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Diimine ligand as a novel chemiluminescence enhancer of luminol-containing compounds
Talanta 77 (2009) 1761–1766
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Diimine ligand as a novel chemiluminescence enhancer of luminol-containing compounds Chaivat Smanmoo, Mutsumi Yamasuji, Tomoko Sagawa, Takayuki Shibata, Tsutomu Kabashima, De-Qi Yuan, Kahee Fujita, Masaaki Kai ∗ Faculty of Pharmaceutical Sciences, School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan
a r t i c l e
i n f o
Article history: Received 15 July 2008 Received in revised form 6 October 2008 Accepted 6 October 2008 Available online 17 October 2008 Keywords: Chemiluminescence enhancer Enhancer Isoluminol Luminol Luminol-containing polymer
a b s t r a c t A series of diimine ligands (DLs) have been synthesized and evaluated for their non-enzymatic chemiluminescence (CL) enhancement of isoluminol or luminol-containing compounds. Of the DLs, N,N’-bis(m-hydroxylbenzylidene)propylenediamine (DL 10) was found to greatly enhance their CLs approximately 40 folds for isoluminol, 10 folds for luminol and 6 folds for a luminol-containing polymer. The CL enhancement of the compounds was observed in the presence of CH3 CN, H2 O2 , tetra-npropylammonium hydroxide (TPA), and Fe (III) ion. The possible mechanism of this CL enhancement was discussed on the basis of the chelate formation of the ligand and the metal ions. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The use of chemiluminescent reagents in immunoassay and DNA-hybridization assay has gained its popularity since no isotopic labeling is required. Besides, it offers a rapid assay with good sensitivity and selectivity for analytes [1]. The most wellknown chemiluminescence (CL) detection system is based on the enzymatic system coupled with enzyme-labeled horseradish peroxidase (HRP) or alkaline phosphatase (ALP) [2–4]. However, these assays are limited by the instability of enzymes [5,6]. Therefore, the non-enzymatic assay for CL detection with extremely high sensitivity is highly required. In order to increase the sensitivity of the non-enzymatic assay, an effective CL enhancer is desirable in the CL reaction. The presence of CL enhancers is beneficial to the CL reaction as they increase the intensity of light emission and prolong the emission [7]. In the last decade, several CL enhancers have been developed for the enzymatic CL reaction, mainly in HRP assays. These CL enhancers are based on the phenol derivative, e.g. lophine and its derivatives, phenylboronic acid derivatives, 6-hydroxybenzothiazole and phenol derivatives [8–12]. Recently, Cui and co-workers [13] extensively studied a number of phenol compounds for the non-enzymatic CL enhancement in the
luminol–KIO4 –H2 O2 system. However, these CL enhancers only twice enhanced the CL of luminol. Recently, we have developed sensitive macromolecular probes for the CL detection of proteins on a solid-phase membrane [14]. The probe exhibits strong CL depending on the number of luminol moieties that are incorporated into the macromolecular dextran backbone. Therefore more suitable CL enhancers for this macromolecular probe are necessary in order to further increase the sensitivity of this non-enzymatic CL detection system. This paper describes the development of a novel solid-phase CL enhancer based on diimine ligands (DL) for the luminol-containing compound. A newly synthesized diimine ligand (DL 10), N,N’-bis(mhydroxybenzylidene)propylenediamine, was found to significantly enhance the CL of isoluminol, luminol and luminol-containing dextran-based polymer. The CL enhancement by this DL was due to the complexation between DL 10 and Fe (III) which is crucial for the primary oxidation of isoluminol or luminol moiety (Fig. 1).
2. Experimental 2.1. Reagents and apparatus
∗ Corresponding author. Tel.: +81 95 819 2438; fax: +81 95 819 2438. E-mail address: [email protected] (M. Kai). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.10.010
Tetra-n-propylammonium hydroxide (TPA, 1.0 M solution in water) was purchased from Sigma–Aldrich, USA. FeCl3 , luminol, isoluminol, several diamines and benzaldehydes were obtained
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Fig. 1. The presented diagram for the CL enhancement of luminol-containing compounds by DL 10.
