Poly-L-histidine coated microfluidic devices for bacterial DNA purification without chaotropic solutions.

Sequential fluid delivery with minimized external equipment is vital towards a point-of-care diagnostic device. In this work, we have further developed the On-chip Electrowetting Valves concept for the sequential delivery of the reagents to the reaction site in a miniaturized capillary-driven microfluidic chip. Specifically, a disposable polymeric microfluidic device was developed containing capillary force driven microchannels. The device was fabricated using laser ablation and inkjet printing and required no external pumping equipment. The assay was conducted on the microchip containing microfluidic channels with embedded electrowetting valves and a porous membrane patterned with capture molecules and colloidal gold labels. To conduct the assay, the microchip was connected with a low voltage supply which was capable of sequentially opening the valves, delivering the sample and the rinsing reagent to generate visual results. Using T7 bacteriophage as a model, we have demonstrated the development of the device, operation of the valves and execution of the automated assay.

High-throughput fabrication of graphically encoded hydrogel microparticles is achieved by combining flow contact lithography in a multichannel microfluidic device and a high capacity 25 mm LED UV source. Production rates of chemically homogeneous particles are improved by two orders of magnitude. Additionally, the custom-built contact lithography instrument provides an affordable solution for patterning complex microstructures on surfaces.

On-site extraction and purification of bacterial nucleic acids from blood samples using an unpowered microfluidic device

One-step DNA purification and amplification on an integrated plastic microdevice for on-site identification of foodborne pathogens

This paper describes paper-based micro fluidic devices that can be programmed by a user to implement special micro fluidic analysis. The micro fluidic devices are simple to fabricate, and are made out of paper and tape. These devices are programmed by pressing paper-based micro fluidic switches. Pressing a paper-based micro fluidic switch closes a small gap between paper-based micro fluidic channels, and allows fluids to wick from one channel to the other. A fluidic de-multiplexer and a programmable micro fluidic array have been implemented using programmable paper-based micro fluidic device, and programmable operation experiments have also been done using red dye solution and NaNO2 solution. The programmable paper-based micro fluidic devices provide a simple and low cost method for controlling the movement of fluids in paper-based channels, and expand the applications of micro fluidic devices.

The aim of this PhD project is to investigate alternative sensing methodologies that can possibly improve the sensing performances of lab-on-chip (LOC) designed for biochemical applications. Suspended microchannel resonator (SMR) for bio-mechanical sensing applications have become very popular as detection of weigths of chemicals integrated in LOC. They exploit laser doppler vibrometry (LDV) for dynamic mode detection. In this thesis two different SMRs designs have been investigated, involving either technological challenges - the use of polymers as material and processing techniques based on laser micromachining - and different sensing phenomena - the use of the parametric resonance rather than the standard harmonic resonance response. The flexibility of two-photon direct laser writing is exploited to optimize a highly- versatile fabrication strategy based on a shell-writing procedure with the aim to reduce fabrication time of big inlet/outlet sections compatible with most microfluidic systems for LOCs. With respect to standard microfabrication techniques, requiring several technological steps to obtain suspended hollow structures, this method allows to fabricate complex SMR sensors in only one fabrication step, by virtue of its intrinsically three- dimensional nature. A SMR fixed-fixed beam has been fabricated and characterized by LDV. A different sensing mechanism based on the parametric resonance instead of the harmonic resonance has been investigated to develop a novel platform for the characterization of biomolecules in free-flow with unique specificity, sensitivity, and speed: to this purpose a PDMS based device was realized by laser machining, a rapid prototype fabrication technique; beside to it, a commercial fused silica capillary tubing was also employed in the realization of a prototype for this sensing mechanism, and both solutions were tested through LDV

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

The impact of the interference on the molecular communication (MC) between a transmitter and receiver pair which are connected through a microfluidic channel containing fluid flow is investigated. The interference modeling and the capacity analysis is performed based on the microfluidic channel geometry, the flow velocity, and the distance. During the analysis, time-scale of biological oscillators is specifically targeted, which is in the range of several minutes to a few hours. The signal-to-interference and noise ratio is shown to be constant with respect to the location of the interfering transmitter. The capacity of the MC link between the designated transmitter and the corresponding receiver is shown to be upper bounded by 1 bit/per channel use when exposed to a single interfering transmitter. For the multiple, i.e., N , transmitters, the decay of pairwise MC capacity is also studied as a factor of N . Finally, placement of the two transmitter and receiver pairs on the opposite sides of the microfluidic channel is studied. Three different microfluidic interference channel configurations, i.e., both-sided interference (microfluidic X channel), one-sided interference (microfluidic Z channel), and interference-free, are proposed based on the distance of the receiver from the interfering transmitter, microfluidic channel cross section, and the fluid flow velocity. For large-scale integration of chemical analysis systems on a microfluidic chip, the provided information-theoretic analysis and the capacity expressions for the MIC can be utilized to analyze the throughput of the chip, which can lead to improvement in efficiency and optimization of the design.

