased.

In order to clarify this fact more clearly, CV experiments were carried out and the results are shown in Table 2. The oxidation potential was measured using a cyclic voltammetric current at a scan rate of 0.1 V / s in an acetonitrile solution containing 0.1 M tetra-n-butylammonium perchlorate as a support electrolyte, and the value was 1 mM ferrocene (Fc / Fc +), and reference was made to the SCE. The reduction potential (E ^{0 '} _red ) value was calculated from the excitation energy (E _{0 -0} ) and the oxidation potential (E ^0' _ox ).

Compound classification E ^{0 '} _ox
(V vs SCE) E ^{0 '} _red *
(V vs SCE) E _{0 -0} *
(eV) 3 0.40 -1.66 2.06 3-Hcy 0.37 -1.99 2.36 3-Cys 0.38 -1.98 2.36 One 0.77 -1.04 1.81 1-Hcy 0.74 -1.28 2.02 1-Cys 0.70 -1.32 2.02

As shown in Table 2, it is confirmed that the reduction potential of the probe 3 is -1.66 V, and after the addition of Hcy, the potential shifts to -1.99 V. The reduction potential of TPA radicals is known to be about -1.7V, so this data can serve as a basis for explaining why 3-Hcy can hardly form an excited state during the ECL process. However, in probe 1, the reduction potential of 1-Hcy produced after the reaction with Hcy is -1.28 V, which is relatively mild and has a negative value lower than the reduction potential of the TPA radical, so that an effectively excited state can be formed .

Test Example 4. Evaluation of reaction rate

Hcy (5 mM) was added to the probes 1, 4 and 5 (10 μM) according to Examples 1 to 3, respectively, and the reaction rate was measured. Measurements were performed in H ₂ O / MeCN (1: 1 v / v, pH 7.4, 10 mM HEPES) (λex = 500 nm). Probe 1 took 170 minutes to reach a maximum phosphorescence intensity of 90% and probe 4 did not take more than 85 minutes to reach 90% of maximum intensity. It was also confirmed that probe 5 was slightly faster than probe 1. This is consistent with the DFT calculation.

Claims (10)

An electrochemiluminescent probe for detecting homocysteine comprising a compound represented by the following formula (1): < EMI ID =
[Chemical Formula 1]

In Formula 1,
L is a non-luminescent ligand;
R ₁ and R ₂ are each independently selected from hydrogen and C _1-10 alkyloxy, and when R ₁ is C _1-10 alkyloxy, R ₂ is not C _1-10 alkyloxy,
R ₃ is hydrogen. delete The method according to claim 1,
Wherein the non-luminescent ligand L is selected from the following structures:

In the above formulas,
_{R 6, R 7, R 8} , R 9, R 10, R 11 and R ₁₂ are the same or different and are each independently hydrogen, C _{1 - 10} alkyl, halo C _{1 - 6} alkyl and C _{5 - 12} Aryl. The method according to claim 1,
The electrochemiluminescent probe for detecting homocysteine according to claim 1, wherein the compound of Formula 1 selectively reacts with homocysteine to increase an electrochemiluminescence signal. The method according to claim 1,
The electrochemiluminescent probe for detecting homocysteine according to claim 1, wherein the compound of formula (I) reacts with homocysteine to form a hexagonal ring and increase the electrochemiluminescence signal. 6. The method of claim 5,
Wherein the LUMO energy level of the reaction product of the compound of formula (1) and homocysteine is lower than the HOMO energy level of the electrochemiluminescent photoactive reagent. The method according to claim 6,
The electrochemiluminescent probe for detecting homocysteine according to claim 1, wherein the electrochemiluminescent compound is tripropylamine or 2- (dibutylamino) ethanol. A method for detecting homocysteine in vivo using the electrochemiluminescent probe according to any one of claims 1 to 7. An electrochemiluminescence sensor comprising an electrochemiluminescent probe according to any one of claims 1 to 7. 10. The electrochemiluminescent sensor according to claim 9, wherein a change in electrochemiluminescence occurs in the blue-shifted region due to the reaction with homocysteine.

Images