2011 16th International Solid-State Sensors, Actuators and Microsystems Conference, 2011
A microfluidic control system consisting of micropump/valves with a re-usable pneumatic actuator ... more A microfluidic control system consisting of micropump/valves with a re-usable pneumatic actuator and a disposable polymer lab-on-a-slide is presented. The lab-on-a-slide was fabricated using low cost methods, such as injection moulding of TOPAS ® cyclic olefin copolymer (COC) slide, lamination of different layers of polymer, and ultrasonic welding of TOPAS ® lid to the slide. The re-usable pneumatic actuator not only simplifies the design of the lab-on-a-slide and reduces the fabrication cost, but also reduces the possibility of cross contamination during replacement of the disposable lab-on-a-slide. The experiment results show the micropump is capable of providing a wide range of flow rate (20-300 µL/min), and microvalves provides a reliable fluidic control in the different parts of the system.
The determination of pharmacokinetic properties of drugs, such as the distribution coefficient (D... more The determination of pharmacokinetic properties of drugs, such as the distribution coefficient (D) is a crucial measurement in pharmaceutical research. Surprisingly, the conventional (gold standard) technique used for D measurements, the shake-flask method, is antiquated and unsuitable for the testing of valuable and scarce drug candidates. Herein, we present a simple microfluidic platform for the determination of distribution coefficients using droplet-based liquid−liquid extraction. For simplicity, this platform makes use of gravity to enable phase separation for analysis and is 48 times faster and uses 99% less reagents than performing an equivalent measurement using the shake-flask method. Furthermore, the D measurements achieved in our platform are in good agreement with literature values measured using traditional shake-flask techniques. Since D is affected by volume ratios, we use the apparent acid dissociation constant, pK′, as a proxy for intersystem comparison. Our platform determines a pK′ value of 7.24 ± 0.15, compared to 7.25 ± 0.58 for the shake-flask method in our hands and 7.21 for the shake-flask method in the literature. Devices are fabricated using injection molding, the batchwise fabrication time is <2 min per device (at a cost of $1 U.S. per device), and the interdevice reproducibility is high.
The 13th International Conference on Solid-State Sensors, Actuators and Microsystems, 2005. Digest of Technical Papers. TRANSDUCERS '05., 2005
Dielectrophoresis (DEP) and flow cytometry are powerful technologies and widely applied in microf... more Dielectrophoresis (DEP) and flow cytometry are powerful technologies and widely applied in microfluidic systems for handling and measuring cells and particles. Here we present a novel microchip with a positive DEP selective filter integrated with two micro flow cytometers for real-time monitoring of cell sorting processes. On the microchip, two flow cytometers are set upstream and downstream of the DEP filter. When a cell pass through the detection windows, the light scattered by the cell is measured by integrated polymer optical elements. The effects of flow rate, applied voltage, conductivity of the sample, and frequency of the electric field on the sorting efficiency of the chip were investigated. Using the chip, viable and nonviable yeast cells were sorted with high efficiency. At 2 MHz, more than 90 % of the viable and less than 10 % of the non-vial cells were captured on the DEP filter.. Furthermore, the microstructure is simple to fabricate and can easily be integrated with other microstructures for lab-on-a-chip applications (164).
Biosensing systems based on detecting changes in cantilever surface stress have attracted great i... more Biosensing systems based on detecting changes in cantilever surface stress have attracted great interest. To achieve high reliability of measurements, high quality and high reproducibility in functionalization of the sensor surface are key points. In this paper, we investigate different methods to clean and regenerate the sensing surface of cantilever biosensors. Perchloric acid potential sweep, potassium hydroxide-hydrogen peroxide, and piranha cleaning are investigated here. Peak-current potential differences from cyclic voltammetry, X-ray photo-electron spectroscopy and fluorescence detection are applied to characterize surface cleanliness. The experimental results show that piranha cleaning is the most reliable and efficient cleaning procedure.
The enantioselectivity of enzymatic reactions in organic (co)solvents has been studied intensivel... more The enantioselectivity of enzymatic reactions in organic (co)solvents has been studied intensively over the last decades. However, the question of how the enantioselectivity of an enzymatic reaction can be influenced by the reaction medium still remains. In order to understand the influence of cosolvent on enzyme enantioselectivity, we have investigated the kinetics of chymotrypsin catalyzed hydrolysis of ethyl (R)and (S)-3-phenyllactate in water-cosolvent mixtures (up to 36%w/w cosolvent). Our experimental results show that the apparent Michaelis constant for either enantiomer of the substrate increases more than twenty-fold. This can be explained quantitatively by a decrease of the relative thermodynamic activity coefficient of the substrate. We found that VZax and V& decrease, but the V,&,/V& ratio remains constant. This implies that there is no overall effect of the cosolvent on the enantioselectivity, whereas the reaction rates change by orders of magnitude. These findings are in agreement with the simple model presented in this paper, that relies on the assumption that the thermodynamic activity coefficients of the different enzyme species are equal in a particular solvent.
Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease... more Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or molecular-based methods are time consuming and usually take a few hours to days to complete. In response to the demand for rapid on line or on site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic bead-based sample preparation and loop-mediated isothermal amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time, will greatly enhance the practical applicability of the LOC system for rapid on-site screening of Salmonella for applications in food safety control, environmental surveillance, and clinical diagnostics.
Cell sorting has become increasingly important in basic research and medical diagnostics. We demo... more Cell sorting has become increasingly important in basic research and medical diagnostics. We demonstrate here that magnetic activated sorting and fluorescent activated sorting can also be envisaged in microfluidic silicon structures. In these structures we take advantage of the laminar flow in microstructures. Paramagnetic particles sheeted by two buffer streams are separated from non-magnetic particles by deflection in a magnetic field gradient. Particles labelled with a fluorochrome are sheeted by two buffer streams, excited and detected by a photo multiplier tube. The photo multiplier tube switches a valve on one of the outlets of the sorter microstructure and selects a particle by forcing it to the collecting outlet. Sorting in these structures is now being optimised with paramagnetic and fluorescent beads, but ultimately it is our intention to sort living cells. One of the major advantages of microfluidic structures is the capability of integrating various functional modules. Simultaneously with optimising our sorter structures, we are investigating various levels of system integration that may improve the performance of the structure. For many cell sorting applications involving biohazardous materials or requiring absolute sterile techniques, a single use microstructure would be advantages. Using silicon processing techniques, metal plating and injection moulding we have produced polymer microfluidic structures. Laminar flow and flow switching have been demonstrated in these polymer microstructures and we are currently investigating their performance in sorting.
In this work, we develop a generic DNA based sensing platform used for characterizing surface fun... more In this work, we develop a generic DNA based sensing platform used for characterizing surface functionalization and detecting DNA hybridization. Silicon nitride cantilever sensors are fabricated with an integrated three-electrode system and integrated in a microfluidic chip. Cantilevers with gold electrodes are functionalized with thiol-modified single stranded DNA (ssDNA) probes to detect target DNA. During functionalization and hybridization, information related to nanomechanical changes on the surface are obtained by optical measurements of changes in cantilever deflection. Simultaneously, the process is monitored electrochemically. The results clearly indicate that the electrochemical cantilever sensor is very sensitive for detecting DNA hybridization at the cantilever surface.
The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical ap... more The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical applications over the last decade. Many applica- tions utilize SYBR Green I dye to follow the accumulation of amplicons in real time. SYBR Green I has, however, a number of limitations that include the inhibition of PCR, preferential binding to GC-rich sequences and effects on melting curve
More than 200 known diseases are transmitted via foods or food products. In the United States, fo... more More than 200 known diseases are transmitted via foods or food products. In the United States, food-borne diseases are responsible for 76 million cases of illness, 32,500 cases of hospitalisation and 5000 cases of death yearly. The ongoing increase in worldwide trade in livestock, food, and food products in combination with increase in human mobility (business-and leisure travel, emigration etc.) will increase the risk of emergence and spreading of such pathogens. There is therefore an urgent need for development of rapid, efficient and reliable methods for detection and identification of such pathogens. Microchipfabrication has had a major impact on electronics and is expected to have an equally pronounced effect on life sciences. By combining micro-fluidics with micromechanics, micro-optics, and microelectronics, systems can be realized to perform complete chemical or biochemical analyses. These so-called 'Labon-a-Chip' will completely change the face of laboratories in the future where smaller, fully automated devices will be able to perform assays faster, more accurately, and at a lower cost than equipment of today. A general introduction of food safety and applied micro-nanotechnology in life sciences will be given. In addition, examples of DNA micro arrays, micro fabricated integrated PCR chips and total integrated lab-on-a-chip systems from different National and EU research projects being carried out at the Laboratory of Applied Micro-Nanotechnology (LAMINATE) group at the National Veterinary Institute (DTU-Vet) Technical University of Denmark and the BioLabchip group at the Department of Micro and Nanotechnology (DTU-Nanotech), Technical University of Denmark (DTU), Ikerlan-IK4 (Spain) and other 16 partners from different European countries will be presented.
