Lanthanide-Based Probes for Oxidative Stress

University of Minnesota Ph.D. dissertation. June 2014. Major: Chemistry. Advisor: Valerie Pierre. 1 computer file (PDF); xxiii, 141 pages.Oxidative stress, or the imbalance of reactive oxidative species and antioxidants, is implicated in a wide variety of physiological functions and diseases. Currently, little is known about the biological concentrations and the exact roles of individual species. In particular, the cellular concentration of hydroxyl radical and the etiology of this reactive oxygen species in disease states are unclear. The photophysical properties of luminescent lanthanide-based imaging agents and the magnetic properties of fluorinated contrast agents make them favorable candidates to monitor oxidative species in biological environments. Luminescent lanthanide-based probes for hydroxyl radical are presented. These probes utilize aromatic acid pre-antennas that sensitize terbium emission upon hydroxylation. The ability of hydroxylated and non-hydroxylated aromatic acids including benzoate, benzamide, isophthalate, isophthalamide, trimesate, and trimesamide to sensitize Tb DO3A was evaluated by time-delayed luminescence spectroscopy. The formation of a weak ternary complex between hydroxytrimeasamide and Tb-DO3A was confirmed by temperature-dependent titrations. The luminescence response of the bimolecular Tb DO3A and trimesamide probe to hydroxyl radical generated by the photolysis of hydrogen peroxide was investigated. The system exhibits excellent selectivity for hydroxyl radical over other biologically relevant reactive oxygen and nitrogen species. Next, fluorinated magnetic resonance imaging contrast agents responsive to hydroxyl radical are described. The 3,5-difluorobenzoic acid probe is water soluble and ratiometrically responds to hydroxyl radical. Upon hydroxylation, a fluoride ion is released. The relative signal intensity of the product and that of the unreacted contrast agent can then be used to monitor the analyte in a ratiometric manner by 19F NMR and 19F MRI. The selectivity of the system towards hydroxyl radical compared to other reactive oxygen and nitrogen species is also measured. Paramagnetic, lanthanide-based contrast agents incorporating the sensing moiety are also evaluated for increased sensitivity of detection compared to the diamagnetic analogs. Additionally, a family of lanthanide-based luminescent complexes based on a macrocyclic core featuring different sensitizing antennas and variable pendant arms are investigated in terms of their biological compatibility. The cellular uptake of Tb-DOTA complexes containing hydroxyisophthalamide (IAM), methoxyisophthalamide (IAM(OMe)), or phenathridine (Phen) antenna were comparable despite their differences in hydrophobicity. The luminescence quenching of Tb-DOTA-IAM(OMe) was also investigated in cell lysate by time-delayed spectroscopy. Pendant arms varying in hydrophobicity and charge were used to evaluate the effect of structural and electronic properties on cellular viability and cell association as measured by a MTT assay and ICP-MS, respectively. Regardless of the amide substituents, complexes based on Tb-DOTAm-IAM(OMe) core exhibited low cytotoxicity and low cellular association. Thus, complexes based on this platform are well-suited for the detection of extracellular analytes

