Design and Optimization Methods for Pin-Limited and Cyberphysical Digital Microfluidic Biochips

<p>Microfluidic biochips have now come of age, with applications to biomolecular recognition for high-throughput DNA sequencing, immunoassays, and point-of-care clinical diagnostics. In particular, digital microfluidic biochips, which use electrowetting-on-dielectric to manipulate discrete droplets (or "packets of biochemical payload") of picoliter volumes under clock control, are especially promising. The potential applications of biochips include real-time analysis for biochemical reagents, clinical diagnostics, flash chemistry, and on-chip DNA sequencing. The ease of reconfigurability and software-based control in digital microfluidics has motivated research on various aspects of automated chip design and optimization.</p><p>This thesis research is focused on facilitating advances in on-chip bioassays, enhancing the automated use of digital microfluidic biochips, and developing an "intelligent" microfluidic system that has the capability of making on-line re-synthesis while a bioassay is being executed. This thesis includes the concept of a "cyberphysical microfluidic biochip" based on the digital microfluidics hardware platform and on-chip sensing technique. In such a biochip, the control software, on-chip sensing, and the microfluidic operations are tightly coupled. The status of the droplets is dynamically monitored by on-chip sensors. If an error is detected, the control software performs dynamic re-synthesis procedure and error recovery.</p><p>In order to minimize the size and cost of the system, a hardware-assisted error-recovery method, which relies on an error dictionary for rapid error recovery, is also presented. The error-recovery procedure is controlled by a finite-state-machine implemented on a field-programmable gate array (FPGA) instead of a software running on a separate computer. Each state of the FSM represents a possible error that may occur on the biochip; for each of these errors, the corresponding sequence of error-recovery signals is stored inside the memory of the FPGA before the bioassay is conducted. When an error occurs, the FSM transitions from one state to another, and the corresponding control signals are updated. Therefore, by using inexpensive FPGA, a portable cyberphysical system can be implemented.</p><p>In addition to errors in fluid-handling operations, bioassay outcomes can also be erroneous due the uncertainty in the completion time for fluidic operations. Due to the inherent randomness of biochemical reactions, the time required to complete each step of the bioassay is a random variable. To address this issue, a new "operation-interdependence-aware" synthesis algorithm is proposed in this thesis. The start and stop time of each operation are dynamically determined based on feedback from the on-chip sensors. Unlike previous synthesis algorithms that execute bioassays based on pre-determined start and end times of each operation, the proposed method facilitates "self-adaptive" bioassays on cyberphysical microfluidic biochips.</p><p>Another design problem addressed in this thesis is the development of a layout-design algorithm that can minimize the interference between devices on a biochip. A probabilistic model for the polymerase chain reaction (PCR) has been developed; based on the model, the control software can make on-line decisions regarding the number of thermal cycles that must be performed during PCR. Therefore, PCR can be controlled more precisely using cyberphysical integration.</p><p>To reduce the fabrication cost of biochips, yet maintain application flexibility, the concept of a "general-purpose pin-limited biochip" is proposed. Using a graph model for pin-assignment, we develop the theoretical basis and a heuristic algorithm to generate optimized pin-assignment configurations. The associated scheduling algorithm for on-chip biochemistry synthesis has also been developed. Based on the theoretical framework, a complete design flow for pin-limited cyberphysical microfluidic biochips is presented.</p><p>In summary, this thesis research has led to an algorithmic infrastructure and optimization tools for cyberphysical system design and technology demonstrations. The results of this thesis research are expected to enable the hardware/software co-design of a new class of digital microfluidic biochips with tight coupling between microfluidics, sensors, and control software.</p>Dissertatio

Development of an acousto-electric biochemical sensor (AEBS) for monitoring biological and chemical processes

