Localized surface plasmon resonance biosensors for real-time biomolecular binding study

Surface Plasmon Resonance (SPR) and localized surface plasmon resonance (LSPR) biosensors have brought a revolutionary change to in vitro study of biological and biochemical processes due to its ability to measure extremely small changes in surface refractive index (RI), binding equilibrium and kinetics. Strategies based on LSPR have been employed to enhance the sensitivity for a variety of applications, such as diagnosis of diseases, environmental analysis, food safety, and chemical threat detection. In LSPR spectroscopy, absorption and scattering of light are greatly enhanced at frequencies that excite the LSPR, resulting in a characteristic extinction spectrum that depends on the RI of the surrounding medium. Compositional and conformational change within the surrounding medium near the sensing surface could therefore be detected as shifts in the extinction spectrum. This dissertation specifically focuses on the development and evaluation of highly sensitive LSPR biosensors for in situ study of biomolecular binding process by incorporating nanotechnology. Compared to traditional methods for biomolecular binding studies, LSPR-based biosensors offer real-time, label free detection. First, we modified the gold sensing surface of LSPR-based biosensors using nanomaterials such as gold nanoparticles (AuNPs) and polymer to enhance surface absorption and sensitivity. The performance of this type of biosensors was evaluated on the application of small heavy metal molecule binding affinity study. This biosensor exhibited ∼7 fold sensitivity enhancement and binding kinetics measurement capability comparing to traditional biosensors. Second, a miniaturized cell culture system was integrated into the LSPR-based biosensor system for the purpose of real-time biomarker signaling pathway studies and drug efficacy studies with living cells. To the best of our knowledge, this is the first LSPR-based sensing platform with the capability of living cell studies. We demonstrated the living cell measurement ability by studying the VEGF signaling pathway in living SKOV-3 cells. Results have shown that the VEGF secretion level from SKOV-3 cells is 0.0137 ± 0.0012 pg per cell. Moreover, we have demonstrated bevacizumab drug regulation to the VEGF signaling pathway using this biosensor. This sensing platform could potentially help studying biomolecular binding kinetics which elucidates the underlying mechanisms of biotransportation and drug delivery.

Localized Surface Plasmon Resonance Biosensors for Real-Time Biomolecular Binding Study

Surface Plasmon Resonance (SPR) and localized surface plasmon resonance (LSPR) biosensors have brought a revolutionary change to in vitro study of biological and biochemical processes due to its ability to measure extremely small changes in surface refractive index (RI), binding equilibrium and kinetics. Strategies based on LSPR have been employed to enhance the sensitivity for a variety of applications, such as diagnosis of diseases, environmental analysis, food safety, and chemical threat detection. In LSPR spectroscopy, absorption and scattering of light are greatly enhanced at frequencies that excite the LSPR, resulting in a characteristic extinction spectrum that depends on the RI of the surrounding medium. Compositional and conformational change within the surrounding medium near the sensing surface could therefore be detected as shifts in the extinction spectrum. This dissertation specifically focuses on the development and evaluation of highly sensitive LSPR biosensors for in situ study of biomolecular binding process by incorporating nanotechnology. Compared to traditional methods for biomolecular binding studies, LSPR-based biosensors offer real-time, label free detection. First, we modified the gold sensing surface of LSPR-based biosensors using nanomaterials such as gold nanoparticles (AuNPs) and polymer to enhance surface absorption and sensitivity. The performance of this type of biosensors was evaluated on the application of small heavy metal molecule binding affinity study. This biosensor exhibited ~7 fold sensitivity enhancement and binding kinetics measurement capability comparing to traditional biosensors. Second, a miniaturized cell culture system was integrated into the LSPR-based biosensor system for the purpose of real-time biomarker signaling pathway studies and drug efficacy studies with living cells. To the best of our knowledge, this is the first LSPR-based sensing platform with the capability of living cell studies. We demonstrated the living cell measurement ability by studying the VEGF signaling pathway in living SKOV-3 cells. Results have shown that the VEGF secretion level from SKOV-3 cells is 0.0137 ± 0.0012 pg per cell. Moreover, we have demonstrated bevacizumab drug regulation to the VEGF signaling pathway using this biosensor. This sensing platform could potentially help studying biomolecular binding kinetics which elucidates the underlying mechanisms of biotransportation and drug delivery.

