Transport or Store? Synthesizing Flow-based Microfluidic Biochips using Distributed Channel Storage

Flow-based microfluidic biochips have attracted much atten- tion in the EDA community due to their miniaturized size and execution efficiency. Previous research, however, still follows the traditional computing model with a dedicated storage unit, which actually becomes a bottleneck of the performance of bio- chips. In this paper, we propose the first architectural synthe- sis framework considering distributed storage constructed tem- porarily from transportation channels to cache fluid samples. Since distributed storage can be accessed more efficiently than a dedicated storage unit and channels can switch between the roles of transportation and storage easily, biochips with this dis- tributed computing architecture can achieve a higher execution efficiency even with fewer resources. Experimental results con- firm that the execution efficiency of a bioassay can be improved by up to 28% while the number of valves in the biochip can be reduced effectively.Comment: ACM/IEEE Design Automation Conference (DAC), June 201

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

Fluigi: an end-to-end software workflow for microfluidic design

One goal of synthetic biology is to design and build genetic circuits in living cells for a range of applications with implications in health, materials, and sensing. Computational design methodologies allow for increased performance and reliability of these circuits. Major challenges that remain include increasing the scalability and robustness of engineered biological systems and streamlining and automating the synthetic biology workflow of “specify-design-build-test.” I summarize the advances in microfluidic technology, particularly microfluidic large scale integration, that can be used to address the challenges facing each step of the synthetic biology workflow for genetic circuits. Microfluidic technologies allow precise control over the flow of biological content within microscale devices, and thus may provide more reliable and scalable construction of synthetic biological systems. However, adoption of microfluidics for synthetic biology has been slow due to the expert knowledge and equipment needed to fabricate and control devices. I present an end-to-end workflow for a computer-aided-design (CAD) tool, Fluigi, for designing microfluidic devices and for integrating biological Boolean genetic circuits with microfluidics. The workflow starts with a ``netlist" input describing the connectivity of microfluidic device to be designed, and proceeds through placement, routing, and design rule checking in a process analogous to electronic computer aided design (CAD). The output is an image of the device for printing as a mask for photolithography or for computer numerical control (CNC) machining. I also introduced a second workflow to allocate biological circuits to microfluidic devices and to generate the valve control scheme to enable biological computation on the device. I used the CAD workflow to generate 15 designs including gradient generators, rotary pumps, and devices for housing biological circuits. I fabricated two designs, a gradient generator with CNC machining and a device for computing a biological XOR function with multilayer soft lithography, and verified their functions with dye. My efforts here show a first end-to-end demonstration of an extensible and foundational microfluidic CAD tool from design concept to fabricated device. This work provides a platform that when completed will automatically synthesize high level functional and performance specifications into fully realized microfluidic hardware, control software, and synthetic biological wetware

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

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

Novel approaches to the low-cost, portable and rapid detection of bacterial pathogens in foods and food-processing environments

