Electrokinetics based microfluidic systems are potentially promising for lab-on-a-chip applications due to their effectiveness in manipulating nanoscale and biological objects, label-free operation, simple fabrication processes, small voltage requirements, and most importantly simple system integration strategy. Among various electrokinetics techniques, AC electrothermal flow (ACEF) is the most promising technique in microfluidic manipulation toward biomedical applications due to its effectiveness in high conductivity biological and physiological fluids. As relatively little is known about the ACEF induced fluid motion at highly conductive samples, the characteristics of electrothermal manipulation of fluid samples with different conductivities were investigated systematically. For low conductivity sample (below 1 S/m), the characteristics of the electrothermal fluid motion was in quantitative agreement with the theory. For high conductivity samples (greater than 1 S/m), the fluid motion appeared to deviate from the model as a result of electrochemical reactions and the temperature effect. Here, a universal electrode approach which directly implements ACEF-induced sample preparation on a SAM based electrochemical sensor for point-of-care diagnostics of urinary tract infections has also been demonstrated. Using uropathogenic E. coli clinical isolates as model systems, we demonstrate that "on-chip" ACEF-induced sample preparation can improve the sensor performance without complicated system integration strategy and presents a pathway for implementing truly lab on a chip, instead of chip in a lab. Finally an integrated chip approach has been proposed for transforming electrochemical sensing system from laboratory research into point-of-care diagnostics with multiple microelectrodes.

The highly specific recognition processes between biomolecules mediate various crucial biological processes. Uncovering the molecular basis of these interactions is of great fundamental and applied importance. This research work focuses on understanding the interactions of several biomolecular recognition systems and processes that can provide fundamental information to aid in the rational design of sensing and molecular recognition tools. Initially, a reliable and versatile platform was developed to investigate biomolecular interactions at a molecular level. This involved several techniques, including biomolecule functionalization to enable attachment to self-assembled monolayers as well as atomic force microscopy (AFM) based force spectroscopy to uncover the binding or rupture forces between the receptor and ligand pairs. It was shown that this platform allowed determination of molecular binding between single molecules with a high specificity. The platform was further adapted to a general sensing formulation utilizing a group of flexible and adaptive nucleic acid recognition elements (RNA and DNA aptamers) to detect specific target proteins. Investigation of interactions at the molecular level allowed characterization of the dynamics, specificity and the conformational properties of these functional nucleic acids in a manner inaccessible via traditional interaction studies. These interactions were then adapted to aptamer-based detecting methods that at the ensemble or bulk scale, specifically taking advantage of mechanisms uncovered in the biophysical study of this system. A quartz crystal microbalance (QCM) was used to detect protein targets at the bulk level and the affinities and binding kinetics of these systems were analyzed. Along with AFM-based force spectroscopy, ensemble-averaging properties and molecular properties of these interactions could be correlated to contribute to bridging the gap across length scales. Finally, more broadly applicable sensing platform was developed to take advantage of the unique properties of aptamers. DNA was employed both as a carrier and as a molecular recognition agent. DNA was used as a template for nanoconstruction and fabricating unique shapes that could enhance the aptamer-based molecular recognition strategies. With aptamers tagged to distinct nanoconstructed DNA, a novel shape-based detecting method was enabled at the molecular level. The results demonstrated that this is a flexible strategy, which can be further developed as ultrasensitive single molecule sensing strategy in complex environments.

This dissertation is aimed towards using stimuli-responsive pNIPAm-co-AAc microgels synthesized via free-radical precipitation polymerization to prepare stimuli-responsive hydrogel microlenses. Chapter 1 gives a detailed background of hydrogels, and their applications using responsive hydrogels. Chapter 2 describes the use of colloidal hydrogel microparticles as microlens elements and the fabrication method to form the hydrogel microlens arrays via Coulombic interactions. Chapter 3 shows the demonstration of tunable microlenses prepared by the method used in Chapter 2. In this chapter the microlenses are subjected to various pH and temperature in aqueous solutions. Chapter 4 describes that the microlens arrays constructed on Au nanoparticle-functionalized glass substrates by self-assembly display dramatic changes in lensing power in response to an impingent frequency-doubled Nd:YAG laser. The microlens photoswitching is highly reversible, with sub-millisecond lens switching times. Chapter 5 describes the development of bioresponsive hydrogel microlenses as a new protein detection technology. The microlens method is shown to be very specific for the target protein, with no detectable interference from nonspecific protein binding. Chapter 6 describes the use of bioresponsive hydrogel microlenses as a label-free biosensing scaffolding. These microstructures simultaneously act as the biosensors scaffolding/immobilization architecture, transducer, amplifier, and also allow for broad tunability of the analyte concentration to which the microlens is sensitive.

Graphene is a single layer of carbon atoms, laid out in a hexagonal lattice. The material has remarkable properties that opened up several new research areas since its discovery in 2004. One promising field is graphene based biosensors, where researchers hope to create new devices that are smaller, cheaper and more reliable than those based on today’s technology. Among several manufacturing methods, graphene grown on silicon carbide is one of the promising ones for biosensing. A chip design has been developed in order to support research into graphene on silicon carbide as a base material for biosensors. Along with the chip, a holder for electrochemical measurements has been designed and an investigation into the requirements of a custom measurement device for the sensor has been undertaken.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [86]-91).

