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A Disposable Microfluidic Virus Concentration Device Based on Evaporation and Interfacial Tension  [PDF]
Jane Yuqian Zhang,Madhumita Mahalanabis,Lena Liu,Jessie Chang,Nira R. Pollock,Catherine M. Klapperich
Diagnostics , 2013, DOI: 10.3390/diagnostics3010155
Abstract: We report a disposable and highly effective polymeric microfluidic viral sample concentration device capable of increasing the concentration of virus in a human nasopharyngeal specimen more than one order of magnitude in less than 30 min without the use of a centrifuge. The device is fabricated using 3D maskless xurography method using commercially available polymeric materials, which require no cleanroom operations. The disposable components can be fabricated and assembled in five minutes. The device can concentrate a few milliliters (mL) of influenza virus in solution from tissue culture or clinical nasopharyngeal swab specimens, via reduction of the fluid volume, to tens of microliters (mL). The performance of the device was evaluated by nucleic acid extraction from the concentrated samples, followed by a real-time quantitative polymerase chain reaction (qRT-PCR). The viral RNA concentration in each sample was increased on average over 10-fold for both cultured and patient specimens compared to the starting samples, with recovery efficiencies above 60% for all input concentrations. Highly concentrated samples in small fluid volumes can increase the downstream process speed of on-chip nucleic acid extraction, and result in improvements in the sensitivity of many diagnostic platforms that interrogate small sample volumes.
Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices  [PDF]
Laura Maria Zanoli,Giuseppe Spoto
Biosensors , 2013, DOI: 10.3390/bios3010018
Abstract: Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.
Bioprocess Control in Microscale: Scalable Fermentations in Disposable and User-Friendly Microfluidic Systems
Matthias Funke, Andreas Buchenauer, Wilfried Mokwa, Stefanie Kluge, Lea Hein, Carsten Müller, Frank Kensy, Jochen Büchs
Microbial Cell Factories , 2010, DOI: 10.1186/1475-2859-9-86
Abstract: This novel microfermentation system was tested in pH-controlled batch as well as in fed-batch fermentations of Escherichia coli. The pH-value in the culture broth could be kept in a narrow dead band of 0.03 around the pH-setpoint, by pneumatically dosing ammonia solution and phosphoric acid to each culture well. Furthermore, fed-batch cultivations with linear and exponential feeding of 500 g/L glucose solution were conducted. Finally, the scale-up potential of the microscale fermentations was evaluated by comparing the obtained results to that of fully controlled fermentations in a 2 L laboratory-scale fermenter (working volume of 1 L). The scale-up was realized by keeping the volumetric mass transfer coefficient kLa constant at a value of 460 1/h. The same growth behavior of the E. coli cultures could be observed on both scales.In microfluidic MTPs, pH-controlled batch as well as fed-batch fermentations were successfully performed. The liquid dosing as well as the biomass growth kinetics of the process-controlled fermentations agreed well both in the microscale and laboratory scale. In conclusion, a user-friendly and disposable microfluidic system could be established which allows scaleable, fully controlled and fully monitored fermentations in working volumes below 1 milliliter.State-of-the-art bioprocesses are based on a large number of small-scale experiments in which the best performing microbial strain and the optimal cultivation conditions are evaluated. These screening experiments are becoming even more important, since modern methods in genetic engineering and molecular biology can generate thousands of different clones. Moreover, biotechnological research - spurred e.g. by the process analytical technology (PAT) initiative of the FDA [1] - additionally identifies the influences of a growing number of media ingredients and process variables. All these clones and parameters are targets for screening and process optimization [2,3]. On the other hand, pressure
Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge  [PDF]
Joshua M. Kogot, Yanting Zhang, Stephen J. Moore, Paul Pagano, Dimitra N. Stratis-Cullum, David Chang-Yen, Marek Turewicz, Paul M. Pellegrino, Andre de Fusco, H. Tom Soh, Nancy E. Stagliano
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026925
Abstract: Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×1010 members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS.
Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens  [PDF]
Qingqing Cao, Madhumita Mahalanabis, Jessie Chang, Brendan Carey, Christopher Hsieh, Ahjegannie Stanley, Christine A. Odell, Patricia Mitchell, James Feldman, Nira R. Pollock, Catherine M. Klapperich
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0033176
Abstract: A rapid, low cost, accurate point-of-care (POC) device to detect influenza virus is needed for effective treatment and control of both seasonal and pandemic strains. We developed a single-use microfluidic chip that integrates solid phase extraction (SPE) and molecular amplification via a reverse transcription polymerase chain reaction (RT-PCR) to amplify influenza virus type A RNA. We demonstrated the ability of the chip to amplify influenza A RNA in human nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens collected at two clinical sites from 2008–2010. The microfluidic test was dramatically more sensitive than two currently used rapid immunoassays and had high specificity that was essentially equivalent to the rapid assays and direct fluorescent antigen (DFA) testing. We report 96% (CI 89%,99%) sensitivity and 100% (CI 95%,100%) specificity compared to conventional (bench top) RT-PCR based on the testing of n = 146 specimens (positive predictive value = 100%(CI 94%,100%) and negative predictive value = 96%(CI 88%,98%)). These results compare well with DFA performed on samples taken during the same time period (98% (CI 91%,100%) sensitivity and 96%(CI 86%,99%) specificity compared to our gold standard testing). Rapid immunoassay tests on samples taken during the enrollment period were less reliable (49%(CI 38%,61%) sensitivity and 98%(CI 98%,100%) specificity). The microfluidic test extracted and amplified influenza A RNA directly from clinical specimens with viral loads down to 103 copies/ml in 3 h or less. The new test represents a major improvement over viral culture in terms of turn around time, over rapid immunoassay tests in terms of sensitivity, and over bench top RT-PCR and DFA in terms of ease of use and portability.
Terminal Continuation (TC) RNA Amplification Enables Expression Profiling Using Minute RNA Input Obtained from Mouse Brain  [PDF]
Melissa J. Alldred,Shaoli Che,Stephen D. Ginsberg
International Journal of Molecular Sciences , 2008, DOI: 10.3390/ijms9112091
Abstract: A novel methodology named terminal continuation (TC) RNA amplification has been developed to amplify RNA from minute amounts of starting material. Utility of the TC RNA amplification method is demonstrated with two new modifications including obviating the need for second strand synthesis, and purifying the amplification template using column filtration prior to in vitro transcription (IVT). Using four low concentrations of RNA extracted from mouse brain (1, 10, 25 and 50 ng), one round TC RNA amplification was compared to one round amplified antisense RNA (aRNA) in conjunction with column filtration and drop dialysis purification. The TC RNA amplification without second strand synthesis performed extremely well on customdesigned cDNA array platforms, and column filtration was found to provide higher positive detection of individual clones when hybridization signal intensity was subtracted from corresponding negative control hybridization signal levels. Results indicate that TC RNA amplification without second strand synthesis, in conjunction with column filtration, is an excellent method for RNA amplification from extremely small amounts of input RNA from mouse brain and postmortem human brain, and is compatible with microaspiration strategies and subsequent microarray analysis.
Rapid and Sensitive MicroRNA Detection with Laminar Flow-Assisted Dendritic Amplification on Power-Free Microfluidic Chip  [PDF]
Hideyuki Arata,Hiroshi Komatsu,Kazuo Hosokawa,Mizuo Maeda
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0048329
Abstract: Detection of microRNAs, small noncoding single-stranded RNAs, is one of the key topics in the new generation of cancer research because cancer in the human body can be detected or even classified by microRNA detection. This report shows rapid and sensitive microRNA detection using a power-free microfluidic device, which is driven by degassed poly(dimethylsiloxane), thus eliminating the need for an external power supply. MicroRNA is detected by sandwich hybridization, and the signal is amplified by laminar flow-assisted dendritic amplification. This method allows us to detect microRNA of specific sequences at a limit of detection of 0.5 pM from a 0.5 μL sample solution with a detection time of 20 min. Together with the advantages of self-reliance of this device, this method might contribute substantially to future point-of-care early-stage cancer diagnosis.
