Studies on the Development of Electrogenerated Chemiluminescent (ECL) Chemosensors Using Cyclometalated Ir(III) Complex

Abstract

The development of effective chemosensors for detecting small-molecule biotargets has been attracted significant attention in biosensing researches for a long time. Among them, ECL-based chemosensors have several advantages over the conventional analytical techniques such as high sensitivity, good reproducibility, and simple analytic process. Part I describes the development of fluorescent chemosensors for phosphate-containing anions and their application to ECL. Through these investigations, we confirmed that it is possible to apply the same principles of designing fluorescent chemosensors to ECL analysis. Moreover, these studies also showed the both high sensitivity and simplicity of ECL-based detecting systems. First, coumarin-based fluorescent chemosensor 1-2Zn was developed for the sensitive sensing of pyrophosphate (PPi). 1-2Zn has bis(2-pyridylmethyl)amine (DPA)-Zn2+ complex as a selective recognition sites for PPi and a coumarin fluorophore as a signaling unit. 1-2Zn showed an improved detection limit for PPi compared to that of the naphthyl-based PPi sensor due to the introduction of coumarin dye. In addition, it showed good selectivity for PPi over other anions. Additionally, the new chemosensor 2-2Zn was developed for the discrimination of PPi, ATP, and GTP in aqueous solution. The fluorescence changes of 2-2Zn for each phosphate-containing anions resulted from the structural change (excimer formation) and from photo-induced electron transfer (PET) quenching. 2-2Zn induced a little red-shifted emission change when PPi was added, while it led to enhanced fluorescence and fluorescence quenching without a wavelength shifts upon the addition of ATP and GTP, respectively. Lastly, the pyrene-based turn-on probe 3 for organophosphate nerve agents was developed. Probe 3 showed highly sensitive and rapid detection capabilities through intramolecular cyclization. In addition to PL study, we carried out the ECL experiment for diethyl chlorophosphate (DCP) for the first time. Though PET quenching efficiency of probe 3 in ECL analysis was lowered due to the oxidation of tertiary amine and the ECL efficiency of pyrene in oxidative reduction process was lower than previously known ECL luminophores such as Ru(bpy)32+, it was still possible to detect and quantify the nerve agents with high sensitivity. In Part II, highly sensitive ECL chemodosimeters for small anions based on cyclometalated Ir(III) complex were presented. For high ECL efficiency, phenylisoquinoline (PIQ) unit which has ideal HOMO/LUMO energy levels was selected as a main ligand of Ir(III) complex. Probe 4 was synthesized for ECL determination of homocysteine, which selectively reacts with Hcy then produces efficient light emission via an electrochemical process. Formyl groups in the main ligands of probe 4 underwent a ring-formation reaction with Hcy, triggering changes in the thermodynamic and photophysical properties of the probe itself. The level of Hcy was successfully monitored in 99.9% aqueous media with a linear correlation between 0-40 μM. The mechanistic explanation has also been suggested based on the electrochemical and theoretical studies. The ECL analytical method would enable point-of-care testing of Hcy levels and is potentially useful for precautionary diagnosis of cardiovascular diseases. To improve the reactivity between probe 4 and Hcy, probes 5 and 6 were developed, bearing an additional methoxy group as an electron-donating group relative to the original probe 4. Specifically, probe 5 showed improve properties compared to probe 4, including a faster reaction time, higher sensitivity, and a higher turn-on ratio. The methoxy groups in probe 5 induced the destabilization of HOMO energy level, which resulted in the improved reaction rate and fully-quenched initial ECL intensity. Probe 5 showed 15-times lower detection limit and 1.9-times faster reaction time as compared to those of probe 4. This study provided the new strategy with which to design Ir(III)-complex-based molecular probe with high sensitivity and reactivity via the modulation of energy levels with additional substituents. In the next step, the ECL turn-on probe 7 for cyanide based on Ir(III) complex was designed and synthesized. Probe 7 possessed phenyl isoquionline groups as main ligands with dicyanovinyl groups as the reaction sites for cyanide. In the presence of cyanide, the red ECL emission of probe 7 was greatly increased with good selectivity. Probe 7 showed the linear correlation in the range of 0 to 0.4 mM when the probe concentration was 10 uM in an aqueous solution. We conducted a quantification test through a standard addition analysis in tap water successfully with high reliability and reproducibility. Theoretical studies were carried out for the rational design of probe by predicting the HOMO/LUMO energy levels and electronic distributions. Lastly, Ir(III) complex 8 was developed for the selective sensing of sulfide. Probe 8 has two different parts of reaction sites, unsaturated acrylated and dinitrobenzenesulfornyl (DNBS) group. The unsaturated acrylate unit reacted with sulfide selectively, inducing phosphorescence and leading to an ECL enhancement in the blue-shifted region. In addition, the DNBS group was well-known PET quencher which could be cleaved by sulfide and other biothiols such as cysteine or homocysteine. Probe 8 showed high sensitivity and good selectivity by given the introduction of two reaction sites in a single molecule. In Part III, the rational design and mechanistic study of Ir(III)-based ECL probes by tuning the LUMO level were presented. Because the LUMO energy level of emitter is closely related to ECL efficiency during TPA coreactant process, it is possible to develop ECL chemosensors in a new way that differs from the methods used to create fluorescent chemosensors by controlling LUMO level with a specific reaction site for the analyte. We selected the (ppy)2Ir(acac) complex as a backbone molecule and introduced the specific functional groups to various position of phenylpyridine. Three ppy-based Ir(III) complexes bearing formyl group as a reaction site for were developed. The formyl groups in pyridine rings strongly stabilized the LUMO level, and the additional substituents served to modulate the HOMO and LUMO levels while maintaining the HOMO-LUMO energy gap of each iridium complex. The OMe_acac complex showed different signal changes in PL and ECL upon the addition of cyanide. An obvious ratiometric change was observed in PL, whereas the ECL signal decreased. The electron-donating methoxy group meta substituted to coordinated Ir(III) metal in OMe_acac made the HOMO and LUMO energy level unstable, leading to the hard generation of excited states during ECL process. In contrast, H_acac and Br_acac showed similar results in terms of PL and ECL dure to their relatively stabilized LUMO levels. Next, we used boronate groups to Ir(III) complexes as a reaction site for H2O2. In this case, the change of HOMO and LUMO levels depended on the substitution location. Probe 9 and 11 showed a phosphorescent change from yellow to green upon the addition of H2O2, whereas probe 10 showed a phosphorescent change from yellow to green under an identical condition. Probe 12 showed a phosphorescent change similar to those of 9 and 11

however, the modification of pyridyl rings had a strong influence on the LUMO energy level, while the modification of the phenyl rings mainly affected to HOMO energy levels. The destabilization of the LUMO level of probe 12 upon the addition of H2O2 degraded the ECL efficiency by blocking the ECL electron transfer process. The ECL intensity of 12 was greatly decreased upon the addition of H2O2 compared to the results with other probes, demonstraing an outcome similar to that of the PL study. This work provided the rational design of ECL probe based on ECL quenching route via the control of the LUMO level, which is a differentiated strategy for the development of general fluorescence-based probes.