Chip-Oriented Fluorimeter Design and Detection System Development for DNA Quantification in Nano-Liter Volumes

The chip-based polymerase chain reaction (PCR) system has been developed in recent years to achieve DNA quantification. Using a microstructure and miniature chip, the volume consumption for a PCR can be reduced to a nano-liter. With high speed cycling and a low reaction volume, the time consumption of one PCR cycle performed on a chip can be reduced. However, most of the presented prototypes employ commercial fluorimeters which are not optimized for fluorescence detection of such a small quantity sample. This limits the performance of DNA quantification, especially low experiment reproducibility. This study discusses the concept of a chip-oriented fluorimeter design. Using the analytical model, the current study analyzes the sensitivity and dynamic range of the fluorimeter to fit the requirements for detecting fluorescence in nano-liter volumes. Through the optimized processes, a real-time PCR on a chip system with only one nano-liter volume test sample is as sensitive as the commercial real-time PCR machine using the sample with twenty micro-liter volumes. The signal to noise (S/N) ratio of a chip system for DNA quantification with hepatitis B virus (HBV) plasmid samples is 3 dB higher. DNA quantification by the miniature chip shows higher reproducibility compared to the commercial machine with respect to samples of initial concentrations from 103 to 105 copies per reaction

New ionization and tagging methods for mass spectrometry

Mass spectrometry (MS) is an essential detection tool in bioanalytical chemistry owing to its exceptional selectivity and sensitivity, paired with rapid analyte identification and quantification. In this thesis, two classical MS and a newly developed ionization method are employed, including matrix-assisted laser desorption/ionization (MALDI) MS, electrospray ionization (ESI) MS and electrostatic spray ionization (ESTASI) MS. To meet the emerging challenges in bioanalytical chemistry, from disease diagnosis to drug development, new MS-based analytical methods ought to be developed with respect to high sensitivity, throughput, speed, as well as sample consumption and experimental simplification. For these purposes, this thesis presents four analytical strategies combined with MS detection for improved analytical performance in different research fields. Two classical analytical tools, thin layer chromatography (TLC) and 384-well plate, were coupled with ESTASI-MS respectively. In ESTASI-TLC-MS, ESTASI was applied to extract and identify a wide range of molecules of different polarities and chemical structures from both hydrophilic or hydrophobic silica plates with high sensitivity and minimal sample consumption in the femtomole range. In 384-well plate ESTASI-MS, the commercial 384-well plate could work as a container and an emitter for sample spray ionization, without any liquid delivery system or any additional interface. This approach provides fast and high throughput analyses for the large batches of reactions, such as enzyme assay and drug metabolism. Tyrosinase-catalyzed tyrosine oxidation in the presence or absence of inhibitors and cytochrome P450-catalyzed metabolic reactions of two drugs were studied respectively. To improve analysis sensitivity, two strategies, mass barcoded gold nanoparticles (Mb-AuNPs) for MALDI-MS signal amplification and microfabricated on-chip spyhole (Ø 10-12 ¿m) emitter for nanoelectrospray (spyhole-nanoESI), were developed, leading to a low sample consumption and high analysis efficiency. Mb-AuNPs combined with magnetic separation were applied for multiplex cow¿s milk allergy diagnosis in a component-resolved manner. IgE antibodies (Abs) could be extracted from a patient¿s blood serum by the formation of a sandwich structure between allergenic proteins-coated Mb-AuNPs and anti-human IgE Abs-functionalized magnetic beads (MBs). Detection of Mb-AuNPs by MALDI-MS provides a limit of detection (LOD) down to picograms per milliliter level for specific IgE Abs from only 1 ¿L of patient¿s blood serum. To take advantages of microfluidics in low sample consumption and easy integration, a novel interface of spyhole-nanoESI was designed for coupling microfluidics with MS and showed an improved sensitivity than standard ESI. This disposable microchip coupled with MS/MS shows potential application in cancer diagnosis by the successful detection of a small cell lung cancer biomarker in 1 ¿L of human serum at the extensive stage, without any complicated sample preparation steps

Fabrication of DNA microarrays on Poly (methylmethacrylate) substrates for biomolecular reporting