Development of Nanostructured Glucose Biosensor

With the development of nanotechnology and nanomaterials, biosensors incorporated with novel nanomaterials and nanostructures have shown significant potential in point-of-care medical devices because of their rapid interaction with target analytes and their miniaturized systems. Nanomaterials and nanostructures with special chemical, physical and biological characteristics are able to enhance biosensors’ performance in terms of sensitivity and selectivity. Therefore, my study focused on development of special nanostructures used for advanced glucose biosensor. Monitoring of blood glucose level is essential for diabetes management. However, current methods require people with diabetes to have blood test with 5-8 times per day. Compared to other methods, optical and magnetic techniques have a potential in developing minimally invasive or non-invasive, and continuous glucose monitoring nanostructured biosensors. Consequently, this thesis presented nanostructured optical and magnetic glucose biosensors by incorporating novel nanomaterials and fabricating nanostructures for the next generation of glucose biosensor in the tears. The glucose biorecognition biomolecule used in the biosensors was Concanavalin A (Con A). Con A is a lectin protein that has strong affinity to glucose. Fluorescence resonance energy transfer (FRET) technique was applied to develop optical glucose biosensors. FRET biosensor is a distance-dependent biosensor. The fluorescence emission of a donor molecule could be used to excite acceptor when the distance between donor and acceptor is close enough (\u3c 20 nm). Three different types of nanostructures were developed and used as the donors of the glucose FRET biosensors. The first type of sensor is a ZnO/quantum dots-based glucose biosensors. Hybrid ZnO nanorod array with decoration of CdSe/ZnS quantum dots were prepared and coated on silicone hydrogel which is a common materials of contact lens. The patterned nanostructured FRET sensor could quickly measure rats’ tear glucose in an extremely small amount (2 µL) of diluted tear sample. The second type of biosensor is based on upconversion nanomaterials. Upconversion NaGdF4: Yb, Er nanoparticles with diameter of about 40±5 nm have been prepared by polyol process and coated on silicone hydrogel to directly sense the tear glucose level on the rats’ eye surface. The results show that the upconversion nanomaterials based lens sensor is able to quickly measure glucose in rats’ blood samples. The third type of sensor utilizes the unique optical properties of carbon nanomaterial, fluorescent carbon dots and graphene oxide nanosheets. The carbon dots with tunable fluorescence were developed by a microwave-assisted process. The carbon dots are used as a fluorescence donor in the biosensor, the chitosan coated graphene oxide acts as the fluorescence acceptor to quench the emission of carbon quantum dots. In the presence of glucose, the emission of carbon quantum dots could be restored as a function of the concentration of glucose. Two linear relationships of the restored emission of the sensor and the concentration of glucose were observed, in the range of 0.2 mM to 1 mM, and 1 mM to 10 mM, respectively. On the other hand, a magnetoresistive (MR) nanostructured glucose biosensor has been developed by exploiting hybrid graphene nanosheets decorated with FeCo magnetic nanopartciles. The Fe3O4/silica core/shell nanoparticles are used as the magnetic label of glucose, which could bind onto the surface of FeCo/graphene nanocomposited sensor. The binding of magnetic label onto the hybrid graphene nanosheets can result in the change of the magnetoresistance. The MR signal as a function of the glucose level of diluted rat blood samples is measured in a range of 2 mM to 10 mM. In summary, novel nanomaterials and nanostructures with special fluorescent and magnetoresistive properties are fabricated for developing nanostructured glucose biosensors, which could bring alternative approaches for convenient management diabetes

Photophysical Detection of Singlet Oxygen

The chemical reactivity of singlet oxygen (1O2) (SO) derives from its electronically excited state. Being a unique reactive oxygen species SO takes part in many important atmospheric, biological physical, chemical, and therapeutic process and attracted current research interest. To understand the mechanistic pathways in various process the detection and quantification of SO is very important. The direct method of detection is very challenging due to its highly reactive nature. Only direct method of determination of phosphorescence of SO at 1270 nm has been utilised but that also puts some limitation due to very low luminescence quantum yield. Indirect method using UV–Vis spectrophotometric, fluorescent and chemiluminescent probes has been extensively studied for this purpose. Elucidation of various mechanistic processes improvised the use of sophisticated spectroscopic detection probe for SO have been discussed in a simple and lucid manner in this article through citation of literature examples. Four major spectroscopic methods i.e. spectrophotometry, fluorescence, emission and chemiluminescence are elaborately discussed with special emphasis to chemical probes having high selectivity and sensitivity for SO

Supramolecular Luminescent Sensors

There is great need for stand-alone luminescence-based chemosensors that exemplify selectivity, sensitivity, and applicability and that overcome the challenges that arise from complex, real-world media. Discussed herein are recent developments toward these goals in the field of supramolecular luminescent chemosensors, including macrocycles, polymers, and nanomaterials. Specific focus is placed on the development of new macrocycle hosts since 2010, coupled with considerations of the underlying principles of supramolecular chemistry as well as analytes of interest and common luminophores. State-of-the-art developments in the fields of polymer and nanomaterial sensors are also examined, and some remaining unsolved challenges in the area of chemosensors are discussed