This work describes the development of mediated amperometric biosensors that are able to monitor the metabolic activity of both single and mixed microbial populations, with applications in toxicity assessment and wastewater treatment plant protection. Biosensor systems have been constructed incorporating either the single-species eubacteria Escherichia coli or Pseudomonas putida, Bioseed®, or a mixture of activated sludge organisms from wastewater treatment plants, as the sensing components immobilised on disposable screen printed electrodes in stirred reaction vials. The biosensor approach is generic allowing for a wide range of microbial cell types to be employed. Appropriate bacterial species can be selected for specific sensor applications in order to confer validity and relevance to the test, hence the biosensor can be tailor-made to assess the toxicity in a particular environment and provide diagnostically valid and relevant results. The biosensors have been used to assess the toxicity of a standard toxicant and toxicant formulations and in blind testing of a range of industrial effluents, in parallel with a number of bioassays including Microtox® and activated sludge respiration inhibition. The biosensor results generally show significant correlation to the appropriate conventional toxicity tests. In this study, an activated sludge based biosensor assay was developed and used to assess the toxicity of industrial process and site effluents with the specific purpose of wastewater treatment plant protection. Data generated compared significantly with those from an activated sludge respiration inhibition test, with added advantages of rapidity, safety and ease of use.

This thesis describes the development of biosensing systems incorporating eukaryotic cells. The ultimate objective of this work was to design devices capable of rapidly assessing the toxicity of effluents and environmental pollutants. Although much work remains to be done in order to achieve this goal, the work reported here demonstrates, in principle, the approaches adopted. The first approach exploited the reducing nature of healthy biological cells. So called 'redox mediated whole cell biosensors' have been described before. In this work, an algal toxicity test of short duration was developed and sensors incorporating cultured fish cells were described for the first time. The sensitivity of biosensors incorporating the green alga Selenastrum capricornutum, to diuron and pentachlorophenol, was found to compare favourably with that from other standard ecotoxicological tests. However, although the sensitivity of biosensors incorporating immobilised BF-2 fish cells was found to compare well with that of other fish cell-based toxicity tests, it appeared that whole organism tests were much more sensitive to the compounlds tested. The second approach involved the genetic manipulation of fish cells in order to incorporate luminescent reporter genes. Although this work is less well advanced, it demonstrates that the luc reporter gene can be successfully inserted into BF -2 fish cells and that these transformed cells can produce a luminescent response when incubated with luciferin substrate. Preliminary investigations have indicated that the sensitivity of luc-transformed BF-2 cells to 4-chlorophenol is comparable to that of some standard whole organism ecotoxicological tests and although much work is still required to validate this approach, it could eventually provide a simple, sensitive and rapid route to toxicity assessment.

Thesis (Ph.D.)--Boston University / The large electromagnetic field enhancement provided by nanostructured noble metal surfaces forms the foundation for a series of enabling optical analytical techniques, such as surface enhanced Raman spectroscopy (SERS), surface enhanced IR absorption spectroscopy (SEIRA), surface enhanced fluorescent microscopy (SEF), to name only a few. Critical sensing applications have, however, other substrate requirements than mere peak signal enhancement. The substrate needs to be reliable, provide reproducible signal enhancements, and be amenable to a combination with microfluidic chips or other integrated sensor platforms. These needs motivate the development of engineerable SERS substrate "chips" with defined near- and far-field responses. In this dissertation, two types of rationally designed SERS substrates - nanoparticle cluster arrays (NCAs) and SERS stamp - will be introduced and characterized. NCAs were fabricated through a newly developed template guided self-assembly fabrication approach, in which chemically synthesized nanoparticles are integrated into predefined patterns using a hybrid top-down/bottom-up approach. Since this method relies on chemically defined building blocks, it can overcome the resolution limit of conventional lithographical methods and facilitates higher structural complexity. NCAs sustain near-field interactions within individual clusters as well as between entire neighboring clusters and create a multi-scale cascaded E-field enhancement throughout the entire array. SERS stamps were generated using an oblique angle metal deposition on a lithographically defined piston. When mounted on a nanopositioning stage, the SERS stamps were enabled to contact biological surfaces with pristine nanostructured metal surfaces for a label-free spectroscopic characterization. The developed engineered substrates were applied and tested in critical sensing applications, including the ultratrace detection of explosive vapors, the rapid discrimination of bacterial pathogens, and the label-free monitoring of the enzymatic degradation of pericellular matrices of cancer cells.

Solid-state nanopores are an emerging class of single-molecule detectors that provide information about molecular identity via the analysis of transient fluctuations in the ionic current flowing across a nanoscale pore in a thin membrane. The transport of biomolecules across a pore is a key step in nanopore-based sensing of DNA, RNA and proteins. The dynamics of biomolecular transport are complex and depend on the strength of many interactions, which can be tuned with temperature. However, temperature is rarely controlled during solid-state nanopore experiments because of the added electrical noise from the temperature control and measurement systems, greatly reducing the signal-to-noise ratio when detecting individual molecules. So far, the use of electric-based heating and cooling strategies has limited the recording bandwidth to the kHz range, restricting the studies to long polymers translocating via the pore relatively slowly. Yet, many molecules translocate through the pore orders of magnitude faster. This research presents the development and testing of an instrument to allow low-noise electrical recording of nanopore signals at MHz bandwidth as a function of temperature. Initial experiments using this custom-built instrument for the study of linear DNA polymers confirm previously observed translocation behaviours, while providing a higher temporal resolution. Overall results show that high-speed nanopore experiments are possible while controlling the temperature up to 70 °C, opening up exciting opportunities to study the unfolding of proteins toward single-molecule protein sequencing and the passage of DNA nanostructures for different bioassays. Future work will focus on realizing microfluidic flow cells and nanopore performance at higher temperature for longer recording times.

No description available.