DNA microarrays require the use of substrates with well-established surface modification / probe attachment chemistries. Glass/quartz have been widely adopted as typical support materials since their surface modification chemistries which involve the use of siloxane –based chemistries have been widely studied however; these chemistry is susceptible to hydrolytic cleavage especially at high or low pH values. Recently, polymers have been sought as alternative microarray support materials but their surface modification strategies are not well characterized compared to glass. This report will entail surface photo-modification of PMMA polymer substrates by UV irradiation which produces functional scaffolds of carboxylic groups that allow covalent attachment of amine-terminated oligonucleotide probes onto these surfaces via carbodiimide coupling chemistries. The photo-modification process for microarray fabrication involves only three steps; (1) broadband UV exposure of the polymer surface; (2) carbodiimide coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface. Since microfluidics offer several advantages such as reduction in reagent cost, reduction in hybridization assay times and parallel processing of samples; we incorporate them in the microarray construction by using poly (dimethylsiloxane) microchannels that are reversibly sealed to the photoactivated PMMA substrates. Parallel sample processing minimizes contamination effects that can give rise to false positives which can be a significant issue especially for diagnostic applications. We demonstrate use of these protocols with linear oligonucleotide probes for screening multiple KRAS 2 mutations possessing high diagnostic value for colorectal cancers whereby a Ligase Detection Reaction/universal zipcode array assays was carried out using parallel detection of two different low abundant DNA point mutations in KRAS 2 oncogenes with allelic composition evaluated at one locus. The same covalent attachment protocols were utilized for immobilizing hairpin probes (molecular beacons) in a microarray format that were used to report on the analysis of complementary DNA (cDNA) specific for fruitless (fru) and ods-site homeobox (OdsH) genes extracted from Drosophila Melanogaster fruit flies. To further improve the analytical sensitivities exhibited by these hairpin probes; we used phthalocyanine dyes for dual labeling of oligonucleotide probes that will be used for reporting on biomolecular association events

Materials engineering of semiconductor quantum dots for biosensing applications

The brightness and photostability of semiconductor quantum dots (QDs) has prompted the exploration of their use in a wide variety of fields. Several examples of QD-based biosensors have been reported but none have actually replaced their preexisting technologies. This work reveals the barriers hindering widespread use of QD based biosensors and examines how QDs can be engineered for improved utility in bioassay designs. The first portion of this project aims to improve Förster Resonance Energy Transfer (FRET) that use QDs as both the donor and acceptor. FRET-based sensors often use fluorescent dyes (FD) or proteins (FPs), but their photo- and chemical instability can be problematic. Contemporary QD-QD FRET systems suffer from unacceptably high background signal due to direct acceptor excitation. Materials engineering is used to create QD donors that are brighter than their QD acceptors to mitigate this effect. First, CdSe/xCdS/xZnS QDs of increasing shell thickness were synthesized and tested in a QD-fluorescent dye system to elucidate the effect of increased donor size on the performance of a FRET sensor. The optimal donors were medium-sized and 8 times brighter than commercially available QDs while retaining ~60% FRET efficiency. When used in a sensor, changes in sensor brightness were visible by eye. Moving towards QD-QD systems, a pH-based aggregation assay was used to test how QD heterostructures comprised of different semiconductor materials perform as FRET donors or acceptors. The fundamental principles uncovered are used to improve contemporary QD-QD FRET sensing and show that sensors can be designed to use color change as a visible, easy-to-decipher readout. Color change-based sensor output is further explored in an allosteric transcription factor-based small-molecule sensor that employs QDs as the sole fluorescent label. A highly modular design is presented that achieves a nanomolar concentration visual limit of detection. The ease of use, and fast, instrument-free readout of the sensor shows promise for its development into a fully integrated point-of-care device, endorsing the value of further developing QD-based in vitro biosensors for clinical or commercial translation.2020-06-04T00:00:00

Dynamic assembly, disassembly and bundling of the bacterial cell division protein FtsZ and YgfE (ZapA)