Ultrasound-Enhanced Chemiluminescence for Bioimaging

Tissue imaging has emerged as an important aspect of theragnosis. It is essential not only to evaluate the degree of the disease and thus provide appropriate treatments, but also to monitor the delivery of administered drugs and the subsequent recovery of target tissues. Several techniques including magnetic resonance imaging (MRI), computational tomography (CT), acoustic tomography (AT), biofluorescence (BF) and chemiluminescence (CL), have been developed to reconstruct three-dimensional images of tissues. While imaging has been achieved with adequate spatial resolution for shallow depths, challenges still remain for imaging deep tissues. Energy loss is usually observed when using a magnetic field or traditional ultrasound (US), which leads to a need for more powerful energy input. This may subsequently result in tissue damage. CT requires exposure to radiation and a high dose of contrast agent to be administered for imaging. The BF technique, meanwhile, is affected by strong scattering of light and autofluorescence of tissues. The CL is a more selective and sensitive method as stable luminophores are produced from physiochemical reactions, e.g. with reactive oxygen species. Development of near infrared-emitting luminophores also bring potential for application of CL in deep tissues and whole animal studies. However, traditional CL imaging requires an enhancer to increase the intensity of low-level light emissions, while reducing the scattering of emitted light through turbid tissue environment. There has been interest in the use of focused ultrasound (FUS), which can allow acoustic waves to propagate within tissues and modulate chemiluminescence signals. While light scattering is decreased, the spatial resolution is increased with the assistance of US. In this review, chemiluminescence detection in deep tissues with assistance of FUS will be highlighted to discuss its potential in deep tissue imaging

Luminescent Transition Metal Complexes: Optical Characterization, Integration into Polymeric Nanoparticles and Sensing Applications

Photoluminescence is a fascinating phenomenon which has a huge impact on our daily life. Many important applications are based on this principle, such as imaging diagnostics, bioanalytic, photocatalysis, solar cells, or optoelectronic devices. The emerging demands for versatile photoluminescent materials have encouraged generations of scientists to develop different types of luminophores, ranging from molecular dyes to luminescent nanomaterials. Among them, luminescent transition metal complexes (TMCs), which consist of one (or more) metal center and several organic or inorganic ligands, are drawing increasing interest due to their unique photophysical and photochemical properties, such as large Stokes shift, long-lived triplet excited state, sharp emission band (f-block metal complexes), and multiple stale oxidation states (d-block metal complexes). These distinct optical properties are not only of great research interest, but also have led to commercial applications, such as imaging agents, optical sensors, light-harvesting materials, optical barcoding, and displays. The demands and desires of optoelectronic devices and higher requirements in bioanalytic make the development of new TMCs necessary, which needs to be examined in detail not only in terms of their chemical but much more importantly their photophysical properties. In this work, a series of new types of luminescent TMCs are involved, including Cr(III)-, Pt(II)-, and Pd(II) complexes. Based on their optical studies, a series of proof-of-concept applications were designed by introducing these metal complexes to different nanomatrix, such as polymeric nanoparticles and metal-organic frameworks (MOFs), resulting various luminescent nanosensors or energy-conversion materials. The major part of this work is based on the [Cr(ddpd)2]3+ complex (ddpd = N, N′‐dimethyl‐N, N´-dipyridine‐2‐ylpyridine‐2,6‐diamine) and his derivatives. Fundamental photophysical studies of these Cr(III) complexes showed that their photoluminescence properties can be significantly enhanced by ligand and solvent deuteration. Moreover, a choice of bulky counter anions can provide an enhancement in the photoluminescence properties as well as the oxygen sensitivity. In addition, based on the photophysical understanding of the [Cr(ddpd)2]3+ complex, a proof-of-concept study of photon upconversion in molecular chromium ytterbium salts was completed. Upon an excitation of the Yb3+ sensitizers at 976 nm, these solid-state salts produced upconverted luminescence of the Cr3+ activator at 780 nm at room temperature. Another proof-of-concept study based on the [Cr(ddpd)2]3+ complex was investigated by designing and developing multianalyte nanosensors for simultaneously measuring temperature (“T”), oxygen (“O”), and pH (“P”) in aqueous phase under one excitation wavelength. Apart from the [Cr(ddpd)2]3+ complexes, four novel Pt(II)- and Pd(II)-complexes bearing tetradentate ligands were also studied regarding their photophysical properties in solutions and in polystyrene nanoparticles (PS-NPs). In PS-NPs, the aggregation-induced Metal-Metal-to-Ligand Charge-Transfer (3MMLCT) state of the fluorinated Pt(II) complex is red-shifted compared to the monomeric emission and performs insensitive to oxygen, allowing the particles as self-referenced oxygen nanosensor in both the luminescence intensity and lifetime domains. Additionally, a triplet–triplet annihilation upconversion (TTA-UC) system was developed based on a crystalline MOF. A Pd(II) porphyrin complex acted as the sensitizer immobilized in the MOF walls, while a 9,10-diphenylanthracene annihilator was filled in the channels. Upon green light excitation at 532 nm, the resulting MOF crystalline showed an upconverted blue emission with delayed lifetime from 4 ns to 373 µs and a triplet–triplet energy transfer efficiency of 82%

