Tone reversal of an AFM lateral force image due to hybridization of oligonucleotides immobilized on polymers.
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Alkenes↗
Biomedical subjects
Ralf Lenigk
Publications and source records attributed to Ralf Lenigk.
Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection.
A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.
Animals↗
Hybridization enhancement using cavitation microstreaming.
Conventional DNA microarray hybridization relies on diffusion of target to surface-bound probes, and thus is a rate-limited process. In this paper, a micromixing technique based on cavitation microstreaming principle that was developed to accelerate hybridization process is explained. Fluidic experiments showed that air bubbles resting on a solid surface and set into vibration by a sound field generated steady circulatory flows, resulting in global convection flows and, thus, rapid mixing. The time to fully mix dyed solutions in a 50-microL chamber using cavitation microstreaming was significantly reduced from hours (a pure diffusion-based mixing) to 6 s. Cavitation microstreaming was implemented to enhance DNA hybridization in both fluorescence-detection-based and electrochemical-detection-based DNA microarray chips. The former showed that cavitation microstreaming results in up to 5-fold hybridization signal enhancement with significantly improved signal uniformity, as compared to the results obtained in conventional diffusion-based biochips for a given time (2 h). Hybridization kinetics study in the electrochemical detection-based chips showed that acoustic microstreaming results in up to 5-fold kinetics acceleration. Acoustic microstreaming has many advantages over most existing techniques used for hybridization enhancement, including a simple apparatus, ease of implementation, low power consumption (approximately 2 mW), and low cost.
Acoustics↗
Plastic biochannel hybridization devices: a new concept for microfluidic DNA arrays.
Conventional DNA hybridization assay kinetics depends solely on the diffusion of target to surface-bound probes, causing long hybridization times. In this study, we examined the possibilities of accelerating the hybridization process by using microfluidic channels ("biochannels") made of polycarbonate, optionally with an integrated pump. We produced two different devices to study these effects: first, hybridization kinetics was investigated by using an eSensor electrochemical DNA detection platform allowing kinetic measurements in homogenous solution. We fabricated an integrated cartridge for the chip comprising the channel network and a micropump for the oscillation of the hybridization mixture to further overcome diffusion limitations. As a model assay, we used an assay for the detection of single-nucleotide polymorphisms in the HFE-H gene. Second, based on the biochannel approach, we constructed a plastic microfluidic chip with a network of channels for optical detection of fluorescent-labeled targets. An assay for the simultaneous detection of four pathogenic bacteria surrogate strains from multiple samples was developed for this device. We observed high initial hybridization velocities and a fast attainment of equilibrium for the biochannel with integrated pump. Experimental results were compared with predictions generated by computer simulations.
Computer Simulation↗
Genotyping on a complementary metal oxide semiconductor silicon polymerase chain reaction chip with integrated DNA microarray.
A novel method for the fast identification of genetic material utilizing a micro-DNA amplification and analysis device (micro-DAAD) consisting of multiple PCR microreactors with integrated DNA microarrays was developed. The device was fabricated in Si-technology and used for the genotyping of Chinese medicinal plants on the basis of differences in the noncoding region of the 5S-rRNA gene. Successful amplification of the genetic material and the consecutive analysis of the fluorescent-labeled amplicons in the micro-DAAD by the integrated oligonucleotide probes were demonstrated. Parallel analysis was performed by loading the four PCR reactors of the micro-DAAD with different samples of 3-microL volume. Temperature sensors and heating elements of the micro-DAAD enable precise temperature control and fast cycling, allowing the rapid completion of a combined amplification and analysis (hybridization) experiment.
Genotype↗
DNA amplification and hybridization assays in integrated plastic monolithic devices.
PCR amplification, DNA hybridization, and a hybridization wash have been integrated in a disposable monolithic DNA device, containing all of the necessary fluidic channels and reservoirs. These integrated devices were fabricated in polycarbonate plastic material by CO2 laser machining and were assembled using a combination of thermal bonding and adhesive tape bonding. Pluronics polymer phase change valves were implemented in the devices to fulfill the valving requirements. Pluronics polymer material is PCR compatible, and 30% Pluronics polymer valves provide enough holding pressure to ensure a successful PCR amplification. By reducing the temperature locally, to approximately 5 degrees C, Pluronics valves were liquefied and easily opened. A hybridization channel was made functional by oligonucleotide deposition, using Motorola proprietary surface attachment chemistry. Reagent transport on the device was provided by syringe pumps, which were docked onto the device. Peltier thermal electrical devices powered the heating and cooling functionality of the device. Asymmetrical PCR amplification and subsequent hybridization detection of both Escherichia coli K-12 MG1655 and Enterococcus faecalis DNAE genes have been successfully demonstrated in these disposable monolithic devices.
DNA↗