A multi-function, disposable, microfluidic module for mutation detection

Recognition of point mutations in a codon 12 of the K-ras gene, most frequently observed, is considered to be useful in the early diagnosis of several types of the human cancers. We have developed a multifunction, disposable, microfluidic module which detects low-abundant point mutations in human genomic DNA in modular architecture. Each functional component including a microfluidic PCR reactor, a passive diffusional micromixer reactor, and a microfluidic LDR reactor was separately designed and fabricated. Fluidic interconnects were also developed to make a fluidic passage between the functional components. Polycarbonate substrates were micro-molded, using hot embossing with micro-milled brass mold inserts to make all microfluidic components. Developed microassembly using passive alignment features, fabricated on all components, was used to assemble the functional components with the fluidic interconnects using an adhesive bonding technique. Thermal simulations were employed to ensure uniform thermal distributions in the microfluidic PCR and LDR reactors, to isolate the mixing junction in order to avoid heat–induced bubble formation in the passive micromixer reactor, and to have minimal thermal crosstalk due to the asymmetric thermal zones in the PCR and the LDR reactors. A control system was developed to control temperatures enabling thermal cycling in the microfluidic PCR and LDR reactor. LDR products were produced using the module within an hour with DNA sample, which had the ratio of 1:200. Total reaction time was about 67 minutes. By applying an enzyme as a purification of PCR products, a LDR analysis can be optimized and minimized to reduce the false positive signals and inconstant results generated by PCR products during the LDR. The purification system allowed us to successfully quantify the amount of mutant alleles in the genomic DNA. The high degree of accuracy in this module can also facilitate the detection of low-frequency point mutation occurred in other functional genes. This module, fabricated using replication technologies of polymers will be able to supply low cost, disposable detection tools for known disease-causing mutations and also expand to other PCR-based detection assays in diagnostic applications

Characterization and Analysis of Real-Time Capillary Convective PCR Toward Commercialization

Almost all the reported capillary convective polymerase chain reaction (CCPCR) systems to date are still limited to research use stemming from unresolved issues related to repeatability, reliability, convenience, and sensitivity. To move CCPCR technology forward toward commercialization, a couple of critical strategies and innovations are discussed here. First, single- and dual-end heating strategies are analyzed and compared between each other. Especially, different solutions for dual-end heating are proposed and discussed, and the heat transfer and fluid flow inside the capillary tube with an optimized dual-end heating strategy are analyzed and modeled. Second, real-time CCPCR is implemented with light-emitting diode and photodiode, and the real-time fluorescence detection method is compared with the post-amplification end-point detection method based on a dipstick assay. Thirdly, to reduce the system complexity, e.g., to simplify parameter tuning of the feedback control, an internal-model-control-based proportional-integral-derivative controller is adopted for accurate temperature control. Fourth, as a proof of concept, CCPCR with pre-loaded dry storage of reagent inside the capillary PCR tube is evaluated to better accommodate to point-of-care diagnosis. The critical performances of improved CCPCR, especially with sensitivity, repeatability, and reliability, have been thoroughly analyzed with different experiments using influenza A (H1N1) virus as the detection sample. Published by AIP Publishing

Simulation-Assisted Analysis and Design of Microfluidic Polymerase Chain Reaction Assay Devices

The Polymerase Chain Reaction (PCR) is a method by which sample DNA can be sequentially copied in order to increase the concentration of the sample DNA so that it can be measured directly. Interestingly, only specific DNA segments selected by the operator are amplified, if those segments are present in the sample then one will see DNA amplification otherwise nothing will be amplified. As a result, assays can be tailored to detect a wide range of disorders including bacterial infections, viral infections, and even genetic abnormalities/disorders. PCR is a thermally actuated process where the chemistry is governed by the temperature of the reaction chamber. By adjusting the temperature of a PCR reactor through a set of three simple steps the concentration of target sample DNA is roughly doubled at the end of each cycle. However, the reactants are very temperature sensitive. As a result precise temperature control is required in order to conduct a successful PCR run. With the advent of microfluidics, PCR has been miniaturized onto lab-on-chip systems. These systems have many advantages over their conventional counterparts (laboratory scale biological assay with bench-top thermal cycling systems) such as reduced cycling times and reduced reagent costs due to smaller sample volumes, increased automation due to on-chip integration with other modules (for other process steps), and reduced power consumption due to better integration with heating systems. However, the design of these microfluidic systems also has its unique challenges. High thermal gradients throughout the system are commonplace; as a result it is difficult to ensure thermal uniformity within the reaction chamber. Furthermore, it is difficult to correlate the measured temperature of external temperature probes with the temperature of the reactor itself. Lastly, it is very difficult to experimentally measure the real-time temperature profile of the PCR chamber, since any sensor perturbs the system greatly. The work presented within this report will focus on the thermal design of typical microfluidic PCR reactors. Our analysis is focused on using simulation-aided-design to offer insights that serve to validate and optimize the performance of these exemplary reactors. In order to do this, we modified OpenFOAM, an open-source CFD simulation suite that relies on the Finite Volume Method, to include a heat generation term as well as PID control. We first focused on running transient simulation in the absence of control in order to optimize the physical design of our example reactor. Subsequently, we investigated the effect of feedback control on the sample system in order to validate the results obtained experimentally. In order to do this we emulated the entire experimental system and matched the control scheme/parameters used experimentally. What we found was that our simulations were able to provide insights that were otherwise unobtainable. We were able to map the full 3D temperature field of the system in a high fidelity transient simulation. Using this data, we determined that placement of the temperature sensor was crucial to the performance of the device. Furthermore, we discovered that manifold layers relatively far away from the reactor chamber had a profound effect on the temperature measured by out temperature sensor. As a result, the design was modified to include an additional heat spreading layer so that the measured temperature would better reflect the temperature of the chamber. Moreover, our open loop simulations illustrated that simple 1D analytical models were not adequate to predict the performance of microfluidic PCR systems. Our second set of simulations focused on studying how PID control affects the temperature profile of the system over time. Originally experimental validation was done on the optimized system. It was found that the design did not successfully amplify the target DNA sequence, likely due to the amplification of primer-dimers. Using our simulation methodology analysis we found that the heating/cooling rate of the system was position dependent. In our specific example, our temperature sensor was closer to the heating element; thus reaching target temperature well before the reactor itself. Our simulations identified transient discrepancy between the measured temperature and the reactor temperature. Using this information we were able to adjust the control scheme of the experimental system which resulted in successful amplification. Overall the simulation-aided-design exercise has been invaluable in troubleshooting and optimizing the design of microfluidic PCR devices

