Microfluidic method for rapid turbidimetric detection of the DNA of Mycobacterium tuberculosis using loop-mediated isothermal amplification in capillary tubes

We describe a microfluidic method for rapid isothermal turbidimetric detection of the DNA of Mycobacterium tuberculosis. Loop-mediated isothermal amplification is accomplished in capillary tubes for amplifying DNA in less than 15 min, and sensitivity and specificity were compared to conventional loop-mediated isothermal amplification (LAMP). The method can detect as little as 1 pg mL−1 DNA in a sample. Results obtained with clinical specimens indicated 90 % sensitivity and 95 % specificity for microfluidic LAMP in comparison to culture methods. No interference occurred due to the presence of nonspecific DNAs. The findings demonstrate the power of the new microfluidic LAMP test for rapid molecular detection of microorganisms even when using bare eyes. © 2014, Springer-Verlag Wien

Integrated modular microfluidic system for forensic Alu DNA typing

Driven by the numerous applications of genome-related research, fully integrated microfluidic systems have been developed that have advanced the capabilities of molecular and, in particular, genetic analyses. A brief overview on integrated microfluidic systems for DNA analysis is given in Chapter 1 followed by a report on micro-capillary electrophoresis (µCE) of Alu elements with laser-induced fluorescence (LIF) detection, in which the monomorphic Alu insertions on the X and Y chromosomes were utilized to detect male DNA in large female DNA background (Y: X = 1:19) without cell sorting prior to the determination. The polymorphic Alu loci with known restricted geographical distribution were used for ethnicity determination. A valveless integrated microsystem that consists of three modules is discussed as well: (1) A solid-phase extraction (SPE) module microfabricated on polycarbonate, for DNA extraction from whole cell lysates (extraction bed capacity ~209 ±35.6 ng/cm² of total DNA). (2) A continuous-flow polymerase chain reaction (CFPCR) module fabricated in polycarbonate (Tg ~150 ºC) in which selected gene fragments were amplified using biotin and fluorescently-labeled primers accomplished by continuously shuttling small packets of PCR reagents and template through isothermal zones. (3) µCE module fabricated in poly(methylmethacrylate), which utilized a bioaffinity selection and purification bed (2.9-µL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to µCE. Biotin-labeled CFPCR products were hydrostatically pumped through the streptavidin-modified bed where they were extracted onto the surface of the poly(methylmethacrylate) micropillars (50-µm width; 100-µm height; total surface area of ~117 mm²). This SPE process demonstrated high selectivity for biotinylated amplicons and utilized the strong streptavidin/biotin interaction (Kd =10-15M) to generate high recoveries. The SPE selected CFPCR products were thermally denatured and single stranded DNA released for size-based separations and LIF detection. The multiplexed SPE-CFPCR-µCE yielded detectable fluorescence signal (S/N≥3; LOD ~75 cells) for Alu DNA amplicons for gender and ethnicity determinations with a separation efficiency of ~1.5 x105 plates/m. Compared to traditional cross-T injection procedures typically used for µCE, the affinity preconcentration and injection procedure generated signal enhancements of 17-40 fold, critical for CFPCR thermal cyclers due to Taylor dispersion associated with their operation

Microchips for Isothermal Amplification of RNA : Development of microsystems for analysis of bacteria, virii and cells