from TCI Co., Japan. Dextran (MW approximately 2 × 106 ) was obtained from Amersham Biosciences, UK. Biotin-AC5 -hydrazide was obtained from Dojindo, Japan. Sodium borohydride and sodium periodate were obtained from Wako Chemicals, Japan. All chemicals were of analytical grade reagents and were used as received without further purification. The water was prepared using MILLIXQ (Millipore Corp., USA). All organic solvents (acetonitrile and dimethyl sulfoxide) were freshly dried over 4 Å molecular sieves. Glasswares for the synthesis of diimine ligand were flame-dried and cooled under nitrogen atmosphere before use. The gel liquid chromatography (GLC) was carried out using a TSK gel T2000SW column (Tosoh, Japan). Elemental analysis for the synthesized compound was analyzed by Tokyo Chemical Industry, Japan. CL measurements were performed with a BLR-201 luminescence reader (Aloka, Japan). The detection of CL imaging was achieved using a Lumino CCD AE-6930 densitograph (Atto Co., Japan) and the data were processed by a computer running Densitometer Analyst version 4.0 software. For NMR experiments, residual proton signals from the deuteriated solvents were used as references of chloroform (1 H 7.25 ppm, 13 C 77 ppm) and DMSO (1 H 2.50 ppm). 2.2. Synthesis of N,N’-bis(m-hydroxylbenzylidene) propylenediammine (DL 10) 3-Hydroxybenzaldehyde (1.06 mL, 10 mmol) and distilled water (30 mL) were added into a 250 mL bottom flask at room temperature. 1,3-Propylenediamine (0.44 mL, 5 mmol) was subsequently added in one portion and the flask was kept at room temperature with vigorous stirring for 6 h. The yellow precipitate was filtered off affording the crude compound which was the recrystallized with methanol to afford DL 10 as yellow crystals (1.29 g, 92%); mp 80 ◦ C; 1 H NMR (400 MHz, CDCl3 ) ı (ppm) 2.09 (2H, quintet, J 6.8 Hz, NCH2 CH2 CH2 N), 3.69 (4H, t, J 6.8, NCH2 CH2 CH2 N), 6.76 (2H, dd, J 2.0 and 9.80, ArCH), 6.95 (2H, d, J 7.25, ArCH), 7.22 (2H, s, ArCH), 7.51 (2H, dd, J 7.80 and 8.0, ArCH), 8.22 (2H, s, CH N), 9.55 (2H, s, OH), 8.22 (2H, s, 2 × CH N); 13 C NMR (75 MHz, CDCl3 ) ı 31.7 (CH2 ), 59.1 (CH3 ), 124.9 (ArCH), 129.4 (ArCH), 131.7 (ArCH), 135.0 (ArCH), 160.0 (C N); HRMS m/z (ESI) calc. for C17 H18 N2 O2 [M+ +Na] 282.1368, found 282.1674. Other DLs were synthesized according to the above procedure. 2.3. Synthesis of luminol-containing dextran-based polymer Dextran (MW approximately 2 × 106 ) 400 mg was dissolved in H2 O (80 mL), followed by the precipitation with 300 mL of methanol. The precipitated dextran was re-dissolved in 60 mL of distilled water before reacting with sodium periodate (317 mg, 1.48 mmol). The oxidation of dextran was monitored by UVspectrophotometer at the wavelength at 310 nm. After 30% oxidation, the oxidized dextran was precipitated with 400 mL of
methanol, and then dissolved in 80 mL of DMSO. To this reaction mixture, biotin-AC5 -hydrazide (30 mg, 0.08 mmol) was added and allowed to stir at room temperature for 3 h. Glacial acetic acid (16 mL) and luminol (240 mg, 1.35 mmol) were subsequently added into the reaction mixture and left stirring at 60 ◦ C overnight. The modified dextran was precipitated with 300 mL of methanol followed by dissolving the modified dextran in ethylene glycol (30 mL). Sodium borohydride (870 mg, 23 mmol) was subsequently added into the reaction mixture and left stirring at room temperature for 4 h. The resultant biotin and luminol-containing dextran was precipitated with 300 mL of methanol. The precipitate was dissolved in 10 mL of Milli-Q water followed by re-precipitation with 300 mL of methanol. The dextran-based polymeric CL compound was then dried in vacuo and its purity was checked by gel liquid chromatography. 2.4. Procedure of liquid-phase CL detection of isoluminol or luminol enhanced by DLs Chemiluminescent reactions were carried out in 10 mm × 75 mm disposable culture tubes. After the addition of 30 L of 5.0 M isoluminol or luminol in CH3 CN, 90 L of 1.0 M TPA in H2 O, 30 L of CH3 CN, 30 L of 10 mM DLs in H2 O, 20 L of 8.8 M H2 O2 in H2 O, 30 L of 10 mM FeCl3 in H2 O into the tube, the tube was immediately placed in a luminescence reader. The signal of all reactions was displayed and integrated for 1.0 min. The kinetics of the CL reaction was monitored on a recorder connected to the luminescence reader. 2.5. CL Imaging of luminol-containing dextran-based polymer enhanced by DL 10 on a nylon membrane Three different amounts of dextran-based polymeric CL compounds (500, 250 and 130 ng) were spotted directly on a nylon membrane and dried in vacuo for 10 min. The nylon membrane was then washed with 2 mL of 100% methanol at 37 ◦ C for 10 min before drying the membrane in vacuo. The membrane was then immersed into a CL emitting reagents (300 L of CH3 CN, 700 L of 1.0 M TPA in H2 O, and 50 L of 10 mM DL 10 in H2 O) for 10 s. Then, 50 L of 8.8 M H2 O2 and 50 L of 10 mM FeCl3 were added into the emitting solution and left standing for 10 s before the CL detection for 1.0 min with CCD camera.
