DNA templated fluorescent gold nanoclusters reduced by Good’s buffer: from blue emitting seeds to red and near infrared emitters

The final published version is available at NRC Research Press via https://doi.org/10.1139/cjc-2014-0600DNA-templated fluorescent gold nanoclusters (AuNCs) have been recently prepared showing higher photostability than the silver counterpart. In this work, we examined the effect of pH, DNA length, DNA sequence, and reducing agent. Citrate, HEPES, and MES produce blue emitters, glucose and NaBH4 cannot produce fluorescent AuNCs, while ascorbate shows blue emission even in the absence of DNA. This is the first report of using Good’s buffer for making fluorescent AuNCs. Dimethylamine borane (DMAB) produces red emitters. Poly-C DNA produces AuNCs only at low pH and each DNA chain can only bind to a few gold atoms, regardless of the DNA length. Otherwise, large nonfluorescent gold nanoparticles (AuNPs) are formed. Each poly-A DNA might template a few independent AuNCs. The blue emitters can be further reduced to form red emitters by adding DMAB. The emission color is mainly determined by the type of reducing agent instead of DNA sequence.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council || Ontario Ministry of Research and Innovation |

Interfacing fluorescent DNA oligonucleotides with graphene oxide and metal oxides: from adsorption to sensing

DNA, apart from being the mode of genomic information storage, has found several uses in catalysis (DNAzymes) and target detection (aptamers). Developing novel biosensors utilizing these properties has therefore been a significant avenue for research in recent decades. Of these avenues, interfacing fluorescent dye-labelled DNA with various nanomaterials has birthed many sensors which have been implemented in several environments such as lake water, food, and even within the cell. In this thesis, we provide an improved understanding of DNA adsorption on such nanomaterials and interpretation of sensor results. In Chapter 1, background information related to DNA, fluorescence and nanomaterials are introduced, with associated examples of different biosensor design. The fundamental questions arising from these sensor designs are also stated, along with thesis objectives. In Chapter 2, a comparison is made between graphene oxide and inorganic metal oxides for aptamer-based fluorescence sensing. It was found that, for graphene oxide, target/aptamer interactions dominate the sensor response. This is in contrast to the metal oxide nanoparticles, where sensing is achieved through the target simply displacing DNA from the nanomaterial surface. In Chapter 3, the properties of carboxyfluorescein-labelled poly-C DNA are explored in detail. Through fluorescence and circular dichroism experiments, it was seen that carboxyfluorescein stabilizes i-motif formation in poly-C DNA, even at neutral pH. This folding was irreversible upon heating. Unfolding of the structure led to improved adsorption on GO demonstrated through fluorescence desorption experiments. In Chapter 4, the anomalously high affinity of poly-C adsorption was investigated using both fluorescence experiments and simulations. It was found that the arrangement of cytosines within the chain did not affect affinity, merely their total number. Through simulations, it was determined that poly-C DNA spreads out on the GO surface due to its lack of intrastrand interactions. This results in more phosphate-backbone hydrogen bond sites and a more favourable bond. At lower pH, i-motif formation drastically reduces poly-C affinity to GO; intrastrand interactions dominate over GO/DNA binding. In Chapter 5, fluorescence polarization was used to characterize labelled DNA interactions with various nanomaterials. First, it was determined that, at low labelled-DNA concentrations, polarization is artificially increased by scattering of incident polarized light. Polarization is also increased with the addition of GO to this DNA. Through a simple mathematical derivation, it was shown that the increase in polarization with this kind of surface was due to low concentration of free DNA, rather than adsorption to the GO surface. This was compared to a low-quenching surface (Yttrium oxide), in which the total polarization observed was dominated by the binding DNA rather than free DNA. Overall, the work presented in this thesis improves the current understanding of both fundamental DNA/nanomaterial interactions, as well as its implementation in fluorescence-based sensor designs. Future biosensor construction can incorporate these concepts for better sensitivity, specificity and signal interpretation