Ph.D., Electrical Engineering -- Drexel University, 200

Portable “lab-on-chip” platform for bovine mastitis diagnosis in raw milk

Tese de Doutoramento em Ciências Veterinárias, Especialidade de Ciências Biológicas e BiomédicasBovine mastitis is an economic burden for farmers mostly because of decreased milk yield, premature culling and cost of veterinary treatments. The identification of mastitis pathogens is of major importance in order for adequate control measures to be taken, to reduce the risk of appearance of chronic infections, and to target antimicrobial therapy. The aim of this study was to develop and validate a sensitive method for magnetic detection of Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus epidermidis in raw milk samples. Mastitic milk samples were collected aseptically from 81 cows with subclinical mastitis, from 12 Portuguese dairy farms. Ninety one quarter milk samples were selected based on bacteriological results. All samples were submitted to PCR analysis. In parallel, these milk samples were mixed with a solution combining specific antibodies and magnetic nanoparticles, to be analyzed using a lab-on-a-chip magnetoresistive cytometer, with microfluidics sample handling. This immunological recognition was able to detect bacterial presence above 100 cfu/ ml, depending on antibody and targeted bacteria. Comparison with PCR results showed sensitivities of 73% and 41%, specificity values of 25% and 57%, and PPV values of 35% and 54% for magnetic identification of streptococci species with an anti-S. agalactiae antibody and an anti-GB Streptococcus antibody, respectively. Regarding staphylococci species, the sensitivity values found were of 57.1% and 79.3%, specificities of 75% and 50%, and PPV values of 40% and 95.8% for magnetic identification with an anti-S. aureus antibody and an anti-Staphylococcus spp. antibody, respectively. Both bacterial genus studies translated a fair expectation for a “cow-side” use application, making this integrated platform of potential use after further improvements for fast bacteriological infection screening. Some constraints are described as well as the method´s limitations in bacterial quantification.RESUMO - Plataforma portátil “lab-on-chip” para diagnosticar mastite bovina em leite crú - A mastite bovina representa um custo económico relevante para os produtores de leite principalmente devido ao decréscimo da produção leiteira, abate prematuro e custos associados ao tratamento veterinário. Consequentemente, a identificação atempada dos agentes etiológicos é crítica para a implementação de medidas de controlo adequadas, redução do risco de infecções crónicas e aplicação de uma terapia microbiana específica. O objectivo deste estudo foi desenvolver e validar um método de detecção magnética capaz de identificar Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus e Staphylococcus epidermidis em amostras de leite crú. As amostras de leite mastítico utilizadas foram recolhidas de 81 animais com mastite subclínica, de 12 explorações leiteiras nacionais. As amostras de leite de 91 quartos de úbere foram selecionadas tendo em conta os resultados bacteriológicos. Todas as amostras foram analisadas por PCR e pelo citómetro magnetoresistivo “lab-on-chip”, tendo sido necessário neste caso, adicionar uma solução com partículas magnéticas funcionalizadas com anticorpos específicos. Este reconhecimento imunológico detectou presença bacteriana acima das 100 ufc/ml, dependendo do anticorpo e da bactéria-alvo. Comparando com os resultados da análise por PCR, este método de detecção magnética apresentou sensibilidades de 73% e 41%, valores de especificidade de 25% e 57%, e valores VPP de 35% e 54% para identificar espécies de Streptococcus com os anticorpos anti-S. agalactiae e anti-GB Streptococcus, respectivamente. No que diz respeito às espécies de Staphylococcus, os valores de sensibilidade encontrados foram de 57.1% e 79.3%, de 75% e 50% para a especificidade, e de 40% e 95.8% para VPP com os anticorpos anti-S. aureus e anti-Staphylococcus spp., respectivamente. Os dois estudos apontam para uma potencial utilização do tipo “cow-side”, tornando a plataforma integrada potencialmente utilizável para uma rápida monitorização de infecção bacteriológica, após melhorias futuras. O método desenvolvido apresenta algumas restrições e limitações relativamente à quantificação bacteriana

A modular multi electrode array system for electrogenic cell characterisation and cardiotoxicity applications

Multi electrode array (MEA) systems have evolved from custom-made experimental tools, exploited for neural research, into commercially available systems that are used throughout non-invasive electrophysiological study. MEA systems are used in conjunction with cells and tissues from a number of differing organisms (e.g. mice, monkeys, chickens, plants). The development of MEA systems has been incremental over the past 30 years due to constantly changing specific bioscientific requirements in research. As the application of MEA systems continues to diversify contemporary commercial systems are requiring increased levels of sophistication and greater throughput capabilities. [Continues.

Single Cell analysis using AtomicForce Microscopy (AFM)