3. Results and discussion 3.1. Characteristics of DLs Recently, we have developed a non-enzymatic CL detection of a luminol-containing dextran-based polymer on a nylon membrane in which TPA, CH3 CN, H2 O2 and FeCl3 were employed in the CL-reaction system [14]. Attempting to enhance the CL of isoluminol and luminol employing a classical CL enhancer, p-iodophenol, led to no enhancement in our reaction system. This enhancer is most effective for the enzymatic CL reaction with HRP and luminol. Therefore, binding of p-iodophenol into the HRP pocket might be one of the responsibilities for the CL enhancement. During the course of study, we found that DL 1 exhibited weak CL enhancement of isoluminol. Encouraging by this finding, a number of DLs were prepared for the investigation of their CL enhancing ability. Isoluminol and luminol were chosen as representative CL compounds and used for subsequent optimized experiments. Ten DLs were obtained with good to excellent yields (75–92%) (Table 1). These DLs could be soluble at 10 mM concentration in water.
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Table 1 Synthetic scheme and yields of DLs.
. Yield (%)a
Diimine ligand (DL)
DL 1
75
DL 6
86
DL 2
90
DL 7
82
DL 3
84
DL 8
87
DL 4
78
DL 9
90
DL 5
79
DL 10
92
Diimine ligand (DL)
a
Structure
Structure
Yield (%)a
Isolated yield.
3.2. The effect of DL concentration for the CL enhancement of isoluminol The different concentrations of various DLs were investigated (Fig. 2). Of investigated concentrations, 10 mM DL 10 exhibited the strongest CL enhancement of isoluminol. After the optimization of Fe (III) concentrations, 10 mM Fe (III) gave the strongest CL enhancement of isoluminol. Increasing concentration of DLs resulted in significant decrease of CL intensity. This may imply the interruption of the catalytic oxidation of isoluminol. The concentration between DL 10 and Fe (III) at 1:1 ratio was optimal since at this ratio the highest CL enhancement of isoluminol was observed. Therefore, this ratio was chosen for further experiments.
the CL reactions were initiated immediately within 3 s and reached the maximum after 30 s and declined slowly to the baseline after 50 s. In the absence of Fe (III), the CL reaction initiated after 3 s and reached the maximum after 150 s, although its CL intensity was
3.3. Kinetic profiles of CL emission of isoluminol in the presence and absence of DL 10 The CL emission-time profiles of the reactions were investigated (Fig. 3). The CL signals of the reactions were shown to be rapidly emitted in the presence of Fe (III), although the CL signals were much weaker in the absence of DL 10. In the presence of Fe (III),
Fig. 2. The effect of DL concentration on the CL enhancement of isoluminol. The CL reaction was performed according to the procedure of the Section 2.4.
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Fig. 3. Time-course for CL emission of isoluminol in the presence of DL 10 and/or FeCl3 . The CL reaction was performed according to the procedure of the Section 2.4, and for non-addition of the reagents, their solvents were used.
much lower. It was clear that DL 10 significantly enhanced the CL of isoluminol. 3.4. A screening of DLs for the CL enhancement of isoluminol and luminol Various DLs (10 mM) were further screened for the CL enhancement of either isoluminol or luminol (5.0 M each) under the optimized conditions (Fig. 4). Of all ligands screened, DLs 9 and 10 strongly enhanced both CL of isoluminol and luminol. The degree of enhancement for isoluminol in the presence of DL 10 was 40 fold higher than that without DL 10. Other DLs moderately enhanced both CL of isoluminol and luminol.
Fig. 5. The effect of various metal ions on the CL enhancement of isoluminol with DL 10. The CL reaction was performed according to the procedure of the Section 2.4, and for non-addition of the reagents, their solvents were used.
3.5. Metal screening for the CL enhancement of isoluminol by DL 10 The CL of (iso)luminol is known to be efficiently catalyzed in the presence of metal ions [15]. Therefore, the effect of various metal ions (10 mM each) on the CL enhancement of isoluminol was investigated in our reaction system (Fig. 5). Of these metal ions screened, Fe (III) was the best metal catalyst for the CL emission of isoluminol in the presence of DL 10. Although in the presence of Ni (II) or Cu (II) ion, the strong CL emission was obtained, the CL enhancement of isoluminol was less degree as compared with Fe (III) catalyst. 3.6. Proposed mechanism for CL enhancement of isoluminol and luminol by DL 10
Fig. 4. Screening of DLs for the CL enhancement of (a) isoluminol and (b) luminol. The CL reaction was performed according to the procedure of the Section 2.4, and for non-addition of the reagents, their solvents were used.
