This thesis focuses on the use of molecularly imprinted polymers as selective sorbents for solid-phase extraction (MISPE). The MISPE methods developed were mainly intended for use with biological samples, such as human urine and blood plasma. These body fluids are complex samples, which often need an effective clean-up step before analysis to reduce the levels of possible interfering substances from the matrix, especially if the analytes are present in trace amounts. Solid-phase extraction (SPE) is a well-established and routinely used method for clean-up and preconcentration of samples from diverse matrices. However, conventional SPE sorbents often lack selectivity, leading to co-extraction of interferences, which negatively affects the following detection method. One of the advantages of MISPE is the built-in selectivity for a target analyte, or class of structurally related analytes, enabling the efficient clean-up that is often required for biological samples. The built-in selectivity of MISPE originates from the preparation of a highly crosslinked copolymer network in the presence of an imprint molecule, i.e. the template. Subsequent removal of this template molecule leads to the creation of defined recognition sites, complementary to the shape and functionality of the template. In this work, molecularly imprinted polymers were synthesized for the first time for several types of target analytes, including diphosphate esters (Papers I-III) and a protein adduct (Paper IV) and evaluated as sorbents for solid-phase extraction. A MISPE method for extracting local anaesthetic drugs from human plasma was also evaluated (Paper V). The development of appropriate methods for using the prepared polymers to extract target analytes directly from body fluids, and the elucidation of factors that influence their performance, were major foci of all the work underlying this thesis. These are not straightforward tasks, since the recognition mechanism of the material is often based on polar interactions, which are not favoured in aqueous environments. In such cases, non-selective adsorption of the analyte(s) to the polymer surface often occurs. In order to use the MIP sorbent most effectively it is important to suppress this non-selective adsorption, without disrupting the selective adsorption of the target analyte(s) to the imprints. Generally in these studies, this strong analyte-polymer surface interaction could be repressed, and selective adsorption enhanced, by carefully optimising the conditions for washing the sorbent, in terms of organic solvent volumes, solvent polarity and the addition of an ionic modifier. The sample matrix, mainly urine, was found to strongly decrease the capacity of the MIP. Hence, this effect was further investigated. It was found that the presence of NaCl in the sample negatively affected the recovery and repeatability of the method. Furthermore, these parameters could be improved by adjusting the sample pH. It was important to control the pH of the sample, in order both to achieve selective extraction and to increase the extraction recoveries. The selectivity of MISPE for the extraction of diphosphate esters from human urine was demonstrated by comparing its performance with that of a conventional SPE sorbent, a mixed-mode-anion exchanger (MAX). Due to its efficient clean-up, MISPE generated extracts that yielded less complex ion chromatograms in subsequent LC/ESI-MS analysis than extracts from the MAX cartridge. Due to its efficient clean-up, MISPE generated extracts that yielded less complex ion chromatograms in subsequent LC/ESI-MS analysis than extracts from the MAX cartridge. Signal suppression from the interfering co-eluting compounds was detected when the MAX extracts were analysed, which was not the case for the MISPE extracts. These findings show the importance of efficient and selective sample preparation, even if a selective detector is used. Development of LC/ESI-MS methods was also an extensive component of this work (Papers I-IV). Different chromatographic conditions have been evaluated for the optimal separation and detection of the investigated compounds. Use of ion-pairing agents and suitable HPLC columns (Hypercarb and C18 Aquasil) for the acidic, polar analytes, was found to give better retention and separation than use of conventional reversed-phase columns. To improve the selectivity and detectability further, selected ion monitoring (SIM) and selected reaction monitoring (SRM) acquisition modes were used for quantification of the investigated compounds. In summary, the aim of this work was to contribute to the knowledge of the recognition mechanisms of molecularly imprinted polymers in aqueous matrices, which is important for extending the use of MISPE for several types of bioanalytical applications.

