|
Lab-on-a-Chip system for on-site diagnosis of selected Phytophthora species
Lydia Schwenkbier, Student, Institute of Photonic Technology
Some of the most important plant pathogens belong to the genus Phytophthora. Within the EU five Phytophthora species are regulated because the European and Mediterranean Plant Protection Organization identified them to present a very high risk. One of these Phytophthora species is P. ramorum that has caused the Sudden Oak Death in North America and the Larix decline in the United Kingdom. To prevent the epidemic spread of these fungus-like plant pathogens fast, reliable and robust diagnostic tools are mandatory. Until now microbiological, serological and PCR-based techniques are mainly used for routine diagnostic. For the specific identification of one regulated P. species, samples must be sent to a specialized laboratory which is time-consuming and means high financial losses for the nursery industry.
In the current project in collaboration with academic and industrial partners a standardized, high specific and sensitive protocol for on-site-analysis is established. The diagnostic system is based on a microfluidic biochip [1]. The plant sample e.g. leaves has to be macerated for the DNA amplification. Then a set of closely phylogenetic related Phytophthora species are detected by a specific DNA sequence on-chip with electrical and optical readout [2]. The miniaturized lab-on-a-chip system enables the identification of different Phytopthora species in parallel and directly in field.
Cartridge based magnetic immunodiagnostic platform for sensitive point-of-care testing (POCT) of biomarkers and viral pathogens
Raiah Gottheil, Researcher, NMI
A microfluidic platform for sensitive and quantitative detection of diagnostic targets suitable for POCT is reported. The analytes are detected through a magnetic bead based fluorescence sandwich immunoassay. The beads constitute both the solid phase for immobilization of capture molecules and for active manipulation by external magnetic fields. The cartridge comprises several chambers containing sample and assay reagents, separated by capillary stop valves. The assay procedure includes incubation of the sample with beads, washing, labeling with fluorescence detection antibody, washing and fluorescence readout. For the incubation with various assay fluids the beads are focused into a tiny aggregate which then is moved through valves. Upon entering a chamber, the aggregate is redispersed into pearl chain microstirrers using rotating magnetic actuation. Precisely controllable manipulation of beads allows for significant reduction of assay time while large sample volumes may be probed. Due to the unique actuation scheme, no active components such as electrodes or pumps are required within the injection moulded cartridge, hence allowing a cost effective system design. High binding constant of capture antibodies and multiple binding schemes are expected to enable detection of low concentration targets. An Interleukin-8 assay used to evaluate the system yielded a LOD of 1.25 pM.
Capillary Film Diagnostics
Nuno Reis, Lecturer, Loughborough University
The high manufacturing costs of disposable microfluidic systems is currently a bottleneck to large-scale adoption of microfluidic devices in point-of-care and home-care diagnostics markets. Such costs are mainly associated with microengineering the physical and optical features onto the devices, which still representing a substantial technical effort for the whole range of manufacturing techniques currently exploited for the fabrication of microfluidic products.
Melt-extrusion had its origins in plastic industry back to the 1930s, and is an extremely cost effective and efficient technique for shaping thermoplastic polymers because it can be operated as a continuous process. We have invented at the University of Cambridge a new melt-extrusion process that allows producing a continuous plastic ribbon containing a number of parallel microchannels embedded into it, called microcapillary film (MCF) [1]. The mean internal diameter of the capillaries can be manipulated from 10 micron up to 1 mm, and the combination of the flat film geometry with the optical properties of the selected thermoplastic material results in a unique transparent-multiplex microcapillary system at ultra-low cost.
This presentation will give an overview on the application of a 10-plex, 200 micron internal diameter MCF material melt-extruded from fluorinated ethylene propylene (FEP) material to simple, low-cost multi-analyte immunoassay detection [2]. Recently published proof-of-concept data demonstrated sensitive, multiplex serology of cancer biomarkers using fluorescence and optical detection, including flatbed scanner or a smart-phone camera [3]. Capillary film diagnostics technology is currently finding major applications in field assays and point-of-care diagnostics.
Fully-Integrated Fluorescence-Activated Cell Sorter
Francesca Bragheri, Researcher, CNR IFN
A current frontier of cellular biology is the manipulation, analysis and sorting of single cells. Populations of cells in culture and in organisms, although considered nominally identical, often present some heterogeneity, whose analysis may unravel the complexity of many biological phenomena [1]. In recent years considerable effort has been devoted to the development of integrated and low-cost optofluidic devices able to handle single cells. Among the different microfabrication technologies, femtosecond laser micromachining (FLM) [2] is ideally suited for this purpose as it provides the integration of both microfluidic and optical functions on the same glass-chip, thus leading to monolithic, robust and portable devices [3].