At this stage, the mechanism for the CL enhancement of (iso)luminol by DL 10 is still not conclusive, since the crystal of the diimine and Fe (III) complex could not be obtained to analyse its structure with an X-ray diffraction instrument. However, the complex between Fe (III) and DL 10 might effectively enhance the catalysis of the generation of hydroxyl or hydroxyl-like radicals which are important species for the primary oxidation of (iso)luminol. This primary oxidation of (iso)luminol may be a crucial step contributing to the CL enhancement of the proposed system. If the complexation between Fe (III) and DL is one of the important factors for the overall CL enhancement of the proposed system, DLs 4 and 10 should exhibit similar degree of CL enhancement since these DLs are isomers. However, DL 10 exhibited with a higher degree of CL enhancement of both isoluminol and luminol. The differences of these DLs are i) the position of the hydroxyl group (ortho- and meta-positions for DLs 4 and 10, respectively). As shown in Fig. 6, the energy-optimized modeling indicated that the bond length between chelated Fe (III) and nitrogen atom is 1.738 Å for DL 4 and 1.875 Å for DL 10. The bond length between Fe (III) and oxygen atom is 1.905 Å and 2.029 Å for DLs 4 and 10, respectively. It indicated that DL 10 provides a bigger room to accommodate Fe (III) than DL 4. This spacious room for Fe (III) accommodation might be responsible for facilitating the proper conformation of DL to perform a stable complexation between Fe (III). This might be an important factor responsible for the overall CL enhancement of the proposed system. The effect of spacer group of diimine moiety was more pronounced for DLs 3 and 9 compared to DLs 4 and 10. The methylene spacer reduces the bond length between the nitrogen or oxygen atoms and the chelated Fe (III) which lead to the shrinkage of the spacer for Fe (III) accommodation. In addition to the position of the hydroxyl group, the
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Fig. 6. Energy-optimized structures of DLs 4 and 10 chelating Fe (III).
length of the spacer group was also an important factor for the CL enhancement of the proposed system. 3.7. CL enhancement of a luminol-containing compound by DL 10 Finally, DL 10 was employed for the CL enhancement of a luminol-containing dextran-based polymer on a nylon membrane (Fig. 7). In the presence of DL 10, the CL of the dextran-based polymeric compound was enhanced six-fold higher as compared with the absence of DL 10. The reason that less degree of CL enhancement was observed may be due to the less exposure of luminol units buried inside the dextran-based macromolecular structure to the CL emitting reagents. In this detection system, a maximum CL emission should be detected within 5 min for flashing-CL mode adapted to a CCD camera. Therefore, a high concentration of H2 O2 in the presence of a strongly alkaline organic base was required in order to emit the CL fast on the membrane. During this solid-phase CL reaction, a yellow precipitation was observed which might be Fe(OH)3 after the addition of FeCl3 without DL 10. However, this precipitation did not occur in the presence of DL 10 because of the chelation of the metal ion with the diimine. 4. Conclusions In conclusion, DL 10 has been first time synthesized and evaluated for its CL enhancement of the (iso)luminol-containing compounds. The possible mechanism for the CL enhancement by DL 10 is proposed on the basis of the complex formation between Fe (III) and the ligand. This complex might be responsible for the primary oxidation of (iso)luminol residues in the compound which is crucial for the CL enhancement of the present system. The flexible spacer of the propylene group in DL 10 might also provide a better conformation of DL 10 to accommodate Fe (III) ion. This finding permits to use the ligand as an enhancer for the sensitive and non-enzymatic CL detection of luminol-containing compounds. Acknowledgements We thank the Japan Society for the Promotion of Science (JSPS) for the financial support and this work was also supported partly from Japanese Ministry of Health, Welfare and Labor. References
Fig. 7. Non-enzymatic CL enhancement (a) with and (b) without DL 10 for the imaging detection of a luminol-containing dextran-based polymer. The polymer (ca. 2,500,000 Da containing ca. 3000 units of luminol in the molecule) was spotted at 500, 250 and 130 ng on a nylon membrane.
[1] T.P. Whitehead, L.J. Kricka, T.J.N. Carter, G.H.G. Thorpe, Clin. Chem. 25 (1979) 1531. [2] R. Iwata, H. Ito, T. Hayashi, Y. Sekine, N. Koyama, M. Yamaki, Anal. Biochem. 231 (1995) 170. [3] I. Bronstein, B. Edwards, J.C.J. Vote, Biolumin. Chemilumin. 4 (1989) 99. [4] A.P. Schaap, H. Akhavan, L.J. Romano, Clin. Chem. 35 (1989 1863).