New Ln(III) complexes as potential optical probes for biological applications

The unique properties of f-f transitions in trivalent lanthanide complexes are the understandable reason of increasing applications in biosensing field, where their long emission lifetimes, the sharp and easily recognizable emission bands in addition to the large shift between the absorbed and emitted radiation besides a short-lived background fluorescence permit the great advantage to isolate their emission signal from the undesired background fluorescence of the biological samples. Furthermore, luminescent complexes of Eu(III) and Tb(III) are the most employed candidates due to the low sensitivity of their excited state to vibrational quenching effects caused by OH, NH, or CH oscillators, frequently present in solution and imaging environments. For these reasons, Eu(III) and Tb(III) complexes have been extensively exploited as sensors of species in physiological conditions, by allowing the detection of relevant clinical biomarkers in biomedical diagnostics and imaging. For these purposes, a high luminescence emission quantum yield and overall luminosity (or brightness) are strongly required and the intensity of the luminescent response, that it is enhanced with heteroaromatic ligands via antenna effect, is usually correlated to the concentration of target analyte. In this PhD project, a library of new water soluble Eu(III) and Tb(III) complexes based on the chiral fragment 1,2-diaminecyclohexane (DACH) has been successfully synthetized, completely characterized (also in solution) and employed for analytical detection of important bio-analytes such as: bicarbonate, L-lactate, serum albumin, and citrate through mainly total luminescence (TL) and circularly polarized luminescence (CPL). These analytes are the main constituents of extracellular fluid, such as human serum

Design and Application of Task-Specific GUMBOS and NanoGUMBOS for Sensing and Separation

The work presented in this dissertation employs task-specific materials for sensing and protein separation applications. These materials were derived from a group of uniform materials based on organic salts (GUMBOS). GUMBOS are organic salts similar to ionic liquids, but have melting points ranging from 25 to 250 °C. As with ionic liquids, the properties of GUMBOS can be easily tuned by changing the counter-ion. Thus, task-specific GUMBOS can be designed and prepared with properties that are beneficial for applications in sensing or protein separation. In this dissertation, the selective responsive behavior of a series of GUMBOS and nanomaterials derived from GUMBOS (nanoGUMBOS) were evaluated. Firstly, binary nanoGUMBOS, containing two cyanine cations, were synthesized and characterized. Based on significant spectral overlap and differences in reactivity towards hydroxyl radicals, the two cyanine cations in the binary nanoGUMBOS were able to generate a ratiometric fluorescence response. These results suggest a promising ratiometric probe for detection and quantification of hydroxyl radicals. This approach of investigating binary nanoprobes will serve as the basis for designing other cyanine-based fluorescent probes for biosensing and imaging. Secondly, a series of cyanine-based GUMBOS were combined to serve as a sensor array for detection of proteins. The cyanine-based sensor elements utilized in this sensor array, exhibit different aggregation behaviors when mixed with the seven proteins, giving various fluorescence responses. The resulting responses exhibited cross-reactive patterns, which can be analyzed to discriminate proteins at a low concentration. Finally, nanoGUMBOS derived from imidazolium ionic liquids and magnetic dysprosium-based anions were designed as magnetic, nanoadsorbent materials for selective hemoglobin isolation. These nanoGUMBOS were successfully applied in selective hemoglobin (Hb) isolation from human whole blood. All studies presented in this dissertation demonstrate promising advantages of GUMBOS-based materials in the field of sensing and protein separation

