Academic literature on the topic 'Rapid Sensing of Bacteria'

This dissertation contains two different research topics. One is a "Nano Scale Device for Plasmonic Nanolithography - Optical Antenna' and the other is a 'Nano Scale Device for Rapid Sensing of Bacteria - SEPTIC'. Since these two different research topics have little analogy to each other, they were divided into different chapters throughout the whole dissertation. The 'Optical Antenna' and 'Nanowell / Microwell / ISFET Sensor' represent the device names of each topic 'Plasmonic Nanolithography' and 'Rapid Sensing of Bacteria' respectively. For plasmonic nanolithography, we demonstrated a novel photonic device - Optical Antenna (OA) - that works as a nano scale object lens. It consists of a number of sub-wavelength features in a metal film coated on a quartz substrate. The device focuses the incident light to form a narrow beam in the near-field and even far-field region. The narrow beam lasts for up to several wavelengths before it diverges. We demonstrated that the OA was able to focus a subwavelength spot with a working distance (also the focal length) of several µm, theoretically and experimentally. The highest imaging resolution (90-nm spots) is more than a 100% improvement of the diffraction limit (FWHM = 210 nm) in conventional optics. A model and 3D electromagnetic simulation results were also studied. Given its small footprint and subwavelength resolution, the PL holds great promise in direct-writing and scanning microscopy. Collaborative work demonstrated a Nanowell (or Microwell) device which enables a rapid and specific detection of bacteria using nano (or micro) scale probe to monitor the electric field fluctuations caused by ion leakage from the bacteria. When a bacteriophage infects a bacterium and injects its DNA into the host cell, a massive and transitory ion efflux from the host cell occurs. SEPTIC (SEnsing of Phage-Triggered Ion Cascade) technology developed by collaboration uses a nanowell device to detect the nano-scale electric field fluctuations caused by this ion efflux. The SEPTIC provides fast (within several minutes), effective (living cell only), phage specific (simple and less malfunction), cheap, compact and robust method for bacteria sensing. We fabricated a number of devices, including 'Nanowell', 'Microwell' and 'ISFET (Ion Selective Field Effect Transistor)', which detect bacteria-phage reactions in frequency domain and time domain. In the frequency domain, detected noise spectrum is characterized by 1/f[beta]. The positive reaction showed much higher [beta] =̃1 than that of background noise or negative reaction ( [beta] =̃0). For the time domain, we observed abnormal pulses (> 8[omega] ) lasting 0.1 ~ 0.3 s which match the duration of ion flux reported by prior literatures. And the ISFET showed the phage-infection-triggered pulse in the form of the deviated drain current. Given the size of nanowell (or microwell, ISFET) and the simplified detection electronics, the cost of bacteria sensing is significantly reduced and the robustness is well improved, indicating very promising applications in clinical diagnosis and bio-defense.

Environmental pollutants pose great risks and adverse effects to humans and therefore arouse global environmental concern. Bacterial sensors capable of assessing the bioavailability and toxicity of pollutants show great advantages in environmental sensing. This project aims at developing a bioluminescent bacteria-based microfluidic sensor for online monitoring of environmental contaminants and toxicity. Microfluidic devices immobilised with Acinetobacter sp. ADP1_lux cells as a model strain have been developed for quantitative bioassays. Three microfluidic devices were developed and tested in order to trap and culture a monolayer of bacterial cells. The terrace device is capable of trapping a monolayer of cells in a chamber for tracking single-cell growth and response. This device utilises a barrier channel lower than the cell diameter. Two flow channels can be used to load bacterial cells, deliver fresh media and inducers and wash away overgrown cells. The device was used to measure the bioluminescence induction of ADP1_lux cells and its capability to track individual cell growth was demonstrated with E.coli cells. Since bioluminescence signals from a monolayer of ADP1_lux cells were too weak to be detected after 2 h induction by 200 µM salicylate, a microwell device was developed to concentrate cells in individual microwells for population-based analysis. Cell loading procedures, dimensions of wells, carbon sources and on-chip cultures that affect bioluminescence light intensities were investigated. This device succeeded in detecting 200 µM salicylate within 1 h. However, long-term cell culture revealed that ADP1_lux cells tend to form biofilms. Cell populations in individual wells varied greatly, making quantification impossible. Therefore, this device is only suitable for rapid detection of high concentrations of contaminants if biofilm forming bacteria cells are used as biosensors. In contrast, in the case of non-adherent cells such as E.coli, a uniform population distribution in each well was achieved after 2-day culture, suggesting this method is applicable to perform long term, quantitative bioassays using suitable, non-adherent cells. To be able to detect low concentrations of contaminants and overcome potential biofilm formation, a new population array device was developed as a proof of concept to control and isolate cell populations. It consists of a network of microfluidic channels and an array of microchambers. The device was characterised with fluorescent dyes and its capability to perform quantitative bioluminescence assays was evaluated by detecting a range of concentrations of salicylate solutions (from 10 µM to 50 µM salicylate) using ADP1_lux cells. A linear correlation between bioluminescence intensities and salicylate concentrations was successfully established within 90 min induction. It is worth noting that the population array device is the first demonstration of bioluminescence detection at the length scale of microns. Therefore, it has potential to perform multiplex detection within a small footprint where different types of whole cell biosensors can be employed simultaneously. To this end, a logarithmic serial dilution device was also developed to enable quantitative, multiplex bioassays to be conducted in the same device. This integrated dilution and population device provides a powerful tool for rapid quantification of multiple contaminants simultaneously in a sample.