Protein microarray technology has an enormous potential for in vitro diagnostics (IVD)1. Miniaturized and parallelized immunoassays are powerful tools to measure dozens of parameters from minute amounts of sample, whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first diagnostic products are already released on the market. However, in order for protein microarrays to become broadly accepted tools in IVD, a number of criteria have to be fulfilled concerning robustness and automation. Robustness and automation are key demands to improve assay performance and reliability of multiplexed assays, and to minimize the time of analysis. These key demands are addressed in this thesis and novel methods and techniques concerning assay automation, array fabrication as well as performance and detection strategies related to protein microarrays are presented and discussed. In the first paper an automated assay format, based on planar protein microarrays is described and evaluated by the detection of several auto-antibodies from human serum and by quantification of matrix metalloproteases present in plasma. Diffusion-rate limited solid phase reactions were enhanced by microagitation, using the surface acoustic wave technology, resulting in a slightly increased signal-to-noise ratio. In the second paper of the thesis, a novel multiplexed immunoassay system was developed by combining a direct immunoassay with a competitive system. This set-up allows quantification of analytes present in widely varying concentrations within a single multiplex assay. In the third paper, a new concept for sample deposition is introduced, addressing contemporary problems of contact or non-contact microarrayers in protein microarray fabrication. In the fourth paper, a magnetic bead-based detection method for protein microarrays is described as a cost-effective alternative approach to the commonly used fluorescence-based confocal scanning systems. The magnetic bead-based detection could easily be performed by using an ordinary flatbed scanner. In addition, applying magnetic force to the magnetic bead-based detection approach enables to run the detection step more rapidly. Finally, in paper five, a microfluidic bead-based immunoassay for multiplexed detection of receptor tyrosine kinases in breast cancer tissue is presented. Since the assay is performed inside a capillary, the amounts of sample and reagent material could be reduced by a factor of 30 or more when compared with the current standard protein microarray assay. / QC 20101112

No description available.

Recent proteome studies have discovered the presence of heat shock protein 10 (HSP-10) as an immunosuppressant in ovarian cancer patients. Due to the severity of ovarian cancer, the development of highly sensitive techniques for the early detection of this cancer is well in demand. In this manuscript, the thickness shear mode (TSM) acoustic wave biosensor will be used for the real-time and label-free detection of HSP-10 in buffer. The TSM sensitivity for HSP-10 is evaluated based on resonance frequency shifts generated by the biosensor. A nucleic acid aptamer, which is specifically engineered by in vitro selection to target HSP-10, is employed as the biosensing element of the biosensor. Alkylthiol-based self-assembling monolayers (SAMs), composed of various linker/diluent molar ratios, are used to immobilize the aptamer onto gold-coated piezoelectric quartz substrates. The TSM biosensing properties for avidin-biotin interactions are also evaluated in order to assess the biosensor response to HSP-10 protein-aptamer interaction.

Recent proteome studies have discovered the presence of heat shock protein 10 (HSP-10) as an immunosuppressant in ovarian cancer patients. Due to the severity of ovarian cancer, the development of highly sensitive techniques for the early detection of this cancer is well in demand. In this manuscript, the thickness shear mode (TSM) acoustic wave biosensor will be used for the real-time and label-free detection of HSP-10 in buffer. The TSM sensitivity for HSP-10 is evaluated based on resonance frequency shifts generated by the biosensor. A nucleic acid aptamer, which is specifically engineered by in vitro selection to target HSP-10, is employed as the biosensing element of the biosensor. Alkylthiol-based self-assembling monolayers (SAMs), composed of various linker/diluent molar ratios, are used to immobilize the aptamer onto gold-coated piezoelectric quartz substrates. The TSM biosensing properties for avidin-biotin interactions are also evaluated in order to assess the biosensor response to HSP-10 protein-aptamer interaction.

