Mark Kielpinski

Development of a lab-on-a-chip device for diagnosis of plant pathogens

A lab-on-a-chip system for rapid nucleic acid-based analysis was developed that can be applied for diagnosis of selected Phytophthora species as a first example for use in plant pathology. All necessary polymerase chain reaction process (PCR) and hybridization steps can be performed consecutively within a single chip consisting of two components, an inflexible and a flexible one, with integrated microchannels and microchambers. Data from the microarray is collected from a simple electrical measurement that is based on elementary silver deposition by enzymatical catalyzation. Temperatures in the PCR and in the hybridization zone are managed by two independent Peltier elements. The chip will be integrated in a compact portable system with a pump and power supply for use on site. The specificity of the lab-on-a-chip system could be demonstrated for the tested five Phytophthora species. The two Pythium species gave signals below the threshold. The results of the electrical detection of the microarray correspond to the values obtained with the control method (optical grey scale analysis).

Micro Flow-Through Thermocycler with Simple Meandering Channel with Symmetric Temperature Zones for Disposable PCR-Devices in Microscope Slide Format

Chip-based flow-through PCR implements the PCR as a continuous process for nucleic acid analytics. The sample is transported in a winding channel through temperature zones required for denaturation, annealing and extension. Main fields of application are the monitoring of continuous processes for rapid identification of contaminants and quality control as well as high throughput screening of cells or microorganisms. A modular arrangement with five heating zones for flow-through PCR is discussed and evaluated. The special heater arrangement allows the implementation of up to 40 cycles on the footprint of a microscope slide, which is placed on top of a 5 zones heating plate. Liquid/liquid two phase flow of PCR reaction mixture and mineral oil have been applied to create a segmented flow process scheme. In that way, the developed system may provide flow-through PCR as a unit operation for the droplet based microfluidics platform. The single use of disposable devices is commonly preferred due to the sensitivity of the PCR process to contaminations. All-glass microfluidic chips and disposable chip devices, made from polycarbonate as a replication with identically geometry, have been fabricated and tested. For the first time, microchannel geometries with nearly circular profile developed by all-glass technology have been transferred to mass fabrication by injection compression molding. Both devices have been successfully applied for the detection of the tumor suppressor gene p53. Although product yield and selectivity of the amplification process do not depend on the chip material, a well defined, reliable segmented flow regime could only be realized in the all-glass chip.

Effects of Fluid and Interface Interaction on Droplet Internal Flow in All-Glass Micro Channels

Segmented flow of aqueous droplets transported by an immiscible separation medium in all-glass micro channels is characterized by an interplay of interface interaction and phase internal flow field formation. As a result, pressure drop, local shear rates and mixing performance are determined by this interplay. Yet, these effects are strongly influenced by the properties of the employed fluids, wettability of the channel walls and experimental conditions like flow rate and fluid phase ratio. We utilize micro Particle Imaging Velocimetry (μPIV) aside direct pressure measurements as a tool to measure and visualize these effects and show their dependency on fluidic properties and experimental conditions. All-glass micro channels have been prepared by wet etching of glass half channels and anodic bonding of two glass substrates. Micro channels have coplanar faces at top and bottom and sidewalls with a half circular shape. Wetting properties of the internal micro channel surfaces are adjusted by treatment with octadecyl-trichlorosilane. Sample droplets that fully seal the channel are embedded in a separation fluid that completely wets the channel. A permanent thin film of separation fluid prevents direct contact between sample fluid and micro channel walls. In contrast to micro channels with rectangular cross-section [1,2], we observe alternating flow circulation in separation medium and droplets under these conditions [3]. As a consequence, the total pressure drop is almost independent of droplet viscosity.Copyright

Chip Systems for Analysis of Nucleic Acids with Integrated Amplification and Detection

