The use of laser desorption as a sample introduction method for solid phase microextraction (SPME) has been investigated in this research project. Three different types of analytical instruments, mass spectrometry (MS), ion mobility spectrometry (IMS) and gas chromatography (GC) were employed as detectors. The coupling of laser desorption SPME to these three instruments was constructed and described in here. <br /><br /> Solid phase microextraction/surface enhanced laser desorption ionization fibers (SPME/SELDI) were developed and have been coupled to two IMS devices. SPME/SELDI combines sampling, sample preparation and sample introduction with the ionization and desorption of the analytes. Other than being the extraction phase for the SPME fiber, the electro-conductive polymer coatings can facilitate the ionization process without the involvement of a matrix assisted laser desorption/ionization (MALDI) matrix. The performance of the SPME coatings and the experimental parameters for laser desorption SPME were investigated with the SPME/SELDI IMS devices. The new SPME/SELDI-IMS 400B device has a faster data acquisition system and a more powerful data analysis program. The optimum laser operation parameters were 250 <em>&mu;J</em> laser energy and 20 <em>Hz</em> repetition rate. Three new SPME coatings, polypyrrole (PPY), polythiophene (PTH) and polyaniline (PAN) were developed and evaluated by an IMS and a GC. The PPY coating was found to have the best performance and was used in most of the experiments. The characteristics of the PPY and the PTH SPME/SELDI fiber were then assessed with both IMS and MS. Good linearity could be observed between the fiber surface area and the signal intensity, and between the concentration and the signal intensities. <br /><br /> The ionization mechanism of poly(ethylene glycol) 400 (PEG) was studied with the SPME/SELDI-IMS 400B device. It was found that the potassiated ions and sodiated ions were both present in the ion mobility spectra. The results obtained with quadrupole time-of-flight (QTOF) MS confirmed the presence of both potassiated and sodiated ions. This result suggested that cationization is the main ionization process when polymers are directly ionized from the PPY coated silica surface. Four PEGs with different average molecular weights and poly(propylene glycol) 400 were also tested with this SPME/SELDI device. The differences between the ion mobility spectra of these polymers could be used for the fast identification of synthetic polymers. <br /><br /> The SPME/SELDI fibers were then coupled to QTOF MS and hybrid quadrupole linear ion trap (QqLIT) MS, respectively. Improved sensitivity could be achieved with QqLIT MS, as the modified AP MALDI source facilitated the ion transmission. The application of method for analysis of urine sample and the bovine serum albumin (BSA) digest were demonstrated with both PPY and PTH fibers. The LOD for leucine enkephalin in urine was determined to be 40 <em>fmol &mu;L^-1</em> with PTH coated fiber; and the LOD for the BSA digest was 2 <em>fmol &mu;L^-1</em> obtained with both PTH and PPY fibers. <br /><br /> A new multiplexed SPME/AP MALDI plate was designed and evaluated on the same QqLIT MS to improve the throughput, and the performance of this technique. The experimental parameters were optimized to obtain a significant improvement in performance. The incorporation of diluted matrix to the extraction solution improved the absolute signal and S/N ratio by 104X and 32X, respectively. The incorporation of reflection geometry for the laser illumination improved the S/N ratio by more than two orders of magnitude. The fully optimized high throughput SPME/AP MALDI configuration generated detection limit improvements on the order of 1000-7500X those achieved prior to these modifications. This system presents a possible alternative for qualitative proteomics and drug screening. <br /><br /> Laser desorption SPME as a sample introduction method for the fast analysis of non-volatile synthetic polymers was also demonstrated here. The coupling of laser desorption SPME to GC/FID and GC/MS was performed, and the advantage of laser desorption over traditional thermal desorption was demonstrated in this research. Laser desorption PEG 400 was observed more effcient than thermal desorption. Good separation was obtained even with a 1-m or 2-m column. These results demonstrate the potential of laser desorption SPME as a sample introduction method for the fast GC analysis of non-volatile compounds such as synthetic polymers.