The construction and optimization of a surface plasmon resonance sensor for the studies of bio-molecular interactions

Bio-molecular interactions exist ubiquitously in all biological systems. This dissertation project was to construct a powerful surface plasmon resonance (SPR) sensor. The SPR system is used to study bio-molecular interactions in real time and without labeling. Surface plasmon is the oscillation of free electrons in metals coupled with surface electromagnetic waves. These surface electromagnetic waves provide a sensitive probe to study bio-molecular interactions on metal surfaces. This project resulted in the successful construction and optimization of a homemade SPR sensor and the development of several new powerful protocols to study bio-molecular interactions. It was discovered through this project that the limitations of earlier SPR sensors are related not only to the instrumentation design and operating procedures, but also to the complex behaviors of bio-molecules on sensor surfaces that were very different from that in solution. Based on these discoveries the instrumentation design and operating procedures were fully optimized. A set of existing sensor surface treatment protocols were tested and evaluated and new protocols were developed in this project. The new protocols have demonstrated excellent performance to study biomolecular interactions. The optimized home-made SPR sensor was used to study protein-surface interactions. These protein-surface interactions are responsible for many complex organic cell activities. The co-existence of different driving forces and their correlation with the structure of the protein and the surface make the understanding of the fundamental mechanism of protein-surface interactions a very challenging task. Using the improved SPR sensor, the electrostatic interaction and hydrophobic interaction were studied separately. The results of this project directly confirmed the theoretical predictions for electrostatic force between the protein and surface. In addition, this project demonstrated that the strength of the protein-surface hydrophobic interaction does not solely depend on the hydrophobicity as reported earlier. Surface structure also plays a significant role.

AFM and SPR studies of protein adsorption at solid/liquid interface

Membrane-like structure formed by surfactant molecules of didodecyldimethylammonium bromide (DDAB) on both HOPG and gold electrodes were studied with AFM and SPR techniques. The study shows that the thickness of the adsorbed layer of DDAB is strongly dependent on the concentration of the vesicle solution. We have also investigated the adsorption of redox protein, Cytochrome c, on graphite electrode with in situ tapping mode AFM. The protein adsorbs spontaneously onto the electrode covered with an adsorbed phosphate layer and forms a uniform monolayer. The adsorbed protein exhibits a reversible electron transfer at 0.17 V (Ag/AgCI) once the electrode potential has been increased to 0.75V. Using surface plasmon resonance spectroscopy we have measured subtle conformational change in protein, Cyt c, due to electron transfer of a single electron on MPA-coated gold electrode. The electron transfer induced change in the resonant angle is about 0.006 deg., which corresponds to ~ 0.2 A decreases in the thickness. This is consistent with that reduced state is more compact than the oxidized state.

Development of a surface-enhanced raman spectroscopy method for the detection of benzodiazepines in urine

Benzodiazepines are among the most prescribed compounds for anti-anxiety and are present in many toxicological screens. These drugs are also prominent in the commission of drug facilitated sexual assaults due their effects on the central nervous system. Due to their potency, a low dose of these compounds is often administered to victims; therefore, the target detection limit for these compounds in biological samples is 10 ng/mL. Currently these compounds are predominantly analyzed using immunoassay techniques; however more specific screening methods are needed. ^ The goal of this dissertation was to develop a rapid, specific screening technique for benzodiazepines in urine samples utilizing surface-enhanced Raman spectroscopy (SERS), which has previously been shown be capable of to detect trace quantities of pharmaceutical compounds in aqueous solutions. Surface enhanced Raman spectroscopy has the advantage of overcoming the low sensitivity and fluorescence effects seen with conventional Raman spectroscopy. The spectra are obtained by applying an analyte onto a SERS-active metal substrate such as colloidal metal particles. SERS signals can be further increased with the addition of aggregate solutions. These agents cause the nanoparticles to amass and form hot-spots which increase the signal intensity. ^ In this work, the colloidal particles are spherical gold nanoparticles in aqueous solution with an average size of approximately 30 nm. The optimum aggregating agent for the detection of benzodiazepines was determined to be 16.7 mM MgCl2, providing the highest signal intensities at the lowest drug concentrations with limits of detection between 0.5 and 127 ng/mL. A supported liquid extraction technique was utilized as a rapid clean extraction for benzodiazepines from urine at a pH of 5.0, allowing for clean extraction with limits of detection between 6 and 640 ng/mL. It was shown that at this pH other drugs that are prevalent in urine samples can be removed providing the selective detection of the benzodiazepine of interest. ^ This technique has been shown to provide rapid (less than twenty minutes), sensitive, and specific detection of benzodiazepines at low concentrations in urine. It provides the forensic community with a sensitive and specific screening technique for the detection of benzodiazepines in drug facilitated assault cases.^