Here a new optofluidic device is presented, which is capable of sorting single cells, with optical forces, on the basis of their fluorescence. Both fluorescence detection and single cell sorting functions are integrated in the microfluidic chip by FLM. The capability of the device to act as a micro fluorescence-activated cell-sorter has been tested first on polystyrene beads, secondly on a cell sample of human transformed fibroblasts. Preliminary results open up new scenarios in the field of cytofluorimetry and fluorescence cell sorting, with the possibility to build miniaturized, portable devices.
High resolution amino group functionalization using a local DBD plasma-printer
Jean-Paul Schalken, Pre Doctoral Student, InnoPhysics B.V.
Modification of a surface by amino (–NH2) functionalities plays an important role in the research for biosensors. Functionalities are able to detect and bind specific chemical groups for lab-on-chip applications. Surface modification, as well as material deposition, is demonstrated to be possible using a local atmospheric pressure dielectric barrier discharge (DBD), which is able to generate a millisecond micro-plasma. This allows micro-patterned surface modification and material deposition on glass and polymer substrates with a resolution down to ~100 µm. Depending on the feed gases, it is also possible to perform a local etching process.
In this research surfaces are functionalized by amino groups using a N2/NH3 plasma gas mixture. The surface properties are characterized by contact angle measurements, as well as by XPS and FTIR analysis, showing the grafted –NH2 groups together with their grafting efficiency (N/C) and selectivity (NH2/N). These measurements are performed both on glass and polymer substrates, demonstrating amino functionalization of FEP (fluorinated ethylene propylene). Also the option of depositing small functionalized dots (~ 1 mm2) on FEP will be demonstrated.
A Microfluidic Platform for the Investigation of Directional Memory in Tip Growing Cells
Mahmood Ghanbari Mardasi, Researcher at Optical-Bio Microsystems Laboratory, Concordia University, Concordia University
In this paper a polydimethylsiloxane (PDMS) microfluidic platform is presented to study the directionality of growth of tip growing cells. The device enables manipulating and trapping of individual cells and consists of one inlet to introduce the cell suspension into the chip, three outlets to conduct the medium or cells out of the chip, a main chamber to guide the cells toward the growth mirochannels and serially arranged microchannels with different geometries connected to the main chamber to conduct the growth of individual tip growing cells. The device was tested by Camellia japonica pollen and experimental results show that pollen tubes can grow through microchannels with complex shapes and their growth rate are comparable to those obtained in conventional bulk assays. Also Contrary to root hairs [1], no sign of internal memory that determines the growth direction of the tube tip was observed in pollen tubes. The device has been optimized for use with pollen, but can be modified simply for the study of other tip growing cells produced from small cell bodies. Due to prominent similarities between pollen tube and nerve cells [2], Insights from this research can be useful in different areas such as medicine, pharmacology, and biomedical diagnostics.
Electrowetting on a micro-porous structure to enable a valve without moving parts in microfluidic systems
Michael Bassler, Principal Investigator, Institut fuer Mikrotechnik Mainz GmbH
Lifton et al. have shown the use of electrowetting in microbatteries by introducing a micro-porous structure, which is actively switched to the wetting state [1]. Here, electrowetting is used on a micro-porous structure inside a fluidic channel and a valve without moving parts is demonstrated. An electrically conductive membrane is functionalized with an insulting layer and a hydrophobic coating and is integrated into a microfluidic channel. Under constant pressure a finite liquid plug is stopped at the initially hydrophobic membrane. After switching to the hydrophilic state the plug passes the membrane until the rear end stops at the membrane. The plug detaches from the membrane after switching back to the hydrophobic state. The system is described by the liquid entry pressure (LEP), which is the pressure required to fully wet the pores and the corresponding liquid detachment pressure (LDP) for the de-wetting step. Both, LEP and LDP, depend on the size and shape of the pores, the surface tension of the liquid and the contact angle of the liquid on the pore surface. An analytical model and experimental results for LEP and LDP will be presented.
A microfluidic flow cytometer for continuous counting and detecting of sub-µm-sized bacteria in a disposable PMMA cartridge
Michael Bassler, Principal Investigator, Institut fuer Mikrotechnik Mainz GmbH
A miniaturized flow cytometer based on the concept of spatially modulated emission was implemented in a disposable microfluidic chip for detecting and counting bacteria and microorganisms. Microfluidic chips were fabricated by hot-embossing from low-cost PMMA material and a fluidic staining module was integrated on the chip to enable an instantaneous and continuous viable cells-count based on fluorescence emission. The detection concept will be described and experimental results on various types of microorganisms and flow cytometry calibration beads will be discussed. The system exhibits extraordinary sensitivity and is capable of detecting sub-µm-sized bacteria. As an application example the total viable cell count in drinking water will be presented.