The use of non-radioactive iodine as a label in biological assays

A microassay for the determination of iodide and iodine-containing compounds based on the Sandell-Kolthoff reaction was developed by O’Kennedy et al., (1989). Non-radioactive iodine was then used to label antibodies for immunoassay. The sensitivity of this immunoassay has been shown to be comparable to that obtained in an ELISA when the enzyme used was horseradish peroxidase. This study was carried out to investigate the feasibility of labelling DNA probes with non-radioactive iodine. Nucleic acids have previously been labelled with 125Iodine and these iodination procedures were adopted. Labelling of nucleic acids was carried out both chemically using the oxidizer Iodo gen, and enzymatically via the incorporation of 5-iodo-2’-deoxycytidine 5 ’-triphosphate by random priming. 2% and 38% incorporation was observed for these methods, respectively. The degree of iodination achieved was determined using the iodide microassay. HPLC of digested nucleic acids was also carried out to measure the incorporation of iodine. The iodide microasssay was modified and then validated in the range 1- 10ng/ml potassium iodide. The catalytic effect of 5-iodocytidine, and related compounds, in the iodide microassay was also considered. The iodide assay was performed on nitrocellulose paper to investigate the feasibility of applying this assay in a dot blot format. The limit of quantification for iodide in this format was 1 .0pg/ml. To use cold-iodine as a direct label for DNA probes a more sensitive iodide microassay would be required. For this reason a number of chemiluminescent systems, which were quenched by iodide, were investigated. However, the lowest limit of quantification obtained for the microassays considered was 10ng/ml iodide. The use of iodine as an indirect label for DNA probes was also examined and polyclonal antibodies to 5-iodocytidine were produced

LASER ABLATION IN LIQUID FOR THE CONTROLLED PRODUCTION OF PHOTOLUMINESCENT GRAPHENE QUANTUM DOTS AND UPCONVERTING NANOPARTICLES

Photoluminescent­ (PL) nanomaterials play an important role in areas including displays, sensing, solar, photocatalysis, and bio applications. Traditional methods to prepare PL materials suffer many drawbacks such as harsh chemical precursors, complicated synthetic steps, and production of many byproducts. Laser ablation in liquid (LAL) has emerged as a promising alternative to prepare materials that is single-step, fast, uses fewer precursors, produces fewer side products, and has simple purification steps. During LAL, a solid target is irradiated with a pulsed laser source. The laser pulses cause plasma plumes of the target material to form which are cooled, condensed, and can react with the surrounding liquid. This dissertation explores LAL as an alternative method to produce two important classes of PL nanomaterials: graphene quantum dots (GQDs) and upconverting nanoparticles (UCNPs). GQDs are a class of carbon PL materials that are lightweight, biocompatible and can be produced from cheap and abundant carbon sources. Their PL properties depend on both radiative recombination of intrinsic states through their carbon backbone as well as defect like states from surface functionalization. LAL of carbon nano-onions in water was used to produce GQDs. These GQDs were systematically compared to those produced by a traditional chemical oxidation method and showed blue shifted emission, higher fractions of hydroxyl-groups, and smaller sizes. Nitrogen doping with controlled chemical composition allowed further tuning of the PL and was achieved by including dopant molecules in the liquid during LAL. Lifetime measurements showed three emissive pathways and provided greater understanding of the roles of intrinsic and defect like emissive states. UCNPs composed of NaYF4:Yb3+/Er3+ are interesting for many bio applications but are challenging to prepare with both high upconversion efficiency and water solubility. Control of the UCNP phase is important for high efficiency. LAL was used to address these issues by irradiating a target of desired phase in an aqueous solution containing capping agents which allowed for formation of water soluble UCNPs of the desired phase. Tuning of laser parameters allowed control of the size, composition, and PL of the UCNPs. This work showcases LAL as a method to efficiently produce PL nanomaterials with controlled properties