The goal of the present work was to develop a microchip for amplification and detection of mRNA by employing nucleic acid sequence-based amplification (NASBA) technology. The technology platform should in principle be adaptable for any clinical analysis using mRNA or ssDNA as a target. To demonstrate the microchip functionality, identification of human papillomavirus (HPV) type 16, the etiological agent for cervical cancer has been used. The work shows for the first time successful real-time amplification and detection employing NASBA in microsystem formats using custom-made instruments. The first silicon-glass chips contained reaction chambers of 10 nl and 50 nl, which decreased the NASBA reaction volume by a factor of 2000 and 400, respectively. Further, experiments employing cyclic olefin copolymer (COC) microchips for simultaneous amplification and detection, automatically distributed the sample into 10 parallel reaction channels with detection volumes of 80 nl. In order to detect the simultaneous amplification in the reaction channels, a second custom-made optical detection system with increased sensitivity, heat regulation and an automatic non-contact pumping mechanism, was made. Dilution series of both artificial HPV 16 oligonucleotides and SiHa cell lines showed that the detection limits for the microchips were comparable to those obtained for experiments performed in conventional routine-based laboratory-systems. For experiments related to the development of a self-contained microchip for NASBA, the detection volume was increased to 500 nl due to the advantage of an increased fluorescence signal. For the NASBA reaction, biocompatible surfaces are critical. It was not possible to amplify any target in microchips with native silicon or COC surfaces. Adsorption measurements indicated clearly that fluorescently labelled mouse IgG bound non-specifically to the hydrophobic native COC surfaces, while PEG coated COC surfaces showed adequate protein resistance. Of the coatings tested for the COC microchips, surfaces modified with PEG showed the best biocompatibility. Successful amplification was obtained with silicon microchips when the surfaces were modified with either SigmaCote™ or SiO2. In order to integrate the NASBA reagents on chip, a thorough evaluation of the reagents to be spotted and dried was performed. Because of the limited number of microchipsavailable, it was necessary to map the most critical parameters on macroscale prior to transfer to the microscale. The DMSO and sorbitol enclosed in the standard NASBA reaction mixture were difficult to dry, and therefore it was necessary to add these compounds to the oligonucleotides or the sample of extracted nucleic acids before the sample was applied on the amplification chip. The standard NASBA reagents consist of the two main solutions, mastermix and enzymes, in addition to the sample. Both the mastermix and the enzymes were stable only when spotted and dried separately. Protectants, such as PEG and trehalose were essential for recovery of enzymatic activity after drying on macroscale. The times for diffusion of modified molecular beacons in dried mastermix and of fluorescently labelled mouse IgG in the dried enzyme solution were ~60 seconds and ~10 minutes, respectively. So far, only dried enzymes with 0.05% PEG protectant have been successfully amplified on chip. Successful amplification using a rehydrated mastermix on microchip still remains. Optimal design and fabrication methods of the microchips were found to be crucial for chip performance. Rough surfaces do not only create background noise for the optical measurements, but it also contributes to generation of bubbles and problems related to manipulation of the sample within the channel network. The silicon microchips were manufactured with optically smooth surfaces. However, low surface roughness was not easily obtained for the COC microchips. Of the fabrication methods evaluated, it was the injection moulded chips which showed the smoothest surfaces, closely followed by the hot embossed chips. Milled and laser ablated chips produced the roughest surfaces. A novel non-contact pumping mechanism based on on-chip flexible COC membranes, combined with actuation pins in the surrounding instrument, was tested and evaluated. The mechanism enabled metering, isolation and movement of nanoliter sized sample plugs in parallel reaction channels. The COC chips with integrated pumps were able to simultaneously move parallel sample plugs along the reaction channels in four different positions. Each reaction channel contained a set of 4 actuation chambers in order to obtain metering, isolation and movement of the sample plug into the detection area. The pump accuracy depended on the evaporation of sample and the deformation of the COC membranes. The results presented in this work are promising with regard to the development of a complete integrated and self-contained mRNA amplification microchip for multi-parallel target testing of clinical samples

Integrated microfluidic systems with sample preparation and nucleic acid amplification

Rapid, efficient and accurate nucleic acid molecule detection is important in the screening of diseases and pathogens, yet remains a limiting factor at point of care (POC) treatment. Microfluidic systems are characterized by fast, integrated, miniaturized features which provide an effective platform for qualitative and quantitative detection of nucleic acid molecules. The nucleic acid detection process mainly includes sample preparation and target molecule amplification. Given the advancements in theoretical research and technological innovations to date, nucleic acid extraction and amplification integrated with microfluidic systems has advanced rapidly. The primary goal of this review is to outline current approaches used for nucleic acid detection in the context of microfluidic systems. The secondary goal is to identify new approaches that will help shape future trends at the intersection of nucleic acid detection and microfluidics, particularly with regard to increasing disease and pathogen detection for improved diagnosis and treatment

Recent developments in nucleic acid identification using solid-phase enzymatic assays

This review (containing 101 references) covers recent achievements in the development of new approaches for enzymatically assisted detection of nucleic acids on microarrays. We discuss molecular techniques including the polymerase chain reaction, reverse transcription, allele specific primer extension and a range of isothermal techniques for the amplification and discrimination of nucleic acids. This also includes their implementation into microfluidic systems. These techniques all show great promise for use in the life sciences by allowing for high throughput, cost effective and highly sensitive and specific analysis of nucleic acids. Importantly, they can be potentially integrated into personalized and point-of-care medicine