Development of a Surface-Enhanced Raman Spectroscopy Method for the Detection of Benzodiazepines in Urine

Benzodiazepines are among the most prescribed compounds for anti-anxiety and are present in many toxicological screens. These drugs are also prominent in the commission of drug facilitated sexual assaults due their effects on the central nervous system. Due to their potency, a low dose of these compounds is often administered to victims; therefore, the target detection limit for these compounds in biological samples is 10 ng/mL. Currently these compounds are predominantly analyzed using immunoassay techniques; however more specific screening methods are needed. The goal of this dissertation was to develop a rapid, specific screening technique for benzodiazepines in urine samples utilizing surface-enhanced Raman spectroscopy (SERS), which has previously been shown be capable of to detect trace quantities of pharmaceutical compounds in aqueous solutions. Surface enhanced Raman spectroscopy has the advantage of overcoming the low sensitivity and fluorescence effects seen with conventional Raman spectroscopy. The spectra are obtained by applying an analyte onto a SERS-active metal substrate such as colloidal metal particles. SERS signals can be further increased with the addition of aggregate solutions. These agents cause the nanoparticles to amass and form hot-spots which increase the signal intensity. In this work, the colloidal particles are spherical gold nanoparticles in aqueous solution with an average size of approximately 30 nm. The optimum aggregating agent for the detection of benzodiazepines was determined to be 16.7 mM MgCl2, providing the highest signal intensities at the lowest drug concentrations with limits of detection between 0.5 and 127 ng/mL. A supported liquid extraction technique was utilized as a rapid clean extraction for benzodiazepines from urine at a pH of 5.0, allowing for clean extraction with limits of detection between 6 and 640 ng/mL. It was shown that at this pH other drugs that are prevalent in urine samples can be removed providing the selective detection of the benzodiazepine of interest. This technique has been shown to provide rapid (less than twenty minutes), sensitive, and specific detection of benzodiazepines at low concentrations in urine. It provides the forensic community with a sensitive and specific screening technique for the detection of benzodiazepines in drug facilitated assault cases.

Multimode Analysis of Nanoscale Biomolecular Interactions

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.

A Multi-Faceted Diagnostic Approach to Lung Infections in Patients with Cystic Fibrosis

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

Nuclear magnetic resonance spectroscopic and computational investigations of chloroperoxidase catalyzed regio- and enantio-selective transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. ^ To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. ^ To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O 2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.^

Nuclear Magnetic Resonance Spectroscopic and Computational Investigations of Chloroperoxidase Catalyzed Regio- and Enantio-Selective Transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.

Biomimetic modeling of the nitrogen-centered radical postulated to occur during the inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides

Ribonucleotide reductases (RNR) are essential enzymes that catalyze the reduction of ribonucleotides to 2'-deoxyribonucleotides, which is a critical step that produces precursors for DNA replication and repair. The inactivation of RNR, logically, would discontinue producing the precursors of the DNA of viral or cancer cells, which then would consequently end the cycle of DNA replication. Among different compounds that were found to be inhibitors of RNR, 2'-azido-2'-deoxynucleotide diphosphates (N3NDPs) have been investigated in depth as potent inhibitors of RNR. Decades of investigation has suggested that the inactivation of RNR by N3NDPs is a result of the formation of a nitrogen-centered radical (N·) that is covalently attached to the nucleotide at C3' and cysteine molecule C225 [3'-C(R-S-N·-C-OH)]. Biomimetic simulation reactions for the generation of the nitrogen-centered radicals similar to the one observed during the inactivation of the RNR by azionuclotides was investigated. The study included several modes: (i) theoretical calculation that showed the feasibility of the ring closure reaction between thiyl radicals and azido group; (ii) synthesis of the model azido nucleosides with a linker attached to C3' or C5' having a thiol or vicinal dithiol functionality; (iii) generation of the thiyl radical under both physiological and radiolysis conditions whose role is important in the initiation on RNR cascades; and (iv) analysis of the nitrogen-centered radical species formed during interaction between the thiyl radical and azido group by electron paramagnetic resonance spectroscopy (EPR). Characterization of the aminyl radical species formed during one electron attachment to the azido group of 2'-azido-2'-deoxyuridine and its stereospecifically labelled 1'-, 2'-, 3'-, 4'- or 5,6-[2H 2]-analogues was also examined. This dissertation gave insight toward understanding the mechanism of the formation of the nitrogen-centered radical during the inactivation of RNRs by azidonucleotides as well as the mechanism of action of RNRs that might provide key information necessary for the development of the next generation of antiviral and anticancer drugs.