Continued outbreaks of foodborne illness involving dairy products in the United States stress the importance for rapid methods of detection of pathogenic microorganisms in food processing environments. Pathogenic microorganisms, such as Salmonella are widespread, and can be found in a variety of foods, ingredients and in industrial environments. The presence of pathogens in dairy products constitutes great risk for increased exposure, illness and reduces overall quality of the foodstream. As a result, emphasis has been placed on adapting or developing sensitive techniques to rapidly detect notable pathogens, such as Salmonella, Listeria monocytogenes and Escherichia coli O157:H7 in both contaminated foods and industrial environments. Common assays employed in the detection of pathogenic microorganisms, though effective in identification, are time consuming and may require several days for processing. The necessity to quickly screen food products and industrial environments has led to an emphasis to develop rapid, sensitive, automated techniques in food processing operations. Numerous methods of identification and detection have been implemented in food processing environments. An optimal approach to the rapid detection of microbial pathogens would incorporate several advantages including: 1) improved time-to-result, 2) low-cost, 3) ease of operation and 4) simple interpretation. Such an approach may enable simple and cost-effective sampling of pathogenic microorganisms, which can be used to improve industrial efficiency. As a possible alternative to existing detection efforts, low-cost diagnostic (LCD) tools, particularly paper-based analytical devices (PADs), may be employed for rapid, sensitive and selective detection. PADs are frequently combined with colorimetric detection, in which chromogenic substrates are used to yield a visual representation of detection. Different enzyme-substrate pairs may be employed to accomplish various goals—from simple “presence/absence” to species-specificity. While “presence/absence” is limited, the use of shared enzymes is advantageous during detection and identification of metabolic state. Depending upon environmental factors, bacteria may exist in active or dormant states; reversion of a pathogen from dormancy to a metabolically active state may result in rapid growth and instances of illness. As the level of enzymatic expression varies between metabolic states, oxidoreductases and alkaline phosphatases (ALP) were investigated as vehicles for colorimetric detection. Oxidoreductases are present in greater amounts in metabolically active bacteria, and are capable of reducing 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) to formazans. Nitrophenyl phosphate (PNPP) is present in dormant bacteria, and cleaves phosphate groups from para-nitrophenyl-phosphate salts, resulting in para-nitrophenol. Combined use of enzymatic substrates, including INT and 5-methylphenazin-5-ium methyl sulfate (PMS) for metabolically active bacteria, and INT and PNPP for dormant bacteria, yielded an improved colorimetric readout visible by eye within 30 min. With detection achieved within 30 min, the two assays, INT-PMS and INT-PNPP, decrease time-to-result, are portable and may be amenable to on-site detection in agricultural, environmental and industrial settings. While the use of non-specific bacterial enzymes may limit some applications, immobilization of bacteria-specific bacteriophage (P22, T4) onto paper can provide an additional layer of specificity. Bacteriophage are robust, and may be easily absorbed onto paper. In this work, immobilized bacteriophage facilitated specific capture of Salmonella Typhimurium on paper, followed by detection of metabolic state with either the INT-PMS or INT-PNPP assay. This combined approach can be applied to the analysis of mixed cultures, given the generally genera-specific nature of the selected bacteriophages. Moreover, the use of chromogenic substrates simplifies assay design, as color change is easily interpreted by the eye or with basic instrumentation. However, despite these advantages, the requirement for a 48-hour absorption period represents a drawback, lengthening time-to-result. An alternative to the use of bacteriophage for cell capture are magnetic ionic liquids (MILs). MILs are magnetoactive “molten salt” solvents, containing a paramagnetic component integrated into the cation or anion moiety of the salt. MILs are considered “green” solvents, and are nonvolatile, nonflammable, with tunable physicochemical properties. Due to their hydrophobic and liquid nature, MILs can be quickly be distributed with agitation (stirring or vortexing) throughout aqueous food samples as liquid micro- or nanodispersions. After encountering and binding bacterial cells, cell-MIL complexes can then be collected magnetically or after density-driven sedimentation for further processing. MIL-based capture of bacteria has been previously combined with real-time polymerase chain reaction (qPCR) for the rapid detection of E. coli. While use of qPCR obviates the need for time-consuming steps such as gel electrophoresis, its inherent complexity and cost may prohibit its use in point-of-care or resource-limited settings. Isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), may have considerable advantages as alternatives to PCR. RPA results in exponential amplification of nucleic acids and operates at a constant, near-physiological temperature (~40°C), eliminating the need for a thermocycler, generating target-specific amplicons in less than 20 min. The combined use of MIL-based extraction and rapid, streamlined pathogen detection using RPA was investigated. The ability of MIL solvents to quickly extract Salmonella Typhimurium was first examined by dispersing MIL into an aqueous suspension, followed by rapid (~30 s) physical enrichment (concentration) and extraction using an applied magnetic field. Following extraction, viable bacteria were desorbed from the MIL extraction phase with exposure to a nutrient-rich broth (Luria Bertani medium), referred here to as a “back-extraction” step. In efforts to improve back-extraction, recovery of the model Gram-negative bacterium Serratia marcescens from the MIL extraction phase was investigated using several back-extraction media varying in ionic strength and nutrient composition. The highest recovery of cells was obtained using a nutrient-rich tryptone medium supplemented with NaCl. This modification of the extraction protocol enabled improvement in MIL-based bacterial concentration, enriching cells by a factor of 5 - 6X within 3–5 min. The improved MIL assay was then examined in conjunction with RPA for rapid detection of Salmonella Typhimurium. MIL-based sample preparation was compared with use of a commercial sample preparation solution, PrepMan® Ultra Sample Preparation Reagent (PMU), for detection of Salmonella Typhimurium in artificially-contaminated pasteurized foods. PMU is commonly coupled with PCR to eliminate or inactivate PCR inhibitors and uses both heating and centrifugation steps. As an established method for sample preparation, use of PMU served as a benchmark method against which our MIL-based process was compared. In aqueous suspensions of Salmonella Typhimurium, detection was achieved as low as 103 CFU mL-1 using the combined MIL-RPA approach, which is equivalent to the previously investigated MIL-qPCR method, and, in our hands, outperformed the PMU method by an order of magnitude. Visualization of amplified products was achieved using gel electrophoresis or lateral flow readouts. Nucleic acid lateral flow immunoassays (NALFIA) require less than 5 min for amplicon visualization, are portable, require minimal technical expertise during interpretation and are easy to implement outside of laboratory settings. The need for electric-based heating elements for RPA incubation was eliminated through the use of low-cost, portable, supersaturated sodium acetate heat packs. This repurposing of consumer-grade hand warmers for nucleic acid amplification is a novel approach and easily incorporated into the MIL-RPA scheme. While MILs have been successfully used for capture and concentration of bacteria from foods prior to culture- or nucleic acid-based detection, little is known about their interactions with bacteria—including modes of physical association or potential antimicrobial activities. Further understanding these interactions may facilitate optimization of MIL-based capture in challenging food matrices, as well as modification of downstream procedures to mitigate the impacts of potential bacterial injury during extraction and concentration. To begin this work, a series of multi-strain panels, including seven representative Salmonella DNA subgroups and eight strains of E. coli O157:H7, were exposed to the Ni(II) MIL and plated in parallel on non-selective and selective media. Calculated enrichment factors (EF) were similar between media types, while individual cell counts were nearly identical, suggesting that the Ni(II) MIL, as applied during our capture and concentration assay, does not cause assay-limiting cellular injury in these two pathogens. Observed variability between EF values may result from differences in the extraction efficiency of the MIL, with some strains exhibiting weaker affinity for the MIL compared to other strains tested, which is an area of ongoing research. Importantly, our results demonstrate capture and recovery of strains representative of all seven Salmonella DNA subgroups and all eight strains of E. coli O157:H7 tested, with comparable recovery on non-selective and selective media. This initial and ongoing research on characterization of MIL-bacterial interactions establishes the foundation for further evaluation of new MIL structures for improving the preconcentration and recovery of viable microorganisms from complex food matrices