Miniaturised nucleic acid analysis systems

The design and implementation of miniaturised systems for analysis of nucleic acids from various biological samples has undergone extensive development. Several advances have been made particularly with the integration of nucleic acid amplification and detection, where amplification is most often polymerase chain reaction (PCR). Sample preparation remains a major obstacle for achieving a quantitative analysis employing full miniaturised integration. Miniaturised devices for nucleic acid sample preparation, amplification and detection have to be further developed in order to achieve a fully integrated system, which ultimately can perform single cells genomic analysis with sample-in-answer-out ability. In this thesis, three miniaturised systems have been presented, which can be used for purification and preconcentration of DNA, pre-amplification and long-term storage of DNA, and amplification with real-time detection of DNA, respectively. The first miniaturised system applies isotachophoresis for pretreatment of DNA, where the DNA sample can be purified and concentrated using a discontinuous electrolyte system. Both qualitative and quantitative information can be acquired simultaneously. The second miniaturised system employs simple isothermal multiple displacement amplification, (MDA) for whole genome amplification (WGA) of human genomic DNA. The miniaturised WGA process showed a high efficiency of 95.8%, and the fidelity of the amplified products is extremely high as suggested by single-nucleotide polymorphisms analysis. For the last system, we developed a bidirectional shunting PCR microdevice equipped with real-time fluorescence detection, which allows higher flexibility and fast thermocycling by combining both advantages of stationary PCR and continuous-flow PCR. Real-time monitoring of RNase P PCR amplification from lower concentration human genomic DNA down to ~24 copy numbers or 12 cells was achieved. The three systems described in this thesis can be readily adapted to current reported miniaturised platforms. Such a fully integrated device capable of quantitative nucleic acid analysis remains an enigma, and with further development will represent significant importance for the development of point-of-care device.Das Design und die Implementierung miniaturisierter Systeme für die Analyse von Nukleinsäuren aus verschiedenen biologischen Proben haben eine beträchtliche Entwicklung erlebt. Fortschritte wurden insbesondere bei der Integration der Nukleinsäure-Vervielfältigung und -Detektion gemacht, wobei die Vervielfältigung meistens auf der Polymerase-Kettenreaktion (Polymerase Chain Reaction, PCR) beruht. Die Probenvorbereitung bleibt ein Haupthemmnis bei dem Versuch, eine quantitative Analyse mit vollständig miniaturisierten Systemen zu verwirklichen. Miniaturisierte Geräte für die Probenvorbereitung, Vervielfältigung und Detektion von Nukleinsäuren müssen weiter entwickelt werden, um ein vollständig integriertes System zu verwirklichen, das letztendlich in der Lage ist, die Genomanalyse einzelner Zellen mit "sample-in-answer-out"-Fähigkeit durchzuführen. In dieser Arbeit werden drei miniaturisierte Systeme präsentiert, die jeweils für die DNA-Aufreinigung und -Vorkonzentrierung, deren Vor-Vervielfältigung und Langzeit-Speicherung bzw. der Vervielfältigung mit Echtzeitdetektion von DNA verwendet werden können. Das erste miniaturisierte System nutzt die Isotachophorese zur Vorbehandlung der DNA, bei der die DNA-Probe in einem diskontinuierlichen Elektrolytsystem gereinigt und aufkonzentriert werden kann. Dabei können sowohl qualitative als auch quantitative Informationen simultan aufgenommen werden. Das zweite miniaturisierte System verwendet die simple Methode der isothermischen Multiplen Displacement Amplification (MDA) für die Vervielfältigung des gesamten Genoms (Whole Genome Amplification, WGA) der humanen, genomischen DNA. Der miniaturisierte WGA-Prozess zeigte eine hohe Effizienz von 95,8% und die Wiedergabetreue des vervielfachten Produkts ist extrem hoch, was durch die Ergebnisse einer Single Nucleotide Polymorphism (SNP) Analyse angedeutet wurde. Für des letzte System entwickelten wir ein kleines bidirektionales shunting-PCR-Instrument, in dem die injizierte DNA durch eine temperierte Zone mehrfach hin und her verschoben wird. Ausgestattet mit einem Fluoreszens Detektor, erreicht man eine höhere Flexibilität und schelle Temperaturzyklen, und kombiniert so die Vorteile der stationären PCR und der Durchfluss-PCR. Eine Echtzeitdetektion der RNase-P-PCR-Vervielfältigung von niedrig konzentrierter, humaner genomischer DNA mit einem Minimum von ~24 Kopien oder 12 Zellen wurde erreicht. Die drei in dieser Arbeit beschriebenen Systeme können direkt an aktuelle miniaturisierte Aufbauten angepasst werden. Ein solches vollständig integriertes, zur quantitativen Nukleinsäure-Analyse fähiges Gerät bleibt ein Mysterium, und zusammen mit weiteren Verbesserungen wäre es von großer Bedeutung für die Entwicklung von point-of-care Geräten

Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform

Configurable and Up-Scalable Microfluidic Life Science Platform for Cell Based Assays by Gravity Driven Sequential Perfusion and Diffusion

Microfluidics has the potential to significantly change the way modern biology is performed, but for this potential to be realized several on-chip integration and operation challenges have to be addressed. Critical issues are addressed in this work by first demonstrating an integrated microfluidic tmRNA purification and real time nucleic acid sequence based amplification (NASBA) device. The device is manufactured using soft lithography and a unique silica bead immobilization method for the nucleic acid micro purification column. The integrated device produced a pathogen-specific response in < 3 min from the chip-purified RNA. Further enhancements in the device design and operation that allow the on-chip integration of mammalian cell handling and culturing produced a novel integrated NASBA array. This system demonstrated for the first time that it is possible to combine on a single micro-device cell culture and real time NASBA. In order to expand the cell based assay capabilities of the integrated NASBA array and simplify the device operation novel hydrodynamics and cell sedimentation within trench structures and gravity driven sequential perfusion and diffusion mechanisms were developed. These mechanisms were characterized and implemented within an iCell array device. iCell array can completely integrate cell based assays with bio-analytical read-out. The device is highly scalable and can enable the configurable on-chip integration of procedures such as adherent and non-adherent cell-culture, cellstimulation, cell-lysis, cell-fixing, protein-immunoassays, bright field and fluorescent microscopic monitoring, and real time detection of nucleic acid amplification. The device uses on-board gravity driven flow control which makes it simple and economical to operate with dilute samples (down to 5 cells per reaction), low reagent volumes (50 nL per reaction), highly efficient cell capture (100% capture rates) and single cell protein and gene expression sensitivity. The key results from this work demonstrate a novel technology for versatile, fully integrated microfluidic array platforms. By multiplexing this integrated functionality, the device can be used from routine applications in a biology laboratory to high content screenings

Loop mediated isothermal amplification based detection of equine respiratory pathogens using a portable, smartphone-based setup

New tools are needed to enable rapid detection, identification, and reporting of infectious viral and microbial pathogens in a wide variety of point-of-care applications that impact human and animal health. We report the design, construction, and characterization of a multiplexed platform for multiplexed analysis of disease-specific DNA sequences that utilizes a smartphone camera as the sensor in conjunction with a handheld “cradle” that interfaces the phone with a silicon-based microfluidic chip embedded within a credit card-sized cartridge. Utilizing specific nucleic acid sequences for four equine respiratory pathogens as representative examples, we demonstrate the ability of the system to utilize a single 15 µL droplet of test sample to perform selective positive/negative determination of target sequences, including integrated experimental controls, in approximately 30 minutes. Our approach utilizes loop mediated isothermal amplification (LAMP) reagents pre-deposited into distinct lanes of the microfluidic chip, which, when exposed to target nucleic acid sequences from the test sample, generates fluorescent products that, when excited by appropriately selected light emitting diodes (LEDs) are visualized and automatically analyzed by a software application running on the smartphone microprocessor. The system achieves detection limits comparable to those obtained by laboratory-based methods and instruments. Assay information is combined with information from the cartridge and the patient to populate a cloud-based database for epidemiological reporting of test results

Molecular Microfluidic Bioanalysis: Recent Progress in Preconcentration, Separation, and Detection

This chapter reviews the state-of-art of microfluidic devices for molecular bioanalysis with a focus on the key functionalities that have to be successfully integrated, such as preconcentration, separation, signal amplification, and detection. The first part focuses on both passive and electrophoretic separation/sorting methods, whereas the second part is devoted to miniaturized biosensors that are integrated in the last stage of the fluidic device