Sepsis is a severe blood infection caused by bacteria entering the blood stream. Sepsis caused by antibiotic resistant bacteria is very dangerous with a high mortality rate. The current clinical diagnostic methods for sepsis require culturing the blood sample prior to other steps of the diagnosis procedure. Culturing the blood samples is a time-consuming step which increases the time required for the diagnostic procedure. Considering the fact that the mortality rate of the sepsis increases as time passes, it is essential to find methods for sepsis diagnosis that do not require culturing the samples. The first step of a new diagnostic method for antibiotic resistant sepsis, we have developed a new method for rapid separation of the bacteria from whole human blood based on centrifugal force. Density and size differences between blood cells and bacteria lead to different sedimentation velocities for each of these cells and microorganisms in a centrifugal field. Spinning blood inoculated with bacteria in our designed hollow disks at the specified speed for a designated period of time creates fairly well-separated layers of blood cells and plasma. Red and white cells have higher sedimentation velocities due to higher densities or larger sizes compared to bacteria, forming a region of dense cells close to the wall of the spinning disk. Bacteria sediment slower than red and white cells, moving to and remaining in plasma. By carefully slowing the spinning speed after separation, we are able to avoid remixing of the blood cell layer and bacteria, thus keeping the bacteria separated from rest of the blood cells. This thesis involves the experimental methods for increasing the recovery of the bacteria from human blood by mechanical and chemical methods. It also explains the theory behind the separation technique used.

Several traditional methods have been used to characterise bacteria, such as biochemical, morphological and molecular tests; however, these methods are time-consuming and not always reliable. Recently, modern analytical techniques have emerged as powerful tools offering high-throughput, reliable and rapid analysis in applications, such as clinical and microbiology studies. A variety of modern analytical techniques have been employed for bacterial characterisation, including matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), liquid chromatography-mass spectrometry (LC-MS), Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy. This thesis focused on developing a robust MALDI-TOF-MS methodology to generate mass spectra profiles for the discrimination of clinically-significant bacteria. The data generated from MALDI-TOF-MS analysis are significantly influenced by a number of experimental factors, namely instrument settings, sample preparation, the choice of matrix, matrix additives and matrix preparation as well as sample-matrix deposition methods. The need to optimise experimental variables for bacterial analysis using MALDI-TOF-MS was evident despite the increased application of this analytical tool for clinical microbiology. Experimental optimisation revealed that the choice of matrix is the most important element in MALDI-TOF-MS analysis. Based on this study, a number of different matrices were used to obtain more reproducible mass spectra to classify bacterial samples using a rapid and effective approach. Studies in this thesis indicated that sinapinic acid (SA) is the best matrix for the analysis of proteins from intact bacteria, while 6-aza-2-thiothymine (ATT) and 2,5-dihydroxybenzoic acid (DHB) produced promising results for the analysis of lipid extracts from bacteria. Analytical techniques in combination with multivariate analysis, such as principal components analysis (PCA) and principal component-discriminant function analysis (PC-DFA), were used for bacterial discrimination. Classification was initially undertaken using MALDI-TOF-MS analysis, and subsequently FT-IR spectroscopy, Raman spectroscopy and LC-MS were performed to confirm the classification results. Two main types of bacteria were used for this analysis: 34 strains from seven Bacillus and Brevibacillus species and 35 isolates from 12 Enterococcus faecium strains. The findings showed that the four analytical techniques provide clear discrimination between bacteria at these different levels. Classification of different Bacillus and Brevibacillus bacteria using MALDI-TOF-MS analysis of extracted lipids was confirmed by LC-MS data. In addition, MALDI-TOF-MS data based on extracted lipids and intact bacterial cell proteins were very similar. MALD-TOF-MS analysis of intact enterococci cells produced successful classification with 78% correct classification rate (CCR) at the strain level. FT-IR and Raman spectroscopic data produced very similar bacterial classification with CCR of 89% and 69% at the strain level, respectively. However, classification based on MALDI-TOF-MS data and that based on spectroscopic data were slightly different (Procrustes distance of 0.81, p < 0.001, at the species level). Overall, the findings in this thesis indicate the potential of MALDI-TOF-MS as a rapid, robust and reliable method for the classification of bacteria based on different bacterial preparations.

The scientific community has gathered an extremely detailed and sophisticated understanding of the genetic and molecular underpinnings of microbial communication. How these microbial communication systems arise and are maintained over evolutionary time-scales however has received relatively little attention. Some major questions remain unanswered such as; what is the function of small diffusible molecules? How does population structure affect the dynamics of social communication and what is the link between the ecology of communication and the virulence of a pathogenic population? Borrowing concepts from evolutionary theory can help to unravel these fundamental questions in the context of microbial communication as it has done in other taxa displaying social behaviours. In addition microbial model organisms in which molecular and genetic tools are abundant lend enormous power to empirical tests of evolutionary theory. This work combines both of these in an attempt to understand the evolution of bacterial communication using the model organism Pseudomonas aeruginosa and its well characterised Quorum Sensing systems. Specifically the focus is in three areas. Firstly this study reveals that the stability of bacterial signalling is vulnerable to perturbations in cost and benefit and genetic conflict. Secondly this study finds that spatial structure (biofilm vs planktonic) influences the outcome of social competition over signalling and reduces population viability. Thirdly this study finds that interspecific and intraspecific competition over public goods impose divergent selective pressures on communication.

Thesis (M. Sc. Med.)--University of Sydney, 2007.<br>Title from title screen (viewed 15 October 2008). Submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Discipline of Medicine, Faculty of Medicine. Includes bibliographical references. Also available in print form.