Synthesis of Phosphate-Crosslinked Starch Nanoparticles for Drug Delivery

Drug delivery agents for chemotherapy drugs have gained significant interest over past few decades due to the need to localize the treatment to cancer cells. So far, polymeric micelles, liposomes, and carbon-based nanomaterials, among others, have shown great promise for this purpose. Starch nanoparticles have emerged as an avenue for drug delivery due to their low toxicity, biocompatibility and low cost. In this work, starch nanoparticles internally crosslinked by sodium trimetaphosphate (STMP) were prepared using a phase inversion emulsion process. From dynamic light scattering, transmission electron microscopy and environmental scanning electron microscopy, the particle size was determined to be 200-500 nm, regardless of STMP concentration used in the synthesis. 31P NMR determined that a wide variety of organic phosphates were present, apart from the desired phosphodiester crosslinking. These included triphosphates, monophosphates and diphosphates. In addition, like typical charged nanogels, these nanoparticles retained significant amounts of water when dispersed in solution. This was related to the electrostatic repulsion between the chains within the nanoparticle. The presence of salt decreased the amount of water retention by screening of this electrostatic repulsion. The prepared nanoparticles were, in general, non-toxic to HeLa cancer cells. In addition, all prepared nanoparticles displayed a high drug loading, with a maximum seen with 30 mol% STMP. This loading was higher at pH 7.6 compared to lower pH. Drug release occurred more readily at lower pH. Finally, it was seen that exposure to typical cell culture environments induced significant release of drug compared to simple buffer environments

Tuple Packing: Efficient Batching of Small Graphs in Graph Neural Networks

When processing a batch of graphs in machine learning models such as Graph Neural Networks (GNN), it is common to combine several small graphs into one overall graph to accelerate processing and remove or reduce the overhead of padding. This is for example supported in the PyG library. However, the sizes of small graphs can vary substantially with respect to the number of nodes and edges, and hence the size of the combined graph can still vary considerably, especially for small batch sizes. Therefore, the costs of excessive padding and wasted compute are still incurred when working with static shapes, which are preferred for maximum acceleration. This paper proposes a new hardware agnostic approach -- tuple packing -- for generating batches that cause minimal overhead. The algorithm extends recently introduced sequence packing approaches to work on the 2D tuples of (|nodes|, |edges|). A monotone heuristic is applied to the 2D histogram of tuple values to define a priority for packing histogram bins together with the objective to reach a limit on the number of nodes as well as the number of edges. Experiments verify the effectiveness of the algorithm on multiple datasets

A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation

Many microbes in their natural habitats are found in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Furthermore, it is estimated that nearly 80% of human infections are associated with biofilms. Biofilms are traditionally defined as three-dimensional, structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. While this view of biofilm largely arises from in vitro studies under static or flow conditions, in vivo observations have indicated that this view of biofilms is essentially true only for foreign-body infections on catheters or implants where biofilms are attached to the biomaterial. In mucosal infections such as chronic wounds or cystic fibrosis or joint infections, biofilms can be found unattached to a surface and as three-dimensional aggregates. In this work, we describe a high-throughput model of aggregate biofilms of methicillin-resistant Staphylococcus aureus (MRSA) using 96-well plate hanging-drop technology. We show that MRSA forms surface-independent biofilms, distinct from surface-attached biofilms, that are rich in exopolymeric proteins, polysaccharides, and extracellular DNA (eDNA), express biofilm-related genes, and exhibit heightened antibiotic resistance. We also show that the surface-independent biofilms of clinical isolates of MRSA from cystic fibrosis and central catheter-related infections demonstrate morphological differences. Overall, our results show that biofilms can form by spontaneous aggregation without attachment to a surface, and this new in vitro system can model surface-independent biofilms that may more closely mimic the corresponding physiological niche during infection. IMPORTANCE The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface. The hanging-drop model of biofilms of methicillin-resistant Staphylococcus aureus (MRSA), the major causative organism of skin and soft tissue infections, shows that these biofilms display morphological and antibiotic response patterns that are distinct from those of their surface-attached counterparts, and biofilm growth is consistent with their in vivo location. The simplicity and throughput of this model enable adoption to investigate other single or polymicrobial biofilms in a physiologically relevant setting

Is Mira a magneto-dusty rotator?