Li, Gang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 102-108). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.v / LIST OF FIGURES --- p.viii / LIST OF TABLES --- p.xi / ABBREVIATIONS --- p.xii / Chapter Chapter One --- Introduction / Chapter 1.1 --- Matrix-assisted Laser Desorption/Ionization (MALDI) --- p.2 / Chapter 1.1.1 --- Laser Desorption --- p.2 / Chapter 1.1.2 --- Matrix-assisted Laser Desorption/Ionization --- p.2 / Chapter 1.2 --- Time-of-flight Mass Spectrometry --- p.6 / Chapter 1.2.1 --- Linear Time-of-flight Mass Spectrometer --- p.6 / Chapter 1.2.2 --- Reflectron Time-of-flight Mass Spectrometer --- p.7 / Chapter 1.2.2.1 --- Linear-field Reflectron --- p.9 / Chapter 1.2.2.2 --- Nonlinear-field Reflectron --- p.12 / Chapter 1.3 --- Structural Analysis Using Time-of-flight Mass Spectrometer --- p.13 / Chapter 1.4 --- Project Objectives --- p.17 / Chapter Chapter Two --- Instrumentation and Experimental / Chapter 2.1 --- Instrumentation --- p.20 / Chapter 2.1.1 --- Laser system --- p.20 / Chapter 2.1.2 --- Flight Tube and Vacuum System --- p.20 / Chapter 2.1.3 --- Ion source --- p.22 / Chapter 2.1.4 --- Deflector and Time Ion Selector --- p.24 / Chapter 2.1.5 --- Two-stage Gridless Reflectron --- p.28 / Chapter 2.1.6 --- "Detectors, Digitizer and Computer System" --- p.28 / Chapter 2.2 --- Experimental --- p.31 / Chapter 2.2.1 --- Sample preparation --- p.32 / Chapter 2.2.2 --- PSD calibration --- p.32 / Chapter Chapter Three --- "Simulation Studies of Time Ion Selector, Collision cells and Curved-field Reflectron" / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Time Ion selector --- p.37 / Chapter 3.3 --- Collision cell --- p.46 / Chapter 3.3.1 --- Simulation of Collision Induced Dissociation (CID) Conditions --- p.46 / Chapter 3.3.2 --- Design and Performance Evaluation of Different Collision Cells --- p.48 / Chapter 3.4 --- Curved-field reflectron (CFR) --- p.58 / Chapter 3.4.1 --- Introduction --- p.58 / Chapter 3.4.2 --- Derivation of Analytical Equations --- p.58 / Chapter 3.4.3 --- Effect of Floating Potential of the Collision Cell --- p.65 / Chapter 3.4.4 --- Effect of R and θ Parameters --- p.65 / Chapter 3.4.5 --- Effect of Length of the Reflectron --- p.70 / Chapter 3.5 --- Conclusions --- p.73 / Chapter Chapter Four --- Construction and Performance Evaluation of Modified Time-of-flight Mass Spectrometer / Chapter 4.1 --- Benchmark Results for the Origin Reflectron Time-of-flight Mass Spectrometer --- p.75 / Chapter 4.2 --- Hardware Modifications of Reflectron Time-of-flight Mass Spectrometer --- p.75 / Chapter 4.2.1 --- Collision Cell --- p.75 / Chapter 4.2.2 --- Curved-field Reflectron --- p.79 / Chapter 4.3 --- Evaluation of the Curved-field Reflectron --- p.81 / Chapter 4.4 --- Evaluation of the field-shaped cylindrical collision cell --- p.85 / Chapter 4.5 --- Conclusions --- p.95 / Chapter Chapter Five --- Concluding Remarks / Chapter 5.1 --- Concluding Remarks --- p.100 / References --- p.101 / Appendix / Appendix 1 User program for time ion selection --- p.108 / Appendix 2 User program for gas collision --- p.111 / Appendix 3 MATHEMATICA program used in calculation for curved-fleld reflectron --- p.114

Since the introduction of electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI), there has been an unprecedented growth of biomolecule analysis using mass spectrometry (MS). One of the most popular applications for mass spectrometry is the field of proteomics, which has emerged as the next scientific challenge in the post-genome era. One critical step in proteomic analysis is sample preparation, a major bottleneck that is attributed to many time consuming and labor-intensive steps involved. Microfluidics can play an important role in proteome sample preparation due to its ability to handle small volumes of sample and reagent, and its capability to integrate multiple processes on a single chip with the potential for high-throughput analysis. However, to utilize microfluidic systems for proteome analysis, an efficient interface between microfluidic chip and mass spectrometry is required. This thesis presents several methods for coupling of microfluidic chips with ESI-MS and MALDIMS. III Three microfluidic-ESI interfaces were developed. The first interface involves fabricating a polymer based microchannel at the rectangular corners of the glass substrates using a single photolithography step. The second interface was build upon the previous interface in which a digital microfluidic platform was integrated with the microchannel in a “top-down” format. The integrated microfluidic system was used for inline quantification of amino acids in dried blood spots that have been processed by digital microfluidics. The third interface was formed by sandwiching a pulled glass capillary emitter between two digital microfluidic substrates. This method is a simpler and more direct coupling of digital microfluidics with ESI-MS as compared to the method used for second interface. Finally, a strategy using a removable plastic “skin” was developed to interface digital microfluidics with MALDI-MS for offline sample analysis. We demonstrated the utility of this format by implementing on-chip protein digestion on immobilized enzyme depots.