Background: Mass disaster events allied with astounding damage of humanity, consequently leading with complexity to recognize human eccentricity. Forensic science has an enormous role in human identification after disaster. As human teeth are admirable source of DNA due to their relatively higher degree of physical and chemical resistance; thus, revival of genetic material is promising with teeth; in case of disasters. Therefore, human teeth can be used as an imperative resource for human identification in mass disasters. Aim and Objectives: This study aims to compare DNA quantity and purity, of extracted human teeth buried in soil and establishes the result that dental tissue can be used as an imperative marker in human identification. Material and Methods: An in vitro experimental study conducted with 30 extracted human teeth. All teeth divided into two groups depending on the time of buried in soil – (i) Group 1 (old group): It was comprised of 14 teeth, buried for 12 months and (ii) Group 2 (new group): It was comprised of 16 teeth, buried for 6 months. Then, DNA isolation, quantification, and purity assessment was and results analyzed by SPSS version 20 using paired and unpaired Student's t-test. Results: This study illustrates that the entire samples were amplifiable in polymerase chain reaction and showing reverently high-quality results. DNA purity was not significantly affected by the storage period of teeth in soil. Conclusion: The study concluded that DNA isolation and assessment of quantity and purity can be successfully done from extracted teeth buried in soil. The quantity and purity of DNA retrieved from those teeth who buried for 6 months was high. The quantity of DNA was significantly affected by the storage period of teeth but the purity or quality of DNA was not. Thus, it established the fact the dental tissue can be used as an imperative marker for human identification.

Microfluidic grooves and channels enable the control of cell positioning and capturing of cells in cell-based biosensor applications. Microfluidic devices also provide a platform for cell-based biological assays. Microfluidic channels have been used to capture cells for various cell-based diagnostics and screening applications. The number of cells and cell locations are important factors which will affect the flow pattern in the microfluidic device and consequently induced shear stresses. Moreover, manual cell counting and cell information extraction is a time consuming and tedious task. Automated techniques reduce human errors and expedite the process. Furthermore, it facilitates extraction of information from obtained images for further fluidic analysis.

This thesis covers devices exploring basic areas of physical and biological research including: surface plasmon enhanced InGaN light-emitting diode, analysis of using AlGaN emitters coupled with thin film heaters to cure onychomycosis infections, tunable organic transistors that use microfluidic source and drain electrodes, as well as an electrical microfluidic pressure gauge for PDMS MEMS. Also analyzed are devices created through the use of novel three-dimensional rapid prototyping techniques, such as: the replication of three-dimensional valves from printed wax molds, chemically robust three-dimensional monolithic SIFEL fluoropolymer microfluidics, microfluidic valves for customized radioactive positron emission dyes, reduction of microfluidic control inputs through the use of pressure multiplexing, bicuspid-inspired microfluidic check valves and microfluidic three-dimensional separation column. Devices created to analyze blood are also treated including: a microfluidic device to extract blood plasma from a fingerstick; inexpensive, portable immunoassay devices and their use in in small cell lung carcinoma and multiple sclerosis; as well as a device to screen metastasizing cancer cells. Devices created to perform polymerase chain reactions are also studied, including: an evaporative cooler for microfluidic channels, thermal management in microfluidics using micro-Peltier junctions in a microfluidic polymerase chain reaction system, and an accessible polymerase chain reaction system.

Nucleic acid sample preparation from whole blood in a paper microfluidic device using isotachophoresis

Several developments over the last few years are being directed toward improving DNA-based analysis to simplify, miniaturize, and reduce the time and cost of analysis, with the objective to allow its use in decentralized settings. One of the most interesting fields is DNA extraction and purification, a key step for ensuring good analytical performance. In this sense, microscale solid phase extraction (μSPE) offers paramount advantages for an improved DNA yield. In this work, we have developed a miniaturized module for DNA purification based on μSPE using a borosilicate glass microfiber filter as the solid phase. We also established a protocol for highly efficient DNA purification from vegetable samples, including leaves and grapes from four different varieties from the PDO Douro and two varieties from the Minho wine regions. The protocol demonstrated excellent performance when compared with a commercial kit with a DNA recovery yield of around 50%.

Intracellular delivery of molecular compounds is required for many in vitro research applications. Ultrasound-induced cavitation has been shown to enhance intracellular delivery of molecular compounds via mechanisms that may include sonoporation or endocytosis. Recently, acoustofluidic approaches have been developed to utilize standing acoustic waves (SAW) for cell manipulation in microfluidic channels. In this study, the effect of SAW on fluorescein release from perfluorocarbon double emulsion droplets in microfluidic channels was explored. Vaporization of perfluorocarbon double emulsion droplets induced by SAW may potentially enhance intracellular delivery of molecular compounds. In this study, fluorescein-loaded double emulsions were passed through a microfluidic device and exposed to 8-MHz SAW. Fluorescein release was quantified by measuring the change in fluorescence of the supernatant before and after treatment. Treatment with SAW in microfluidic channels increased the fluorescence by 8.8-fold compared to the baseline level. Fluorescein release was also higher after treatment with SAW compared to samples that passed through the microfluidic device without exposure to SAW (p = 0.03). These results suggest that SAW and perfluorocarbon double emulsions in microfluidic channels could potentially enhance the efficiency and consistency of intracellular molecular delivery in vitro.

A microfluidic device (MD) has been developed which features a porous silica (PS) monolithic disk synthesized from tetramethyl orthosilicate, incorporated into the device post-fabrication and sealed in place with a second PS monolithic layer, synthesized from potassium silicate. This dual porous silica (DPS) structure provides a pathway for sample introduction to the MD and offers an ideal platform for solid phase extraction (SPE) methodologies which can be rapidly and efficiently integrated into a chip-based format. All silica disk manufacture and functionalization was carried out in batch to provide a readily scalable method of production. Application of this design for processing samples was demonstrated using two alternative nucleic acid purification chemistries, yielding polymerase chain reaction amplifiable DNA extracted from 150μL of human urine in less than 35 min. It is proposed that this DPS system could be further developed for a diverse range of chip-based SPE applications, providing an interface facilitating sample delivery and enabling SPE on-chip. Furthermore, to the author's knowledge it is the first reporting of two different types of PS amalgamated in a single MD.