Although solid-phase microextraction (SPME) technique has gained wide applications from in vitro environmental investigations to in vivo pharmacokinetic studies, there are still challenges for utilizing SPME to track fast concentration change over time at a specific location in a heterogeneous system, such as studying the tissue- specific metabolism or bioaccumulation of pharmaceuticals in a living animal. In this case, the technique must be adaptable for in situ analysis with highly temporal and spatial resolutions. The goal of the research presented was not only to address this issue but also to develop new analytical methods that were more effective for in vivo study using SPME. In order to improve the temporal resolution, fast SPME sampling technique based on pre-equilibrium extraction must be adopted. However, more efforts need to be placed into calibration so as to guarantee the accuracy of the analysis. In this thesis, firstly, the kinetic calibration was proposed for adsorptive SPME fibres that were widely used for biological samples, which paved the way for performing fast sampling for in vivo dynamic monitoring. Secondly, the kinetic calibration was applied for in vivo pharmacokinetic study with beagles, with which not only solid experimental evidence was obtained for the calibration theory, but also an example was shown to address the quantitative capability of in vivo SPME. The developed method showed comparable sensitivity to traditional blood analysis (linear range 5 – 2000 µg/L and limit of detection: 5µg/L). Furthermore, the traditional kinetic calibration based on isotopically labelled standards was simplified to a single time-point calibration, and a single standard calibration was developed for multiple analytes. Therefore, the fast in vivo sampling could be accomplished in a simple but accurate measure; compared to the established equilibrium SPME technique, statistically no significant difference (P<0.05) was observed by using one-way ANOVA and the post-hoc Turkey’s test for multiple comparisons. The second aspect of the thesis addressed the spatial resolution of SPME for in situ analysis. Firstly, the sampling of the SPME with high spatial resolution was modeled with multilayered gel system with the mini-sized SPME fibres. The feasibility of the SPME for in vitro application was demonstrated by sampling in an onion bulb with heterogeneous structure. Afterwards, the miniaturized fibre was successfully applied to the in situ analysis of the concentration distribution of Ochratoxin A in semisolid cheese samples with acceptable sensitivity (Detection limit was 1.5 ng/mL and the linear range was 1.5-500 ng/mL) and comparable accuracy to the standard methods such as liquid extraction and microdialysis. Finally, the in vivo application of the space- and time- resolved SPME was implemented to study the tissue-specific bioaccumulations of pharmaceuticals in fish adipose fins and muscle tissues. The results were validated by the standard method liquid extraction, and they were also comparable to the literature results. The research presented here demonstrated the application potential of the time-and space- resolved SPME for in situ dynamic and static analysis in a living system such as a beagle or fish, and in a non-living system such as a cheese piece or an onion bulb.

Photoluminescence spectroscopy has emerged as a powerful technique for characterizing the structure and optical properties of single-walled carbon nanotubes (SWCNT). While SWCNT diameter and chirality information is now routinely available from photoluminescence spectral analysis, the other primary structural parameter, length, has not been measurable except through tedious microscopy. This thesis extends the use of photoluminescence to obtain length information on ensembles of SWCNT in suspension through analysis of their optical anisotropy when aligned by the shear of a flowing fluid. The theoretical background and custom-built instrumentation are described and demonstrated to yield analyses comparable to the standard method of atomic force microscopy. A unique benefit of the new method is the resolution of correlations between length and (n,m) structural indices through spectral analysis of the data. The common sample preparation steps of sonication and centrifugation are found to alter the sample length distribution in a diameter-dependent manner. The shear aligned photoluminescence anisotropy method provides a new means for quickly determining the lengths of SWCNT in bulk suspensions and more thoroughly investigating their structural properties. Photoluminescence characterization techniques such as this depend on radiative decay of SWCNT excited states, which occurs with efficiencies below 10%. Nonradiative relaxation is clearly dominant, yet the detailed decay pathways and their relationship to nanotube structure remain essentially unknown. It is currently suspected that optically forbidden states, such as spin triplet states, are a major factor in the low luminescence efficiency of SWCNT. Experimental studies are described involving energy transfer from optically excited porphyrin sensitizers in an attempt to selectively populate such unexplored SWCNT triplet states. Efficient energy transfer is clearly observed in non-covalent SWCNT-porphyrin complexes. Analysis suggests that singlet rather than triplet interactions are dominant in this system. These studies demonstrate efficient electronic coupling between excited states of the nanotube and porphyrin that make such complexes potentially useful as artificial light-harvesting chromophores.