A Low-Cost Microfluidic Chip for Rapid Genotyping of Malaria-Transmitting Mosquitoes  [PDF]
Changchun Liu, Michael G. Mauk, Robert Hart, Mariangela Bonizzoni, Guiyun Yan, Haim H. Bau
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0042222
Abstract: Background Vector control is one of the most effective measures to prevent the transmission of malaria, a disease that causes over 600,000 deaths annually. Around 30–40 Anopheles mosquito species are natural vectors of malaria parasites. Some of these species cannot be morphologically distinguished, but have behavioral and ecological differences. Emblematic of this is the Anopheles gambiae species complex. The correct identification of vector species is fundamental to the development of control strategies and epidemiological studies of disease transmission. Methodology/Principal Findings An inexpensive, disposable, field-deployable, sample-to-answer, microfluidic chip was designed, constructed, and tested for rapid molecular identification of Anopheles gambiae and Anopheles arabiensis. The chip contains three isothermal amplification reactors. One test reactor operates with specific primers to amplify Anopheles gambiae DNA, another with specific primers for Anopheles arabiensis DNA, and the third serves as a negative control. A mosquito leg was crushed on an isolation membrane. Two discs, laden with mosquito tissue, were punched out of the membrane and inserted into the two test chambers. The isolated, disc-bound DNA served as a template in the amplification processes. The amplification products were detected with intercalating fluorescent dye that was excited with a blue light-emitting diode. The emitted light was observed by eye and recorded with a cell-phone camera. When the target consisted of Anopheles gambiae, the reactor containing primers specific to An. gambiae lit up while the other two reactors remained dark. When the target consisted of Anopheles arabiensis, the reactor containing primers specific to An. arabiensis lit up while the other two reactors remained dark. Conclusions/Significance The microfluidic chip provides a means to identify mosquito type through molecular analysis. It is suitable for field work, allowing one to track the geographical distribution of mosquito populations and community structure alterations due to environmental changes and malaria intervention measures.
Variability of Solar Five-Minute Oscillations in the Corona as Observed by the Extreme Ultraviolet Spectrophotometer (ESP) on the Solar Dynamics Observatory Extreme Ultraviolet Variability Experiment (SDO/EVE)  [PDF]
Leonid Didkovsky,Alexander Kosovichev,Darrell Judge,Seth Wieman,Tom Woods
Physics , 2012, DOI: 10.1007/s11207-012-0186-3
Abstract: Solar five-minute oscillations have been detected in the power spectra of two six-day time intervals from soft X-ray measurements of the Sun observed as a star using the Extreme Ultraviolet Spectrophotometer (ESP) onboard the Solar Dynamics Observatory (SDO) Extreme Ultraviolet Variability Experiment (EVE). The frequencies of the largest amplitude peaks were found matching within 3.7 microHz the known low-degree (l = 0--3) modes of global acoustic oscillations, and can be explained by a leakage of the global modes into the corona. Due to strong variability of the solar atmosphere between the photosphere and the corona the frequencies and amplitudes of the coronal oscillations are likely to vary with time. We investigate the variations in the power spectra for individual days and their association with changes of solar activity, e.g. with the mean level of the EUV irradiance, and its short-term variations due to evolving active regions. Our analysis of samples of one-day oscillation power spectra for a 49-day period of low and intermediate solar activity showed little correlation with the mean EUV irradiance and the short-term variability of the irradiance. We suggest that some other changes in the solar atmosphere, e.g. magnetic fields and/or inter-network configuration may affect the mode leakage to the corona.
Amplification of Nonlocal Effects in Nonlinear Quantum Mechanics by Extreme Localization  [PDF]
George Svetlichny
Physics , 2004,
Abstract: Due to its connection to the diffeomorphism group, nonlinear quantum mechanics may play an important role in quantum geometry. The Doebner-Goldin nonlinearity (arising from representations of the diffeomorphism group) amplifies nonlocal signaling effects under extreme localization, suggesting that even if greatly suppressed at low energies, such effects may be significant at the Planck scale. This offers new perspectives on Planck-scale physics.
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