Analytical methods for the detection of nucleic acids are suitable for a variety of applications as, for example, the analytical applications in that field are diagnosis of infectious or hereditary diseases, analysis of microbial contaminations, determination of family relations, or identification of genetically modified organisms. In many cases, the available number of DNA or RNA molecules is not sufficient for common detection methods based on hybridization processes. The standard procedure for nucleic acid amplification is polymerase chain reaction (PCR). This thermally controlled, enzymatically catalyzed, and cyclically performed process enables the synthesis of small amounts of nucleic acids. In this chapter, a microchip thermocycler with integrated heaters and temperature sensors for droplet-based on-chip PCR is described. The chip is coupled with a temperature controlling unit and a device for measuring the fluorescence intensity. Different amounts of initial DNA down to 2 molecules/μl could be successfully detected. The applied template DNA was obtained from different Phytophthora species, a group of phytopathogenic organisms with worldwide significance. Small numbers of cells could also be applied and added directly to the PCR mixture. This was studied successfully with human brain microvascular endothelial (HBME) cells and with spores from Phytophthora. Furthermore, another lab-on-a-chip system is presented that combines on-chip PCR and microarray technology within one chip. The chip consists of a DNA chip with integrated electrode structures and a silicone-based chip with incorporated microchannels and reaction chambers. For temperature management, there are heating elements and temperature sensors connected to the chip. Fluidic transportation is implemented by small peristaltic pumps that are installed to the system. Detection is based on enzymatically catalyzed silver deposition. In case of successful hybridization, the microarray spots turn gray or black, and the electric resistance at the electrode gap will decrease. So the hybridization can be analyzed either by optical-based measurement of the gray scale value or by electrical readout of the electrodes. Advantages of lab-on-a-chip systems are low necessary amounts of reagents and power supply, short reaction times, and small dimensions of the systems. These make the lab-on-a-chip system transportable and, therefore, well suited for point-of-care applications.

Application of Self-Control in Droplet-Based Microfluidics

Droplet-based microfluidics provide a powerful platform for high-throughput operations applied in micro analytics, micro reaction technology and live sciences. Todays research interests focus on the development of highly integrated fluidic networks for sample processing according to a microchemical or microanalytical protocol. Normally, fluidic networks with integrated fluidic loops and bypasses are very complicated systems that require a huge effort for external control and integration of actor components. In contrast, in droplet-based microfluidics interface generated forces can be used to temporarily seal bypasses or to generate well defined pressure gradients at strictures. This potential can be used to implement self-control and self-synchronization at functional nodes in order to minimize the effort for external control and actors integration. Here we report on progress in development of functional nodes for self-synchronized 1:1 coalescence of two independently generated droplet sequences at a Y-shaped junction and on approaches for droplet aliquotation at a Y-shaped bifurcation. The droplet connector automatically balances the time delay between two droplets arriving at the junction. On this account, strictures are integrated into the Y-junction and an additional bypass connects the arriving channels. The first arriving droplet stops at the stricture until its fusion partner arrives. The droplet splitter performs an 1:1 aliqoutation of all elements of a droplet sequence. The main challenges are the balancing of pressure differences at the outlets and the correct aliquotation of droplets independent of their volume at a wide range of flow rates. The splitter design is based on the rule that forces required for splitting are always lower than the forces required for complete droplet inflow into only one of the outlet channels without splitting.Copyright

5 Zones Thermocycler for Flow-Through On-Chip PCR

A microfluidic chip system for flow-through PCR reactions with an optimized thermal profile consisting of five temperature zones was presented. It allows the implementation of one PCR cycle in a half channel loop. In contrast, conventional systems with a three zones arrangement require a complete channel loop per cycle. Therefore, this arrangement increases the level of integration and allows the implementation of a 40 cycles flow-through thermocycler on the footprint of a microscope slide. To obtain a high throughput of samples in a small volume (10–100 nl), the fluidic chip device was designed to operate at segmented-flow conditions for PCR. That way, each droplet may contain a single sample that is independently processed while transported through the microchannel. To achieve stable fluidic conditions, the surface of the micro-channels was modified. In addition to the successful flow-through PCR reaction in the micro reactor, the detection of the tumor suppressor p53 for clinical diagnostics was demonstrated.© 2008 ASME