Since the introduction of electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI), there has been an unprecedented growth of biomolecule analysis using mass spectrometry (MS). One of the most popular applications for mass spectrometry is the field of proteomics, which has emerged as the next scientific challenge in the post-genome era. One critical step in proteomic analysis is sample preparation, a major bottleneck that is attributed to many time consuming and labor-intensive steps involved. Microfluidics can play an important role in proteome sample preparation due to its ability to handle small volumes of sample and reagent, and its capability to integrate multiple processes on a single chip with the potential for high-throughput analysis. However, to utilize microfluidic systems for proteome analysis, an efficient interface between microfluidic chip and mass spectrometry is required. This thesis presents several methods for coupling of microfluidic chips with ESI-MS and MALDIMS. III Three microfluidic-ESI interfaces were developed. The first interface involves fabricating a polymer based microchannel at the rectangular corners of the glass substrates using a single photolithography step. The second interface was build upon the previous interface in which a digital microfluidic platform was integrated with the microchannel in a “top-down” format. The integrated microfluidic system was used for inline quantification of amino acids in dried blood spots that have been processed by digital microfluidics. The third interface was formed by sandwiching a pulled glass capillary emitter between two digital microfluidic substrates. This method is a simpler and more direct coupling of digital microfluidics with ESI-MS as compared to the method used for second interface. Finally, a strategy using a removable plastic “skin” was developed to interface digital microfluidics with MALDI-MS for offline sample analysis. We demonstrated the utility of this format by implementing on-chip protein digestion on immobilized enzyme depots.

The use of laser desorption as a sample introduction method for solid phase microextraction (SPME) has been investigated in this research project. Three different types of analytical instruments, mass spectrometry (MS), ion mobility spectrometry (IMS) and gas chromatography (GC) were employed as detectors. The coupling of laser desorption SPME to these three instruments was constructed and described in here. <br /><br /> Solid phase microextraction/surface enhanced laser desorption ionization fibers (SPME/SELDI) were developed and have been coupled to two IMS devices. SPME/SELDI combines sampling, sample preparation and sample introduction with the ionization and desorption of the analytes. Other than being the extraction phase for the SPME fiber, the electro-conductive polymer coatings can facilitate the ionization process without the involvement of a matrix assisted laser desorption/ionization (MALDI) matrix. The performance of the SPME coatings and the experimental parameters for laser desorption SPME were investigated with the SPME/SELDI IMS devices. The new SPME/SELDI-IMS 400B device has a faster data acquisition system and a more powerful data analysis program. The optimum laser operation parameters were 250 <em>&mu;J</em> laser energy and 20 <em>Hz</em> repetition rate. Three new SPME coatings, polypyrrole (PPY), polythiophene (PTH) and polyaniline (PAN) were developed and evaluated by an IMS and a GC. The PPY coating was found to have the best performance and was used in most of the experiments. The characteristics of the PPY and the PTH SPME/SELDI fiber were then assessed with both IMS and MS. Good linearity could be observed between the fiber surface area and the signal intensity, and between the concentration and the signal intensities. <br /><br /> The ionization mechanism of poly(ethylene glycol) 400 (PEG) was studied with the SPME/SELDI-IMS 400B device. It was found that the potassiated ions and sodiated ions were both present in the ion mobility spectra. The results obtained with quadrupole time-of-flight (QTOF) MS confirmed the presence of both potassiated and sodiated ions. This result suggested that cationization is the main ionization process when polymers are directly ionized from the PPY coated silica surface. Four PEGs with different average molecular weights and poly(propylene glycol) 400 were also tested with this SPME/SELDI device. The differences between the ion mobility spectra of these polymers could be used for the fast identification of synthetic polymers. <br /><br /> The SPME/SELDI fibers were then coupled to QTOF MS and hybrid quadrupole linear ion trap (QqLIT) MS, respectively. Improved sensitivity could be achieved with QqLIT MS, as the modified AP MALDI source facilitated the ion transmission. The application of method for analysis of urine sample and the bovine serum albumin (BSA) digest were demonstrated with both PPY and PTH fibers. The LOD for leucine enkephalin in urine was determined to be 40 <em>fmol &mu;L^-1</em> with PTH coated fiber; and the LOD for the BSA digest was 2 <em>fmol &mu;L^-1</em> obtained with both PTH and PPY fibers. <br /><br /> A new multiplexed SPME/AP MALDI plate was designed and evaluated on the same QqLIT MS to improve the throughput, and the performance of this technique. The experimental parameters were optimized to obtain a significant improvement in performance. The incorporation of diluted matrix to the extraction solution improved the absolute signal and S/N ratio by 104X and 32X, respectively. The incorporation of reflection geometry for the laser illumination improved the S/N ratio by more than two orders of magnitude. The fully optimized high throughput SPME/AP MALDI configuration generated detection limit improvements on the order of 1000-7500X those achieved prior to these modifications. This system presents a possible alternative for qualitative proteomics and drug screening. <br /><br /> Laser desorption SPME as a sample introduction method for the fast analysis of non-volatile synthetic polymers was also demonstrated here. The coupling of laser desorption SPME to GC/FID and GC/MS was performed, and the advantage of laser desorption over traditional thermal desorption was demonstrated in this research. Laser desorption PEG 400 was observed more effcient than thermal desorption. Good separation was obtained even with a 1-m or 2-m column. These results demonstrate the potential of laser desorption SPME as a sample introduction method for the fast GC analysis of non-volatile compounds such as synthetic polymers.