Colorectal cancer (CRC) is one of the most prevalent types of cancer, causing significant morbidi... more Colorectal cancer (CRC) is one of the most prevalent types of cancer, causing significant morbidity and mortality worldwide. CRC is curable if diagnosed at an early stage. Mutations in the oncogene KRAS play a critical role in early development of CRC. Detection of activated KRAS is of diagnostic and therapeutic importance. In this study, KRAS gene fragments containing mutations in codon 12 were amplified by multiplex PCR using a 5'-Cy5-labeled reverse primer in combination with 3'-mutation-specific forward primers that were linked with four unique nucleotide-sequence tags at the 5'-end. The Cy5-labeled reverse primer was extended under PCR amplification to the 5'-end of the mutation-specific forward primers and thus included the complimentary sequence of the tag. PCR products were hybridized to tag-probes immobilized on various substrates and detected by a scanner. Our results indicate that all mutations at codon 12 of KRAS derived from cancer cells and clinical samples could be unambiguously detected. KRAS mutations were accurately detected when the mutant DNA was present only in 10% of the starting mixed materials including wild-type genomic DNA, which was isolated from either cancer cells or spiked fecal samples. The immobilized tag-probes were stable under multiple thermal cycling treatments, allowing re-use of the tag-microarray and further optimization to solid PCR. Our results demonstrated that a novel oligonucleotide-tagged microarray system has been developed which would be suitable to be used for detection of KRAS mutations and clinical diagnosis of CRC.
A bioimpedance platform is presented as a promising tool for non-invasive real-time monitoring of... more A bioimpedance platform is presented as a promising tool for non-invasive real-time monitoring of the entire process of three-dimensional (3D) cell culturing in a hydrogel scaffold. In this study, the dynamics involved in the whole process of 3D cell culturing, starting from polymerisation of a bare 3D gelatin scaffold, to human mesenchymal stem cell (MSC) encapsulation and proliferation, was monitored over time. The platform consists of a large rectangular culture chamber with four embedded vertical gold plate electrodes that were exploited in two-and three terminal (2T and 3T) measurement configurations. By switching between the different combinations of electrode couples, it was possible to generate a multiplexing-like approach, which allowed for collecting spatially distributed information within the 3D space. Computational finite element (FE) analysis and electrochemical impedance spectroscopic (EIS) characterisation were used to determine the configurations' sensitivity field localisation. The 2T setup gives insight into the interfacial phenomena at both electrode surfaces and covers the central part of the 3D cell culture volume, while the four 3T modes provide focus on the dynamics at the corners of the 3D culture chamber. By combining a number of electrode configurations, complementary spatially distributed information on a large 3D cell culture can be obtained with maximised sensitivity in the entire 3D space. The experimental results show that cell proliferation can be monitored within the tested biomimetic environment, paving the way to further developments in bioimpedance tracking of 3D cell cultures and tissue engineering.
lines for EIS analysis are presented and demonstrated to provide porosity information in physiolo... more lines for EIS analysis are presented and demonstrated to provide porosity information in physiological buffer that agrees well with a more conventional weight-based approach. We also propose frequency ranges that may serve as means of single-frequency measurements for fast scaffold characterization combined with in vitro monitoring of 3D cell cultures.
Dual Enlargement of Gold Nanoparticles: From Mechanism to Scanometric Detection of Pathogenic Bacteria
Small, 2011
A mechanism of dual enlargement of gold nanoparticles (AuNPs) comprising two steps is described. ... more A mechanism of dual enlargement of gold nanoparticles (AuNPs) comprising two steps is described. In the first step, the AuNPs are enlarged by depositing Au atoms on their crystalline faces. In this process, the particles are not only enlarged but they are also observed to multiply: new Au nuclei are formed by the budding and division of the enlarged particles. In the second step, a silver enhancement is subsequently performed by the deposition of silver atoms on the enlarged and newly formed AuNPs to generate bimetallic Au@Ag core-shell structures. The dual nanocatalysis greatly enhances the electron density of the nanostructures, leading to a stronger intensity for colorimetric discrimination as well as better sensitivity for quantitative measurement. Based on this, a simple scanometric assay for the on-slide detection of the food-born pathogen Campylobacter jejuni is developed. After capturing the target bacteria, gold-tagged immunoprobes are added to create a signal on a solid substrate. The signal is then amplified by the dual enlargement process, resulting in a strong color intensity that can easily be recognized by the unaided eye, or measured by an inexpensive flatbed scanner. In this paper, dual nanocatalysis is reported for the first time. It provides a valuable mechanistic insight into the development of a simple and cost-effective detection format.
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Papers by Anders Wolff