Replication of biological cells for the purpose of imaging and analysis under electron and scanning probe microscopy has facilitated the opportunity to study and examine some molecular processes and structures of living cells in a manner that were not possible before. The difficulties faced in direct cellular analysis when using and operating Atomic Force Microscopy (AFM) in situ for morphological studies of biological cells have led to the development of a novel method for biological cell studies based on nanoimprint lithography. The realization of the full potential of high resolution AFM imaging has revealed some very important biological events such as exocytosis and endocytosis. In this work, a soft lithography Bioimprint replication technique, which involved simple fabrication steps, was used to form a hard replica of the cell employing a newly developed biocompatible polymer that has fast curing time at room temperature essential for this process. The structure and topography of the rat muscle cell and the endometrial (Ishikawa) cancer cell were investigated in this study. Cells were cultured and incubated in accordance with standard biological culturing procedures and protocols approved by the Human Ethics Committee, University of Otago. An impression of the cell profile was created by applying a layer of the polymer onto the cells attached to a substrate and rapidly cured under UV-light. Fast UV radiation helps to lock cellular processes within seconds after exposure and replicas of the cancer cells exhibit ultra-cellular structures and features down to nanometer scale. Elimination of the AFM tip damping effects due to probing of the soft biological tissue allows imaging with unprecedented resolution. Highxx resolution AFM imagery provides the opportunity to examine the structure and topography of the cells closely so that any abnormalities can be identified. Craters that resemble granules and features down to 100 nm were observed. These represent steps on a transitional series of sequential structures that indicate either an endocytotic or exocytotic processes, which were evident on the replicas. These events, together with exocytosis, play a very significant part in the tumorigenesis of these cancer cells. By forming cell replica impressions, not only have they the potential to understand biological cell conditions, but may also benefit in synthesizing three dimensional (3-D) scaffolds for natural growth of biological cells and providing an improvement over standard cell growth conditions. Further examinations by observing the characteristic behaviour of the plasma membrane when the cells were induced by certain compound such as cobalt chloride (CoCl2) under control and stimulated conditions have brought in the opportunity to examine the effect of this stimulant in inducing apoptosis in many different kinds of cells. Numbers of pores formed on the cells membrane were found to increase significantly after the cells where induced with CoCl2 that correlated well with the level of vascular endothelial growth factor (VEGF) receptors expression, which contributed to tumour growth. This indicates CoCl2 has exaggerated the expression of the VEGF growth factor. Investigations were also done to the cells using functionalized nanoparticles as bio-markers to establish the connection between exocytosis with nanopores found on the membrane surfaces of the cells. These microbeads were found attached to sites surrounding the nucleus of the cell and higher numbers of visible beads would confirm that there was an up-regulation of the VEGF expression in cells induced by CoCl2. All these can contribute to expanding the knowledge about exocytosis and fundamental physiology of cells, and also assist in understanding diseases especially cancer

Design and development of a microfluidic platform for use with colorimetric gold nanoprobe assays

Due to the importance and wide applications of the DNA analysis, there is a need to make genetic analysis more available and more affordable. As such, the aim of this PhD thesis is to optimize a colorimetric DNA biosensor based on gold nanoprobes developed in CEMOP by reducing its price and the needed volume of solution without compromising the device sensitivity and reliability, towards the point of care use. Firstly, the price of the biosensor was decreased by replacing the silicon photodetector by a low cost, solution processed TiO2 photodetector. To further reduce the photodetector price, a novel fabrication method was developed: a cost-effective inkjet printing technology that enabled to increase TiO2 surface area. Secondly, the DNA biosensor was optimized by means of microfluidics that offer advantages of miniaturization, much lower sample/reagents consumption, enhanced system performance and functionality by integrating different components. In the developed microfluidic platform, the optical path length was extended by detecting along the channel and the light was transmitted by optical fibres enabling to guide the light very close to the analysed solution. Microfluidic chip of high aspect ratio (~13), smooth and nearly vertical sidewalls was fabricated in PDMS using a SU-8 mould for patterning. The platform coupled to the gold nanoprobe assay enabled detection of Mycobacterium tuberculosis using 3 8l on DNA solution, i.e. 20 times less than in the previous state-of-the-art. Subsequently, the bio-microfluidic platform was optimized in terms of cost, electrical signal processing and sensitivity to colour variation, yielding 160% improvement of colorimetric AuNPs analysis. Planar microlenses were incorporated to converge light into the sample and then to the output fibre core increasing 6 times the signal-to-losses ratio. The optimized platform enabled detection of single nucleotide polymorphism related with obesity risk (FTO) using target DNA concentration below the limit of detection of the conventionally used microplate reader (i.e. 15 ng/μl) with 10 times lower solution volume (3 μl). The combination of the unique optical properties of gold nanoprobes with microfluidic platform resulted in sensitive and accurate sensor for single nucleotide polymorphism detection operating using small volumes of solutions and without the need for substrate functionalization or sophisticated instrumentation. Simultaneously, to enable on chip reagents mixing, a PDMS micromixer was developed and optimized for the highest efficiency, low pressure drop and short mixing length. The optimized device shows 80% of mixing efficiency at Re = 0.1 in 2.5 mm long mixer with the pressure drop of 6 Pa, satisfying requirements for the application in the microfluidic platform for DNA analysis.Portuguese Science Foundation - (SFRH/BD/44258/2008), “SMART-EC” projec

Microfluidics for Biosensing

There are 12 papers published with 8 research articles, 3 review articles and 1 perspective. The topics cover: Biomedical microfluidics Lab-on-a-chip Miniaturized systems for chemistry and life science (MicroTAS) Biosensor development and characteristics Imaging and other detection technologies Imaging and signal processing Point-of-care testing microdevices Food and water quality testing and control We hope this collection could promote the development of microfluidics and point-of-care testing (POCT) devices for biosensing