High-precision velocimetry in the m/s-range for fluorescent beads and cells in a microfluidic flow cytometer
Michael Bassler, Principal Investigator, Institut fuer Mikrotechnik Mainz GmbH
Conventional flow cytometers operate at high cell velocities up to 10 m/s in quartz flow cells with rectangular cross sections of a few hundred micrometers. Here, a microfluidic flow cytometer is presented using microfluidic channels fabricated in low-cost PMMA with a rectangular cross section of 20 µm x 500 µm. The cytometer is based on the spatially modulated fluorescence emission method. The unique capability of the concept to measure the individual particle velocity with high precision at extreme velocities up to a few m/s is demonstrated. Velocity distributions for fluorescent microbeads and fluorescently tagged cells were measured under various flow conditions. Experimental results on the dynamics of the lateral particle and cell migration to the Segreé-Silberberg equilibrium position in the flow profile will be described and discussed. Optimum flow conditions in for flow cytometry in microchannels will be described.
Battery-driven, low-cost heating system for performing a fully automated DNA extraction & LAMP assay in the centrifugal LabTube platform
Melanie Hoehl, Graduate Student, Robert Bosch GmbH Corporate Research
DNA extraction and amplification are key elements of DNA based analysis. Currently, DNA extraction requires extensive handling and pipetting steps. To automate those processing workflows we recently introduced the LabTube platform [1]. The LabTube is a new microfluidic platform for Lab-on-a-Chip applications and is based on modules integrated in a falcon tube. The sample and reagent processing workflow can be automated be by applying process specific centrifugation protocols. In order to optimize the extraction efficiency (e.g. pre-heating of lysis or wash buffers) and to incorporate downstream analysis methods (such as DNA amplification or immunoassays) a cost efficient heating system in the disposable LabTube platform is desirable. Here we introduce a disposable battery-driven heating system for isothermal DNA amplification (LAMP) which can be integrated as a building block into the LabTube platform. We demonstrate fully automated DNA extraction in a standard laboratory centrifuge followed by subsequent automatic LAMP amplification of verotoxin producing Escherichia coli (VTEC ) DNA. The heating system consists of an SMD resistor and a NTC as heating and sensing elements, which are controlled and driven by a microcontroller and a battery. The LAMP reagents are stored in the elution chamber and the amplification starts immediately after the eluate is purged into the chamber. Furthermore, the heating system can be parallelized and enables the control of multiple independent heating zones within one LabTube. It is widely deployable, such as for other heating applications, for electrochemical reactions and/or for quality control.
All polymer based microfluidic devices for biomedical applications
Javier Gonzalo Ruiz, Researcher, IMB-CNM (CSIC)
Methodologies beyond traditional diagnostic, like point-of-care (PoC) and/or non-invasive monitoring, could allow a substantial reduction of healthcare costs compared to classical detection strategies performed at centralized laboratories. Nevertheless, and in spite of the volume of research done about diagnosis methodologies, there is a clear lack of technology transfer to the diagnosis market, due to the high manufacturability costs.
Description of novel materials and fabrication processes should pave the efficient transfer of the advances in diagnosis technology to the real world. The use of polymer-based flexible substrates, such as Cyclo Olefin Polymer (COP) or pressure sensitive adhesive (PSA), would accomplish these objectives. Those materials not only allow the fabrication of low-cost sensors, but possible the fabrication of fluidic systems using of low-cost processes, including rapid prototyping.
Two different approaches are presented for the production of biomedical devices. The first one is a COP-based microfluidic device for the detection of cardiac markers in human serum. The assay use of magnetic particles, coupled to electrochemical sensor as transduction system. The second one is a simple and easy to use COP/PSA-based fluidic device for the non-invasive monitoring of lactate evolution in sweat. In this case, sample loading is based on capillarity as the pumping force and real-time enzymatic detection is accomplished electrochemically.
Detection of rifampicin resistance in Mycobacterium tuberculosis isolated from clinical samples by using Flow-through Hybridization technology with unique system of Membrane Array as an initial indicator of multidrug resistance
Rubina Ghani, Associate Professor, Baqai Medical University
Tuberculosis (TB) is one of the major infectious causes of morbidity and mortality worldwide. Mycobacteria comprise a diverse group of bacteria that are widespread in nature, some of which cause significant disease in humans. TB is difficult to control due to the time taken for the microbiological diagnosis; typically culture on solid media takes 6–8 weeks. Members of the Mycobacterium tuberculosis complex (MTBC) are the most important human pathogens of the genus Mycobacterium.(1)
Advances in the field of molecular biology have provided rapid diagnostic tools that have reduced the turnaround times for detecting MTBC and drug resistance in cultures and directly in clinical specimens from weeks to days(2-4). The most potent first-line antituberculous drugs used for standard treatment of TB are isoniazid (INH) and rifampicin (RMP). Resistance of M. tuberculosis to both these drugs is termed multidrug resistance (MDRTB), and represents an important public health problem in many countries. Knowledge of the susceptibility patterns of M. tuberculosis isolates is important for the effective management of patients and for disease control. Second-line drugs used for treatment of MDRTB are more toxic, less effective, and more expensive.
