Synthesis of ruthenium and bodipy based luminophores and their applications in electrochemiluminescent detection platforms
Date
2019
Authors
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Publisher
University of Delaware
Abstract
Electrogenerated chemiluminescence (ECL) – the unique combination of electrochemistry and spectroscopy – is a powerful analytical technique that offers high sensitivity and selectivity, low noise to signal ratios and wide dynamic range for sensing and detection applications. Because of these incredible properties, ECL has already been applied to a broad range of areas ranging from fundamental studies to a variety of practical applications; however, it is still not as commonly used as some other detection methods such as fluorescence or ELISA and not fully integrated into general screening platforms to date. This dissertation focuses on the improvement and development of novel modular platforms that are based on ECL and describes the utility of inexpensive devices for studying basic electrochemical reactions such as ECL and water splitting. ☐ After a brief introduction to ECL and relevant chemistries in Chapter 1, Chapter 2 focuses on the development of new luminophores that are ECL active, suitable with physiological environments and can label various biomolecules; which is necessary to increase ECL‘s incorporation as a detection method into a wide range of biosensing applications. Specifically, synthesis and characterization of a series of [Ru(bpy)3]2+ and BODIPY architectures functionalized with maleimide and NTA groups that are designed to label cysteine and histidine appended proteins were performed. All of the emitters were easily synthesized via multistep processes and purified using techniques like column chromatography, recrystallization and distillation. [Ru(bpy)3]2+ complexes were highly soluble in aqueous systems; BODIPY probes, on the other hand, required an organic co-solvent (DMSO) to be dissolved in aqueous buffer solutions. The photophysics of the synthesized luminophores was studied thoroughly in Milli-Q water, methanol and Tris buffer solutions depending on luminophores‘ solubility. When excited at 436 nm, [Ru(bpy)3]2+–PEG1–Maleimide and [Ru(bpy)3]2+–PEG8–Maleimide emit at the same wavelength –approximately at 615 nm – in both of the aqueous systems (Milli-Q water and Tris buffer) and show slightly higher quantum yields in the buffer solutions. [Ru(bpy)3]2+–NTA displayed a red shifted luminesce (642 nm) compared to maleimide terminated [Ru(bpy)3]2+ complexes which can be attributed to the introduction of three carboxylic acid groups on the NTA functional group and no enhancement of the quantum yield efficiency was observed in Tris buffer relative to its Milli-Q water solution. In the case of BODIPY derivatives, all three compounds (BODIPY–PEG1–Maleimide, BODIPY–PEG8–Maleimide and BODIPY–NTA) displayed emission signals approximately at 543 nm with irradiation at 488 nm in methanol and Tris buffer. Due to the poor solubility of each BODIPY derivative in aqueous media, higher quantum yield efficiencies were observed in methanol solutions. ☐ In Chapter 3, a novel ECL based detection platform was developed to monitor complex association/dissociation interactions between biomolecules to complement commonly used existing fluorescence-based high throughput screening (HTS) assays. Our ECL platform was established on a basis of binding between a transcription factor (TF) binding domain DNA sequence and a NF-B mutant tagged with an electroactive luminophore. In developing this platform, wt- NF-B and cys- NF-B protein mutants were conjugated with a maleimide appended tris(bipyridine)ruthenium(II) ([Ru(bpy)3]2+–PEG1–Maleimide) luminophore. Photophysical characterization of the NF-B bioconjugates demonstrates that both labeled proteins absorb at approximately 460 nm, characteristic of the MLCT absorption band of [Ru(bpy)3]2+ based complexes and luminesce with quantum yields of cys- NF-B—[Ru] = 0.021 and wt- NF-B—[Ru] = 0.019. In addition, Q-TOF mass spectrometry analysis ensured the successful attachment of the luminophores to the NF-B mutants. To construct the platform to which NF-B could bind, amine terminated TF-DNA was tethered to carboxylic acid functionalized magnetic beads (MBs) using carbodiimide coupling chemistry. Analytical surface characterization techniques including XPS and TOF-SIMS demonstrated the efficacy with which the TF-DNA was covalently linked to the surface of the MBs. Upon incubation of NF-B —[Ru] mutants onto MB—TF-DNA at an electrode surface, an ECL signal was generated consistent with TF-DNA••• NF-B binding. Addition of a competitive NF-B binder to the system resulted in approximately 30% attenuation of the average ECL signal. Overall, these results demonstrate that ECL can be used to study the binding of luminophore labeled NF-B at an electrode interface and suggest that such platforms may potentially find utility for identification of small molecule antagonists of DNA••• NF-B binding. ☐ The proposed sensing platform based on ECL in Chapter 3 along with many others, commercially available or reported in the literature, involves labeling biomolecules. Although these platforms are widely successful, label-based detections can cause issues such as a change in natural physiochemical properties of the biomolecules, complex purification processes, and extra synthesis steps. In Chapter 4, a modular ECL platform was developed to serve as a basis for label free detections which may be useful to address these problems. In an effort to build this platform, commercially available, azide terminated magnetic microparticles were directly labeled with the alkyne terminated tris(bipyridine)ruthenium(II) ([Ru(bpy)3]2+–Alkyne) ECL emitter via Cu-Catalyzed Azide Alkyne Cycloadditon [CuAAC] chemistry. The completion of the modification reaction was monitored with FTIR spectroscopy by observing the disappearance of the characteristic azide band at 2101 cm−1. XPS analysis also provided additional evidence for the successful attachment of [Ru(bpy)3]2+–Alkyne luminophores to the surface of the magnetic beads. Photophysical characterization studies revealed that unmodified magnetic beads do not display any luminescence features prior to being labeled with [Ru(bpy)3]2+–Alkyne luminophores, as expected. After the click reaction, however, excitation at 460 nm resulted in an emission signal that is centered approximately at 605 nm. Additionally, ECL properties of these modified magnetic beads were studied. Simultaneous time-based CV-ECL spectra were collected in the presence of tri-npropylamine (TPrA) co-reactant. The ECL data of the modified particles was well aligned with the fluorescence spectrum of modified magnetic particles. ☐ Finally, in Chapter 5, a low-cost solderless breadboard was built with simple circuit design and utilized as an electrolyzer (as an alternative to expensive potentiostats) to study various electrochemical reactions such as ECL and water splitting in order to more readily incorporate electrochemistry in undergraduate teaching laboratories. For ECL experiments, a well-known and extensively studied [Ru(bpy)3)]2+ emitter with TPrA coreactant was taken as a model system. First, the qualitative demonstration of ECL phenomenon was presented. Upon application of a constant potential to the electrolyte solution (2.5 V), orange emission at the anode surface was observed visibly. Application of different constant potentials – ranging from 0.0 to 3.0 V – to this system resulted in variations of the brightness of ECL emission. Furthermore, by combining a fluorometer with the breadboard electrolyzer, the correlation of ECL intensity with applied potential and concentrations of luminophore and coreactant were studied quantitatively. In addition to the ECL experiments, overall water splitting was also performed using an inexpensive and efficient Co-P catalyst to produce H2 and O2 gases from a basic 1.0 M KOH electrolyte solution. Increasing the applied potential resulted in faster evolution rates for both H2 and O2 gases.