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[5] G.H.G. Thorpe, L.J. Kricka, S.B. Moseley, T.P. Whitehead, Clin. Chem. 31 (1985) 1335. [6] T.P. Whitehead, G.H.G. Thorpe, T.J.N. Carter, Nature 305 (1983) 158. [7] Y. Dotsikas, Y.L. Loukas, Talanta 71 (2007) 906. [8] L.J. Kricka, G.H.G. Thorpe, R.A.W. Stott, Pure Appl. Chem. 59 (1987) 651. [9] T.J.N. Carter, C.J. Groucutt, R.A. W Scott, European Patent, EP 87959, 1982. [10] L.J. Kricka, M. Cooper, X. Ji, Anal. Biochem. 240 (1996) 119. [11] D.N. Lee, H.J. Park, D.H. Kim, S.W. Lee, S.J. Park, B.H. Kim, W.Y. Lee, Bull. Korean Chem. Soc. 23 (2002) 13.
[12] F. Kennedy, N.M. Shavaleev, T. Koullourou, Z.R. Bell, J.C. Jeffery, S. Faulkner, M.D. Ward, Dalton Trans. (2007) 1492. [13] H. Xu, C.-F. Duan, C.-Z. Lai, M. Lian, Z.-F. Zhang, L.-J. Liu, H. Cui, Luminescence 21 (2006) 195. [14] H. Zhang, C. Smanmoo, T. Kabashima, J. Lu, M. Kai, Angew. Chem. Int. Ed. 46 (2007) 8226. [15] S. Hanaoka, J.-M. Lin, M. Yamada, Anal. Chim. Acta 409 (2000) 65.
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Diimine ligand as a novel chemiluminescence enhancer of luminol-containing compounds Chaivat Smanmoo, Mutsumi Yamasuji, Tomoko Sagawa, Takayuki Shibata, Tsutomu Kabashima, De-Qi Yuan, Kahee Fujita, Masaaki Kai ∗ Faculty of Pharmaceutical Sciences, School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan
a r t i c l e
i n f o
Article history: Received 15 July 2008 Received in revised form 6 October 2008 Accepted 6 October 2008 Available online 17 October 2008 Keywords: Chemiluminescence enhancer Enhancer Isoluminol Luminol Luminol-containing polymer
a b s t r a c t A series of diimine ligands (DLs) have been synthesized and evaluated for their non-enzymatic chemiluminescence (CL) enhancement of isoluminol or luminol-containing compounds. Of the DLs, N,N’-bis(m-hydroxylbenzylidene)propylenediamine (DL 10) was found to greatly enhance their CLs approximately 40 folds for isoluminol, 10 folds for luminol and 6 folds for a luminol-containing polymer. The CL enhancement of the compounds was observed in the presence of CH3 CN, H2 O2 , tetra-npropylammonium hydroxide (TPA), and Fe (III) ion. The possible mechanism of this CL enhancement was discussed on the basis of the chelate formation of the ligand and the metal ions. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The use of chemiluminescent reagents in immunoassay and DNA-hybridization assay has gained its popularity since no isotopic labeling is required. Besides, it offers a rapid assay with good sensitivity and selectivity for analytes [1]. The most wellknown chemiluminescence (CL) detection system is based on the enzymatic system coupled with enzyme-labeled horseradish peroxidase (HRP) or alkaline phosphatase (ALP) [2–4]. However, these assays are limited by the instability of enzymes [5,6]. Therefore, the non-enzymatic assay for CL detection with extremely high sensitivity is highly required. In order to increase the sensitivity of the non-enzymatic assay, an effective CL enhancer is desirable in the CL reaction. The presence of CL enhancers is beneficial to the CL reaction as they increase the intensity of light emission and prolong the emission [7]. In the last decade, several CL enhancers have been developed for the enzymatic CL reaction, mainly in HRP assays. These CL enhancers are based on the phenol derivative, e.g. lophine and its derivatives, phenylboronic acid derivatives, 6-hydroxybenzothiazole and phenol derivatives [8–12]. Recently, Cui and co-workers [13] extensively studied a number of phenol compounds for the non-enzymatic CL enhancement in the
luminol–KIO4 –H2 O2 system. However, these CL enhancers only twice enhanced the CL of luminol. Recently, we have developed sensitive macromolecular probes for the CL detection of proteins on a solid-phase membrane [14]. The probe exhibits strong CL depending on the number of luminol moieties that are incorporated into the macromolecular dextran backbone. Therefore more suitable CL enhancers for this macromolecular probe are necessary in order to further increase the sensitivity of this non-enzymatic CL detection system. This paper describes the development of a novel solid-phase CL enhancer based on diimine ligands (DL) for the luminol-containing compound. A newly synthesized diimine ligand (DL 10), N,N’-bis(mhydroxybenzylidene)propylenediamine, was found to significantly enhance the CL of isoluminol, luminol and luminol-containing dextran-based polymer. The CL enhancement by this DL was due to the complexation between DL 10 and Fe (III) which is crucial for the primary oxidation of isoluminol or luminol moiety (Fig. 1).