The effective determination of the hydrodynamic sizes of nanomaterials functionalized with various coatings is a topic of intense research. Analytical ultracentrifugation, a lesser known characterization method that provides hydrodynamic size via a parameter termed the sedimentation coefficient, provides this information in a manner unsurpassed by alternative methods. In the first part of this thesis, I present the application of analytical ultracentrifugation to ligand-stabilized nanocrystals of cadmium selenide, iron oxide, and gold in organic solvents. This method was able to provide distinct sedimentation coefficients for these model nanocrystals, some of which were different in diameter by only an angstrom. Further, we show that the sedimentation coefficient has a significant dependence on the density of the composite material, which is based on the size of the nanomaterial and surface coating. Therefore, we introduced a descriptive size-dependent term into conventional models that accounts for both the inorganic core and organic coating to more accurately represent the overall density. We also explored the effects of different approaches for analytical ultracentrifugation data analysis on the sedimentation coefficients obtained and found that all methods yielded the same value. Finally, we elucidated experimental parameters for the ultracentrifuge that specifically affect nanomaterial samples and established best practices for studying sedimentation for these types of materials. The results from this work allow for better agreement between experimental and theoretical sedimentation coefficients, which is useful for accurately evaluating hydrodynamic sizes of nanomaterials. In the second part of this thesis, the relationship between protein surface charge and gold nanoparticle aggregation is explored by using pH variation and chemical modification. Lysozyme and alpha-lactalbumin share a common three-dimensional fold but have significantly different isoelectric points (pI) and surface charge distributions. Myoglobin is also a small globular protein with a pK intermediate between alpha-lactalbumin and lysozyme, and a variant with a specific sulfhydryl group has been generated. These proteins have been conjugated to gold nanoparticles under varying pH conditions and following chemical modifications that impact the protein's pI. Changes in UV-visible spectra, TEM distribution, and dynamic light scattering indicate that significant aggregation depends on a positively charged protein surface.

Although solid-phase microextraction (SPME) technique has gained wide applications from in vitro environmental investigations to in vivo pharmacokinetic studies, there are still challenges for utilizing SPME to track fast concentration change over time at a specific location in a heterogeneous system, such as studying the tissue- specific metabolism or bioaccumulation of pharmaceuticals in a living animal. In this case, the technique must be adaptable for in situ analysis with highly temporal and spatial resolutions. The goal of the research presented was not only to address this issue but also to develop new analytical methods that were more effective for in vivo study using SPME. In order to improve the temporal resolution, fast SPME sampling technique based on pre-equilibrium extraction must be adopted. However, more efforts need to be placed into calibration so as to guarantee the accuracy of the analysis. In this thesis, firstly, the kinetic calibration was proposed for adsorptive SPME fibres that were widely used for biological samples, which paved the way for performing fast sampling for in vivo dynamic monitoring. Secondly, the kinetic calibration was applied for in vivo pharmacokinetic study with beagles, with which not only solid experimental evidence was obtained for the calibration theory, but also an example was shown to address the quantitative capability of in vivo SPME. The developed method showed comparable sensitivity to traditional blood analysis (linear range 5 – 2000 µg/L and limit of detection: 5µg/L). Furthermore, the traditional kinetic calibration based on isotopically labelled standards was simplified to a single time-point calibration, and a single standard calibration was developed for multiple analytes. Therefore, the fast in vivo sampling could be accomplished in a simple but accurate measure; compared to the established equilibrium SPME technique, statistically no significant difference (P<0.05) was observed by using one-way ANOVA and the post-hoc Turkey’s test for multiple comparisons. The second aspect of the thesis addressed the spatial resolution of SPME for in situ analysis. Firstly, the sampling of the SPME with high spatial resolution was modeled with multilayered gel system with the mini-sized SPME fibres. The feasibility of the SPME for in vitro application was demonstrated by sampling in an onion bulb with heterogeneous structure. Afterwards, the miniaturized fibre was successfully applied to the in situ analysis of the concentration distribution of Ochratoxin A in semisolid cheese samples with acceptable sensitivity (Detection limit was 1.5 ng/mL and the linear range was 1.5-500 ng/mL) and comparable accuracy to the standard methods such as liquid extraction and microdialysis. Finally, the in vivo application of the space- and time- resolved SPME was implemented to study the tissue-specific bioaccumulations of pharmaceuticals in fish adipose fins and muscle tissues. The results were validated by the standard method liquid extraction, and they were also comparable to the literature results. The research presented here demonstrated the application potential of the time-and space- resolved SPME for in situ dynamic and static analysis in a living system such as a beagle or fish, and in a non-living system such as a cheese piece or an onion bulb.

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