Feasibility of Mainstream Nitrite Oxidizing Bacteria Out-Selection and Anammox Polishing for Enhanced Nitrogen Removal

Short-cut nitrogen removal avoids nitrite oxidation to nitrate by nitrite oxidizing bacteria (NOB) and allows a) reduction of formed nitrite to nitrogen gas via heterotrophic denitrification and/or b) oxidation of remaining ammonia with formed nitrite to nitrogen gas via anaerobic ammonia oxidation (anammox). The precondition for achieving shortcut nitrogen removal is suppression of NOB, which is favored by warm and high ammonia strength conditions found in internally generated ammonia-rich waste streams through anaerobic digestion of waste solids referred to as sidestreams or reject water. The discovery of anammox bacteria in the mid-1990s, which are capable of transforming NH4+ to nitrogen gas utilizing NO2- as a substrate, has made suppression of NOB even more critical for nitrogen removal processes that take advantage of the lower energy and cost requirements of this nitrogen conversion compared to traditional nitrogen removal processes. Deammonification relies on ammonia oxidizing bacteria (AOB) to partially convert NH4+ to NO2- and anammox bacteria (AMX) to convert the remaining NH4+ and NO2- to nitrogen gas. The challenges of retaining slow growing AMX initially limited the expansion of benefits from autotrophic nitrogen removal; however, granular sludge and attached growth systems have proven highly effective in achieving deammonification in sidestream processes. Owing to the benefits that include energy and chemical savings, short-cut nitrogen removal has emerged as a viable technology for sidestream treatment. Consequently, mechanisms of NOB suppression to perform short-cut nitrogen removal are generally quite well understood for sidestream applications, which has allowed for the development of robust process control strategies. To date, the concept of deammonification has successfully been implemented in 100 full-scale treatment facilities treating high ammonia strength waste streams around the world. Due to the success of sidestream short-cut nitrogen removal systems, there is great interest in applying this form of nitrogen removal to mainstream processes. Since the dilute and cold conditions of mainstream are not well-suited for suppression of NOB, short-cut nitrogen removal, in particular deammonification, has yet to be implemented in full-scale. The successful implementation of mainstream deammonification would revolutionize and disrupt the way in which biological nitrogen removal is achieved at wastewater treatment facilities. It represents a paradigm shift for the industry, offering the opportunity for sustainable wastewater treatment, energy neutral or even energy positive facilities and dramatic reductions in treatment costs, which has widespread environmental, economic and societal benefits. This dissertation deals with the pilot-scale investigation of short-cut nitrogen removal in low ammonia strength wastewater with temperatures \u3c25 \u3e°C. An A-B process pilot-scale system was operated over a two year period. The A-stage was a high-rate activated sludge system for carbon removal and the B-stage consisted of an activated sludge system that targeted NOB out-selection which was followed by a fully anoxic anammox MBBR. In this study, by employing a combination of intermittent aeration, high DO (\u3e1.5 mg/L), residual effluent NH4+ (\u3e2 mg/L), and aggressive SRT (\u3c 5 days at 23-25 °C) and HRT (\u3c 4hr), NOB out-selection was achieved in the continuous-flow activated sludge process. The development of novel aeration and SRT control strategies based on advanced instrumentation, control, and automation for achieving NOB out-selection in an activated sludge process and nitrogen polishing in subsequent anammox MBBR was shown. A very fast startup time (less than 2 weeks) for anammox MBBR was achieved by seeding anammox granules obtained from a full-scale, sidestream anammox treatment process. Anammox MBBR proved highly stable during the study and a very high maximum nitrogen conversion rate (\u3e 1 gN/m2/d) was demonstrated. Therefore, this study shows carbon re-direction (potentially for energy production) in a high rate A-stage does not cause carbon limitation in the B-stage for nitrogen removal if control strategies and anammox-based nitrogen polishing is used as investigated in this study

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids

Modeling, Identification and Control at Telemark University College

Master studies in process automation started in 1989 at what soon became Telemark University College, and the 20 year anniversary marks the start of our own PhD degree in Process, Energy and Automation Engineering. The paper gives an overview of research activities related to control engineering at Department of Electrical Engineering, Information Technology and Cybernetics

Modeling, Identification and Control at Telemark University College

Master studies in process automation started in 1989 at what soon became Telemark University College, and the 20 year anniversary marks the start of our own PhD degree in Process, Energy and Automation Engineering. The paper gives an overview of research activities related to control engineering at Department of Electrical Engineering, Information Technology and Cybernetics