2. Experimental 2.1. Reagents and apparatus
∗ Corresponding author. Tel.: +81 95 819 2438; fax: +81 95 819 2438. E-mail address: [email protected] (M. Kai). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.10.010
Tetra-n-propylammonium hydroxide (TPA, 1.0 M solution in water) was purchased from Sigma–Aldrich, USA. FeCl3 , luminol, isoluminol, several diamines and benzaldehydes were obtained
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Fig. 1. The presented diagram for the CL enhancement of luminol-containing compounds by DL 10.
from TCI Co., Japan. Dextran (MW approximately 2 × 106 ) was obtained from Amersham Biosciences, UK. Biotin-AC5 -hydrazide was obtained from Dojindo, Japan. Sodium borohydride and sodium periodate were obtained from Wako Chemicals, Japan. All chemicals were of analytical grade reagents and were used as received without further purification. The water was prepared using MILLIXQ (Millipore Corp., USA). All organic solvents (acetonitrile and dimethyl sulfoxide) were freshly dried over 4 Å molecular sieves. Glasswares for the synthesis of diimine ligand were flame-dried and cooled under nitrogen atmosphere before use. The gel liquid chromatography (GLC) was carried out using a TSK gel T2000SW column (Tosoh, Japan). Elemental analysis for the synthesized compound was analyzed by Tokyo Chemical Industry, Japan. CL measurements were performed with a BLR-201 luminescence reader (Aloka, Japan). The detection of CL imaging was achieved using a Lumino CCD AE-6930 densitograph (Atto Co., Japan) and the data were processed by a computer running Densitometer Analyst version 4.0 software. For NMR experiments, residual proton signals from the deuteriated solvents were used as references of chloroform (1 H 7.25 ppm, 13 C 77 ppm) and DMSO (1 H 2.50 ppm). 2.2. Synthesis of N,N’-bis(m-hydroxylbenzylidene) propylenediammine (DL 10) 3-Hydroxybenzaldehyde (1.06 mL, 10 mmol) and distilled water (30 mL) were added into a 250 mL bottom flask at room temperature. 1,3-Propylenediamine (0.44 mL, 5 mmol) was subsequently added in one portion and the flask was kept at room temperature with vigorous stirring for 6 h. The yellow precipitate was filtered off affording the crude compound which was the recrystallized with methanol to afford DL 10 as yellow crystals (1.29 g, 92%); mp 80 ◦ C; 1 H NMR (400 MHz, CDCl3 ) ı (ppm) 2.09 (2H, quintet, J 6.8 Hz, NCH2 CH2 CH2 N), 3.69 (4H, t, J 6.8, NCH2 CH2 CH2 N), 6.76 (2H, dd, J 2.0 and 9.80, ArCH), 6.95 (2H, d, J 7.25, ArCH), 7.22 (2H, s, ArCH), 7.51 (2H, dd, J 7.80 and 8.0, ArCH), 8.22 (2H, s, CH N), 9.55 (2H, s, OH), 8.22 (2H, s, 2 × CH N); 13 C NMR (75 MHz, CDCl3 ) ı 31.7 (CH2 ), 59.1 (CH3 ), 124.9 (ArCH), 129.4 (ArCH), 131.7 (ArCH), 135.0 (ArCH), 160.0 (C N); HRMS m/z (ESI) calc. for C17 H18 N2 O2 [M+ +Na] 282.1368, found 282.1674. Other DLs were synthesized according to the above procedure. 2.3. Synthesis of luminol-containing dextran-based polymer Dextran (MW approximately 2 × 106 ) 400 mg was dissolved in H2 O (80 mL), followed by the precipitation with 300 mL of methanol. The precipitated dextran was re-dissolved in 60 mL of distilled water before reacting with sodium periodate (317 mg, 1.48 mmol). The oxidation of dextran was monitored by UVspectrophotometer at the wavelength at 310 nm. After 30% oxidation, the oxidized dextran was precipitated with 400 mL of
methanol, and then dissolved in 80 mL of DMSO. To this reaction mixture, biotin-AC5 -hydrazide (30 mg, 0.08 mmol) was added and allowed to stir at room temperature for 3 h. Glacial acetic acid (16 mL) and luminol (240 mg, 1.35 mmol) were subsequently added into the reaction mixture and left stirring at 60 ◦ C overnight. The modified dextran was precipitated with 300 mL of methanol followed by dissolving the modified dextran in ethylene glycol (30 mL). Sodium borohydride (870 mg, 23 mmol) was subsequently added into the reaction mixture and left stirring at room temperature for 4 h. The resultant biotin and luminol-containing dextran was precipitated with 300 mL of methanol. The precipitate was dissolved in 10 mL of Milli-Q water followed by re-precipitation with 300 mL of methanol. The dextran-based polymeric CL compound was then dried in vacuo and its purity was checked by gel liquid chromatography. 2.4. Procedure of liquid-phase CL detection of isoluminol or luminol enhanced by DLs Chemiluminescent reactions were carried out in 10 mm × 75 mm disposable culture tubes. After the addition of 30 L of 5.0 M isoluminol or luminol in CH3 CN, 90 L of 1.0 M TPA in H2 O, 30 L of CH3 CN, 30 L of 10 mM DLs in H2 O, 20 L of 8.8 M H2 O2 in H2 O, 30 L of 10 mM FeCl3 in H2 O into the tube, the tube was immediately placed in a luminescence reader. The signal of all reactions was displayed and integrated for 1.0 min. The kinetics of the CL reaction was monitored on a recorder connected to the luminescence reader. 2.5. CL Imaging of luminol-containing dextran-based polymer enhanced by DL 10 on a nylon membrane Three different amounts of dextran-based polymeric CL compounds (500, 250 and 130 ng) were spotted directly on a nylon membrane and dried in vacuo for 10 min. The nylon membrane was then washed with 2 mL of 100% methanol at 37 ◦ C for 10 min before drying the membrane in vacuo. The membrane was then immersed into a CL emitting reagents (300 L of CH3 CN, 700 L of 1.0 M TPA in H2 O, and 50 L of 10 mM DL 10 in H2 O) for 10 s. Then, 50 L of 8.8 M H2 O2 and 50 L of 10 mM FeCl3 were added into the emitting solution and left standing for 10 s before the CL detection for 1.0 min with CCD camera.