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This paper, contain three parts, focus on develop synthesis method for novel functional nanocluster, base on its special optical and reactive property. In the first part, lysozyme VI was mixed with HAuCl4 under acidic condition(pH~3) and yield blue fluorescent Au8 nanocluster. When this Au8 cluster was incubate under alkaline condition (pH~12) a size evolution from Au8 to Au25 took place and monitored with fluorescence spectrum, MALDI MS, DLS. On the other hand, on the role of structural characteristic, fluorescence of Au8 cluster can be quenched by GSH as GSH induced core-etching reaction took place. Take advantage of this reaction, GSH in RBC can be quantitatively analysis by Au8 cluster. Compare with standard medical analysis method, this assay got comparable quantitative result and advantage in environmental friendly, low cost and low sample demand. Follow the first part, it was knowing that Hg2+quench fluorescence of Au25 but Au8 cluster. The second part of research development a ratiometric fluorescence assay for Hg2+ in drinking water sample, by control size evolution of nanocluster mentioned in first part by adding Ag+ into protein direct synthesis nanocluter process. Under optimize condition of bimetallic nanocluster synthesis, two separately fluorescence peak locate under 613 nm and 471 nm represent bigger size and smaller nanoclusters. Using fluorescence intensity ratio of two wavelength, concentration of Hg2+ in water sample can be determined by standard addition method with high reproducibility. Limit of detection was determined to be 0.1 and 0.4 nM, both lower than EPA permitted level in drinking water. On the third part, SA and HAuCl4 was mixed under water contained acetonitrile solution and SA caped nanocluster was form. This nanocluster after self essemble possess special crystalline and surface phenomenon and fit matrix for high reproducible laser desorption/ionization mass spectrometry. Compare with traditional matrix SA, the cluster matrix show 6.6% in RSD with 33%. And the desorption/ionization efficiency of nanocluster matrix is also much higher. Insulin, myoglobin and HSA sample can be quantitative with this matrix with mean RSD lower than 10%.

A method for the oxidation of lipoproteins in vitro was developed using the free radical initiator, 2,2?-azobis-(2-amidinopropane) dihydrochloride (AAPH). Following in vitro oxidation, the susceptibility to oxidation of the serum samples was studied using density gradient ultracentifugation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Shifts in mean buoyant density of the lipoprotein particles, specifically low density lipoprotein (LDL) and high density lipoprotein (HDL), were observed in the density profile following centrifugation. The degree of shift in the density proved to be proportional to the extent of oxidation. Changes in apolipoproteins were studied with MALDI-TOF-MS. Observed variations in the mass spectra include m/z shifts due to chemical modifications and change in isoform distributions. The oxidation procedure and analysis techniques were applied to a clinical application to study the effects of table grape consumption on lipoprotein susceptibility to oxidation. The main objective of the research, to show feasibility that these methods could be used in a clinical setting, was achieved.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI)is one of several proteomics technologies that can be used in biomarker discovery studies. Such studies often have the goal of finding protein markers that predict early onset of cancers such as cervical cancer. The reproducibility of SELDI has been shown to be an issue in the literature. There are numerous sources of error in a SELDI experiment starting with sample collection from patients to the signal processing steps used to estimate the protein mass and abundance values present in a sample. This dissertation is concerned with all aspects of signal processing related to SELDI's use in biomarker discovery projects. In chapter 2, we perform a comprehensive study of the most popular preprocessing algorithms available. Next, in chapter 3, we study the basic statistics of SELDI data acquisition. From here, we propose a quadratic variance measurement model for buffer+matrix only spectra. This model leads us to develop a modified Antoniadis-Sapatinas wavelet denoising algorithm that demonstrates superior performance when compared to MassSpecWavelet, one of the leading techniques for preprocessing SELDI data. In chapter 4, we show that the quadratic variance model 1) extends to real pooled cervical mucus QC data from a clinical study, 2) predicts behavior and reproducibility of peak heights, and 3) finds four times as many reproducible peaks as the vendor-supplied preprocessing programs. The quadratic variance measurement model for SELDI data is fundamental and promises to lead to improved techniques for analyzing the data from clinical studies using this instrument.

Thesis (Ph.D.)--Drexel University, 2009. / Includes abstract and vita. Includes bibliographical references (leaves 204).