The protein FtsZ is a tubulin-like GTPase, which plays an essential role in prokaryotic cell division. In vivo it assembles into a dynamic ring (the Z-ring) at the future site of cell division on the inner surface of the cytoplasmatic membrane. The Z-ring then serves as a scaffold which recruits all other division proteins to form the cytokinetic machinery. The constriction of the ring facilitates the separation of two daughter cells. In vitro, FtsZ polymerizes in the presence of GTP to form single-stranded protofilaments. It is assumed that FtsZ association and assembly reactions studied in vitro will play an important role in understanding the mechanism of Z-ring assembly and disassembly in vivo. In this work, we therefore have studied the dynamics of FtsZ polymerization in vitro, especially the bundling induced with YgfE. YgfE, the putative Escherichia coli ZapA orthologue, is one of these division proteins recruited by FtsZ. It acts to enhance lateral associations between FtsZ fibres to form larger bundles. In this study, we have demonstrated that YgfE exists as a tetramer in solution at the concentrations reported in this study, and the bundling activity is exerted more efficiently on preformed FtsZ protofilaments. We also investigate the importance of the tetramerisation of YgfE on function. A number of mutant forms of ZapA were generated with the aim of disrupting the dimer:dimer interface. Using those mutants we show that tetramerisation is a requirement for both FtsZ bundling, and GTPase modulation activities. In addition, a novel technique, continuous channel flow linear dichroism (LD) spectroscopy, has been first developed in this work. LD is a simple spectroscopy technique for structural characterization of long biomacromolecules in solutions. Continuous channel flow Flow LD has overcome the limitation of Couette flow LD due to the time required to assemble and fill the cell

Genome wide gene expression analysis of two ENU mouse models of major mental illness

Major mental illness is now recognised as one of the leading causes of adult morbidity. Of the adult onset psychiatric disorders, the functional psychoses (schizophrenia, bipolar disorder and recurrent major depression) are the most severe and most common in the general population. Evidence suggests that certain genetic factors influence an individual’s susceptibility to developing these disorders when combined with appropriate social and environmental conditions. Several good candidate genes have been identified. Of relevance to this study is Disrupted in Schizophrenia 1 (DISC1) which was identified in a large Scottish family that carried a balanced translocation (t1:11) and had a history of major mental illness. In 2008, two ENU mutant mouse models with missense mutations in exon 2 of Disc1 were characterised and found to have behavioural and neuroanatomical phenotypes consistent with schizophrenia and major depression. The primary aim of this thesis is to further analyse these mouse models by performing whole genome gene expression studies and secondary protein analysis to identify genes involved in the aetiology of schizophrenia and major depression. My initial analysis used Illumina BeadChip microarray technology to identify 368 genes that were differentially expressed in ENU mutant animals under different biological conditions, compared to appropriate control animals. Nine biological groups were compared including one embryonic group at E13, and three groups treated with appropriate anti-psychotic or anti-depressant drugs. Of the 368 genes identified as differentially expressed, 46 were chosen for validation by qRT-PCR based on fold-change, p-value, functional significance, overenrichment of GO terms, pathway analysis and previous implications in major mental illness. NRXN1, NRXN3 and CDH11 were found to be significantly up-regulated in the schizophrenia mouse model with EGR4 significantly down-regulated compared to C57BL/6J wild-type controls. These findings were also replicated in an independent sample using wildtype littermates. The mental retardation gene PAK3 was up-regulated in the schizophrenia mouse model and expression levels were corrected to a level not significantly different to wild-type, when treated with the PDE4 inhibitor Rolipram. Semi-quantitative western blotting also confirmed the disregulation of EGR4 and PAK3 at the protein level in these animals. RNA expression profiles were also characterised for each of the genes above, and DISC1, through development. In summary this thesis describes the striking disregulation of four prominent genetic candidates of major mental illness in an independent animal model. A first functional link between DISC1 and NRXN1 is described suggesting, for the first time, a DISC1- dependant mechanism for regulating neurexin gene expression

Structures of ferroportin in complex with its specific inhibitor vamifeport

A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe$^{2+}$ transport and induces internalization and degradation of FPN. Dysregulation of the FPN/hepcidin axis leads to diverse pathological conditions, and consequently, pharmacological compounds that inhibit FPN-mediated iron transport are of high clinical interest. Here, we describe the cryo-electron microscopy structures of human FPN in complex with synthetic nanobodies and vamifeport (VIT-2763), the first clinical-stage oral FPN inhibitor. Vamifeport competes with hepcidin for FPN binding and is currently in clinical development for β-thalassemia and sickle cell disease. The structures display two distinct conformations of FPN, representing outward-facing and occluded states of the transporter. The vamifeport site is located in the center of the protein, where the overlap with hepcidin interactions underlies the competitive relationship between the two molecules. The introduction of point mutations in the binding pocket of vamifeport reduces its affinity to FPN, emphasizing the relevance of the structural data. Together, our study reveals conformational rearrangements of FPN that are of potential relevance for transport, and it provides initial insight into the pharmacological targeting of this unique iron efflux transporter