3. Results and discussion 3.1. Characteristics of DLs Recently, we have developed a non-enzymatic CL detection of a luminol-containing dextran-based polymer on a nylon membrane in which TPA, CH3 CN, H2 O2 and FeCl3 were employed in the CL-reaction system [14]. Attempting to enhance the CL of isoluminol and luminol employing a classical CL enhancer, p-iodophenol, led to no enhancement in our reaction system. This enhancer is most effective for the enzymatic CL reaction with HRP and luminol. Therefore, binding of p-iodophenol into the HRP pocket might be one of the responsibilities for the CL enhancement. During the course of study, we found that DL 1 exhibited weak CL enhancement of isoluminol. Encouraging by this finding, a number of DLs were prepared for the investigation of their CL enhancing ability. Isoluminol and luminol were chosen as representative CL compounds and used for subsequent optimized experiments. Ten DLs were obtained with good to excellent yields (75–92%) (Table 1). These DLs could be soluble at 10 mM concentration in water.
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Table 1 Synthetic scheme and yields of DLs.
. Yield (%)a
Diimine ligand (DL)
DL 1
75
DL 6
86
DL 2
90
DL 7
82
DL 3
84
DL 8
87
DL 4
78
DL 9
90
DL 5
79
DL 10
92
Diimine ligand (DL)
a
Structure
Structure
Yield (%)a
Isolated yield.
3.2. The effect of DL concentration for the CL enhancement of isoluminol The different concentrations of various DLs were investigated (Fig. 2). Of investigated concentrations, 10 mM DL 10 exhibited the strongest CL enhancement of isoluminol. After the optimization of Fe (III) concentrations, 10 mM Fe (III) gave the strongest CL enhancement of isoluminol. Increasing concentration of DLs resulted in significant decrease of CL intensity. This may imply the interruption of the catalytic oxidation of isoluminol. The concentration between DL 10 and Fe (III) at 1:1 ratio was optimal since at this ratio the highest CL enhancement of isoluminol was observed. Therefore, this ratio was chosen for further experiments.
the CL reactions were initiated immediately within 3 s and reached the maximum after 30 s and declined slowly to the baseline after 50 s. In the absence of Fe (III), the CL reaction initiated after 3 s and reached the maximum after 150 s, although its CL intensity was
3.3. Kinetic profiles of CL emission of isoluminol in the presence and absence of DL 10 The CL emission-time profiles of the reactions were investigated (Fig. 3). The CL signals of the reactions were shown to be rapidly emitted in the presence of Fe (III), although the CL signals were much weaker in the absence of DL 10. In the presence of Fe (III),
Fig. 2. The effect of DL concentration on the CL enhancement of isoluminol. The CL reaction was performed according to the procedure of the Section 2.4.
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Fig. 3. Time-course for CL emission of isoluminol in the presence of DL 10 and/or FeCl3 . The CL reaction was performed according to the procedure of the Section 2.4, and for non-addition of the reagents, their solvents were used.
much lower. It was clear that DL 10 significantly enhanced the CL of isoluminol. 3.4. A screening of DLs for the CL enhancement of isoluminol and luminol Various DLs (10 mM) were further screened for the CL enhancement of either isoluminol or luminol (5.0 M each) under the optimized conditions (Fig. 4). Of all ligands screened, DLs 9 and 10 strongly enhanced both CL of isoluminol and luminol. The degree of enhancement for isoluminol in the presence of DL 10 was 40 fold higher than that without DL 10. Other DLs moderately enhanced both CL of isoluminol and luminol.
Fig. 5. The effect of various metal ions on the CL enhancement of isoluminol with DL 10. The CL reaction was performed according to the procedure of the Section 2.4, and for non-addition of the reagents, their solvents were used.