We investigate the possibility that a magnetic field may be present in the star $o-$Ceti (hereafter, Mira) and that the field plays a role in the star's mass loss. The model presented here is an application of an earlier derived theory that has been successfully employed for intermediate and high-mass evolved stars, and is now extended to the low-mass end. The modelling shows that it is possible to obtain a hybrid magnetohydrodynamic-dust-driven wind scenario for Mira, in which the role of a magnetic field in the equatorial plane of the star is dynamically important for producing a stellar wind. The wind velocity and the temperatures obtained from the model appear consistent with findings elsewhere.Comment: 5 pages, 2 figure

Antimicrobial and Antibiofilm Activity of Synergistic Combinations of a Commercially Available Small Compound Library With Colistin Against Pseudomonas aeruginosa

Biofilm-associated Pseudomonas aeruginosa infections remain a significant clinical challenge since the conventional antibiotic treatment or combination therapies are largely ineffective; and new approaches are needed. To circumvent the major challenges associated with discovery of new antimicrobials, we have screened a library of compounds that are commercially available and approved by the FDA (Prestwick Chemical Library) against P. aeruginosa for effective antimicrobial and anti-biofilm activity. A preliminary screen of the Prestwick Chemical Library alone did not yield any repositionable candidates, but in a screen of combinations with a fixed sub-inhibitory concentration of the antibiotic colistin we observed 10 drugs whose bacterial inhibiting activity was reproducibly enhanced, seven of which were enhanced by more than 50%. We performed checkerboard assays of these seven drugs in combination with colistin against planktonic cells, and analysis of their interactions over the complete combination matrix using the Zero Interaction Potency (ZIP) model revealed interactions that varied from highly synergistic to completely antagonistic. Of these, five combinations that showed synergism were down-selected and tested against preformed biofilms of P. aeruginosa. Two of the five combinations were active against preformed biofilms of both laboratory and clinical strain of P. aeruginosa, resulting in a 2-log reduction in culturable cells. In summary, we have identified synergistic combinations of five commercially available, FDA-approved drugs and colistin that show antimicrobial activity against planktonic P. aeruginosa (Clomiphene Citrate, Mitoxantrone Dihydrochloride, Methyl Benzethonium Chloride, Benzethonium Chloride, and Auranofin) as well as two combinations (Auranofin and Clomiphene Citrate) with colistin that show antibiofilm activity

HRFlexToT: A High Dynamic Range ASIC for Time-of-Flight Positron Emission Tomography

Time-of-Flight positron emission tomography scanners demand fast and efficient photo sensors and scintillators coupled to fast readout electronics. This article presents the high resolution flexible Time-over-Threshold (HRFlexToT), a 16-channel application-specific-integrated circuit for silicon photomultipliers (SiPM) anode readout manufactured using XFAB 0.18- \mu \text{m} CMOS technology. The main features of the HRFlexToT are a linear Time-over-Threshold with an extended dynamic range (10 bits) for energy measurement, low power consumption (≈ 3.5 mW/ch), and an excellent timing response. The experimental measurements show an energy linearity error of ≈ 3% and an energy resolution of about 12% at 511 keV. Single-photon time resolution measurements performed using an Fondazione Bruno Kessler (FBK) SiPM NUV-HD ( 4 \times 4 mm2 pixel, 40- \mu \text{m} cell) and a Hamamatsu SiPM S13360-3050CS are around 142 and 167 ps full width at half maximum (FWHM), respectively. Coincidence time resolution (CTR) measurements with small cross-section pixelated crystals (LSO:Ce,Ca 0.4%, 2 \times 2 \times 5 mm3) coupled to the same Hamamatsu S13360-3050CS and FBK NUV-HD sensors yield a CTR of 117 ps and 119 ps, respectively. Measurements performed with a large cross-section monolithic crystal (LFS crystal measuring 25 \times 25 \times 20 mm3) and a Hamamatsu SiPM array S13361-6050NE-04 show a CTR of 324 ps FWHM after time-walk and time-skew correction