Laser-Based Nano Fabrication and Nano Lithography

The improvement of fabrication resolutions is an eternal challenge for miniaturizing and enhancing the integration degrees of devices. Laser processing is one of the most widely used techniques in manufacturing due to its high flexibility, high speed, and environmental friendliness. The fabrication resolution of laser processing is, however, limited by the diffraction limit. Recently, much effort has been made to overcome the diffraction limit in nano fabrication. Specifically, combinations of multiphoton absorption by ultrafast lasers and the threshold effect associated with a Gaussian beam profile provide fabrication resolutions far beyond the diffraction limit. The use of the optical near-field achieves nano ablation with feature sizes below 100 nm. Multiple pulse irradiation from the linearly polarized ultrafast laser produces periodic nanostructures with a spatial period much smaller than the wavelength. Unlimited diffraction resolutions can also be achieved with shaped laser beams. In the meanwhile, lasers are also widely used for the synthesis of nano materials including fullerenes and nano particles. In view of the rapid advancement of this field in recent years, this Special Issue aims to introduce the state-of-the-art in nano fabrication and nano lithography, based on laser technologies, by leading groups in the field

Engineering single-molecule, nanoscale, and microscale bio-functional materials via click chemistry

To expand the design envelope and supplement the materials library available to biomaterials scientists, the copper(I)-catalyzed azide-alkyne cycloaddition (CuCAAC) was explored as a route to design, synthesize and characterize bio-functional small-molecules, nanoparticles, and microfibers. In each engineered system, the use of click chemistry provided facile, bio-orthogonal control for materials synthesis; moreover, the results provided a methodology and more complete, fundamental understanding of the use of click chemistry as a tool for the synergy of biotechnology, polymer and materials science. Fluorophores with well-defined photophysical characteristics (ranging from UV to NIR fluorescence) were used as building blocks for small-molecule, fluorescent biosensors. Fluorophores were paired to exhibit fluorescence resonant energy transfer (FRET) and used to probe the metabolic activity of carbazole 1,9a-dioxygenase (CARDO). The FRET pair exhibited a signicant variation in PL response with exposure to the lysate of Pseudomonas resinovorans CA10, an organism which can degrade variants of both the donor and acceptor fluorophores. Nanoparticle systems were modified via CuCAAC chemistry to carry affinity tags for CARDO and were subsequently utilized for affinity based bioseparation of CARDO from crude cell lysate. The enzymes were baited with an azide-modified carbazolyl-moiety attached to a poly(propargyl acrylate) nanoparticle. Magnetic nanocluster systems were also modified via CuCAAC chemistry to carry fluorescent imaging tags. The iron-oxide nanoclusters were coated with poly(acrylic acid-co-propargyl acrylate) to provide a clickable surface. Ultimately, alternate Cu-free click chemistries were utilized to produce biohybrid microfibers. The biohybrid microfibers were synthesized under benign photopolymerization conditions inside a microchannel, allowing the encapsulation of viable bacteria. By adjusting pre-polymer solutions and laminar flow rates within the microchannel, the morphology, hydration, and thermal properties of the fibers were easily tuned. The methodology produced hydrogel fibers that sustained viable cells as demonstrated by the encapsulation and subsequent proliferation of Bacillus cereus and Escherichia coli communities