3.5. Metal screening for the CL enhancement of isoluminol by DL 10 The CL of (iso)luminol is known to be efficiently catalyzed in the presence of metal ions [15]. Therefore, the effect of various metal ions (10 mM each) on the CL enhancement of isoluminol was investigated in our reaction system (Fig. 5). Of these metal ions screened, Fe (III) was the best metal catalyst for the CL emission of isoluminol in the presence of DL 10. Although in the presence of Ni (II) or Cu (II) ion, the strong CL emission was obtained, the CL enhancement of isoluminol was less degree as compared with Fe (III) catalyst. 3.6. Proposed mechanism for CL enhancement of isoluminol and luminol by DL 10
Fig. 4. Screening of DLs for the CL enhancement of (a) isoluminol and (b) luminol. The CL reaction was performed according to the procedure of the Section 2.4, and for non-addition of the reagents, their solvents were used.
At this stage, the mechanism for the CL enhancement of (iso)luminol by DL 10 is still not conclusive, since the crystal of the diimine and Fe (III) complex could not be obtained to analyse its structure with an X-ray diffraction instrument. However, the complex between Fe (III) and DL 10 might effectively enhance the catalysis of the generation of hydroxyl or hydroxyl-like radicals which are important species for the primary oxidation of (iso)luminol. This primary oxidation of (iso)luminol may be a crucial step contributing to the CL enhancement of the proposed system. If the complexation between Fe (III) and DL is one of the important factors for the overall CL enhancement of the proposed system, DLs 4 and 10 should exhibit similar degree of CL enhancement since these DLs are isomers. However, DL 10 exhibited with a higher degree of CL enhancement of both isoluminol and luminol. The differences of these DLs are i) the position of the hydroxyl group (ortho- and meta-positions for DLs 4 and 10, respectively). As shown in Fig. 6, the energy-optimized modeling indicated that the bond length between chelated Fe (III) and nitrogen atom is 1.738 Å for DL 4 and 1.875 Å for DL 10. The bond length between Fe (III) and oxygen atom is 1.905 Å and 2.029 Å for DLs 4 and 10, respectively. It indicated that DL 10 provides a bigger room to accommodate Fe (III) than DL 4. This spacious room for Fe (III) accommodation might be responsible for facilitating the proper conformation of DL to perform a stable complexation between Fe (III). This might be an important factor responsible for the overall CL enhancement of the proposed system. The effect of spacer group of diimine moiety was more pronounced for DLs 3 and 9 compared to DLs 4 and 10. The methylene spacer reduces the bond length between the nitrogen or oxygen atoms and the chelated Fe (III) which lead to the shrinkage of the spacer for Fe (III) accommodation. In addition to the position of the hydroxyl group, the
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Fig. 6. Energy-optimized structures of DLs 4 and 10 chelating Fe (III).
length of the spacer group was also an important factor for the CL enhancement of the proposed system. 3.7. CL enhancement of a luminol-containing compound by DL 10 Finally, DL 10 was employed for the CL enhancement of a luminol-containing dextran-based polymer on a nylon membrane (Fig. 7). In the presence of DL 10, the CL of the dextran-based polymeric compound was enhanced six-fold higher as compared with the absence of DL 10. The reason that less degree of CL enhancement was observed may be due to the less exposure of luminol units buried inside the dextran-based macromolecular structure to the CL emitting reagents. In this detection system, a maximum CL emission should be detected within 5 min for flashing-CL mode adapted to a CCD camera. Therefore, a high concentration of H2 O2 in the presence of a strongly alkaline organic base was required in order to emit the CL fast on the membrane. During this solid-phase CL reaction, a yellow precipitation was observed which might be Fe(OH)3 after the addition of FeCl3 without DL 10. However, this precipitation did not occur in the presence of DL 10 because of the chelation of the metal ion with the diimine. 4. Conclusions In conclusion, DL 10 has been first time synthesized and evaluated for its CL enhancement of the (iso)luminol-containing compounds. The possible mechanism for the CL enhancement by DL 10 is proposed on the basis of the complex formation between Fe (III) and the ligand. This complex might be responsible for the primary oxidation of (iso)luminol residues in the compound which is crucial for the CL enhancement of the present system. The flexible spacer of the propylene group in DL 10 might also provide a better conformation of DL 10 to accommodate Fe (III) ion. This finding permits to use the ligand as an enhancer for the sensitive and non-enzymatic CL detection of luminol-containing compounds. Acknowledgements We thank the Japan Society for the Promotion of Science (JSPS) for the financial support and this work was also supported partly from Japanese Ministry of Health, Welfare and Labor. References
Fig. 7. Non-enzymatic CL enhancement (a) with and (b) without DL 10 for the imaging detection of a luminol-containing dextran-based polymer. The polymer (ca. 2,500,000 Da containing ca. 3000 units of luminol in the molecule) was spotted at 500, 250 and 130 ng on a nylon membrane.
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