Development of Lap-on-a chip technology for the analysis of Natural waters
Ahmed Fallatah, Student, University Of Hull
Current methodology for monitoring water quality normally requires the collection of samples from remote locations, which have then to be stabilized and transported to a laboratory for analysis utilizing large and expensive instrumental systems. This procedure is slow, costly and sample can degrade or become contaminated, therefore, developing a small-scale portable analysis system which will allow the analysis of water quality to be recorded in-situ, will enhance the speed of response to adverse changes in the quality of water.
This poster will describe the development of novel methodology based on microfluidics, which exploits the advantages of miniaturisation for sample introduction and separation of a selected range of inorganic ions that can be used for in- situ water monitoring. Various approaches will be described to achieve these aims including electrophoresis, separation, electrodialysis and preconcentration and the development of a novel suppressed conductivity detection system. The presentation will include a novel sample introduction method, in which ions are extracted into a separation channel through a potassium silicate monolith or electrodialysis membrane by applying a voltage. The results of anion extraction through potassium silicate monolith show very promising results but still require more optimisation.
A New Process for Fabricating Three-Dimensional Micro/Nano Channels
Sara Azimi, PhD student, National University of Singapore
A novel process has been developed for fabrication of combined micro- and nano-channels which can be used to make three-dimentional silicon molds or create arrays of buried, hollow micro/nano channels and cavities in porous silicon and silicon dioxide (i.e. glass). This process is based on high-energy ion irradiation, such as 100 keV to 2 MeV protons, of p-type silicon wafers. Ion irradiation alters the hole current flow during subsequent electrochemical anodization, allowing the anodization rate to be slowed/stopped for low/high fluences and forming multilevel surfaces in a single etch step which can be used as a mold for imprinting in a polymer. For low fluences, we use a recently reported current transport mechanism observed during electrochemical anodization of ion irradiated p-type silicon, which dominates the total current flowing and hence the anodization behavior is localized areas [1]. Using this mechanism in conjunction with high temperature oxidation enables fabrication of buried, multilevel micro- and nano-channels in porous silicon and glass in a single etch step. This is important in fields such as microfluidics and Lab-on-a-chip (LOC) systems, as it provides a much simpler process for fabrication of complex micro/nano structures than what is currently available.
Simple Methods for forming Protein Monolayers
Nobuyoshi Maeji, Chief Scientific Officer, Anteo Diagnostics
Miniaturization and/or label free detection methods create a need for stringent uniformity at the surface interface between the synthetic surface and antibodies and other capture agents. However, direct immobilization of antibodies to synthetic surfaces, such as silicon wafers, ceramics, or plastics damage proteins and often a compromise needs to be made between having an antibody mono-layer and maintaining its stability and function.
Mix&Go™, a novel chelation-based surface chemistry, was developed to enable protein binding on most surfaces used in point-of-care devices. Mix&Go’s metal polymers bind any surfaces having electron donating potential to form a thin, stable, and activated surface. Each chelation point alone binds weakly but multiple chelation points together enable gentle yet strong protein binding.
Mix&Go represents a “one-size-fits-all” surface chemistry approach in situations where maximum antibody performance within mono-layers is critical. Here the effects of a one-step, one-hour Mix&Go activation of gold colloids in Localized Surface Plasmon Resonance are discussed. In addition, increased protein stability on silica and increased assay performance with decreasing surface area on polystyrene surfaces are shown.
A Distinctive Analysis of Fluid Flow Behavior in an AC Electroosmotic Micropump
Nurul Md Yunus, Researcher, Universiti Putra Malaysia
Under project grant “Development of a Low Power AC Electrokinetic Micropump”, Universiti Putra Malaysia, an alternating current electroosmotic micropumping device on glass substrate has been analysed. An asymmetric electrode array made of platinum was used as a micropump. It consisted of pairs of electrodes; the larger electrode of a pair was 15 µm wide and the smaller electrode 5µm. The gap between a large and a small electrode was 5 µm. The gap between each pair was 15 µm. The total length of the electrodes was 3 mm. The velocity of the AC electroosmotic micropump was studied experimentally and theoretically using linear analysis. Two different theoretical approaches were used: firstly on the basis of the "Ramos slip velocity" and secondly the "Coupled ACEO numerical" model. Microsphere tracer particles were suspended in a Potassium Chloride (KCl) solution with a conductivity of 14.5 µS/m to determine pumping performance. The experimental results were obtained for a different range of voltages and frequencies and they were in good agreement with the theoretical predictions, produced using computer simulation with MATLAB. The Ramos slip velocity model described the data better than the coupled ACEO model.
|
