Steven Vermeir

A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays

Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assays performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate this potential, on-chip adhesion islands are fabricated to immobilize MCF-7 human breast cancer cells. Viability studies are performed to assess the functionalization efficiency.

Digital Microfluidic High‐Throughput Printing of Single Metal‐Organic Framework Crystals

The first microfluidic method for accurately depositing monodisperse single MOF crystals is presented, enabling unprecedented high-throughput, yet flexible single-crystal printing. Individual droplets of MOF precursor solutions are actuated over a matrix of hydrophilic-in-hydrophobic micropatterns for the controlled generation of femtoliter droplets. As such, thousands of monodisperse single MOF crystals are printed per second in a desired pattern, without the use of impractically expensive equipment.

Biofunctionalization of electrowetting-on-dielectric digital microfluidic chips for miniaturized cell-based applications

In this paper we report on the controlled biofunctionalization of the hydrophobic layer of electrowetting-on-dielectric (EWOD) based microfluidic chips with the aim to execute (adherent) cell-based assays. The biofunctionalization technique involves a dry lift-off method with an easy to remove Parylene-C mask and allows the creation of spatially controlled micropatches of biomolecules in the Teflon-AF(®) layer of the chip. Compared to conventional methods, this method (i) is fully biocompatible; and (ii) leaves the hydrophobicity of the chip surface unaffected by the fabrication process, which is a crucial feature for digital microfluidic chips. In addition, full control of the geometry and the dimensions of the micropatches is achieved, allowing cells to be arrayed as cell clusters or as single cells on the digital microfluidic chip surface. The dry Parylene-C lift-off technique proves to have great potential for precise biofunctionalization of digital microfluidic chips, and can enhance their use for heterogeneous bio-assays that are of interest in various biomedical applications.

Design and optimization of a double-enzyme glucose assay in microfluidic lab-on-a-chip.

An electrokinetic driven microfluidic lab-on-a-chip was developed for glucose quantification using double-enzyme assay. The enzymatic glucose assay involves the two-step oxidation of glucose, which was catalyzed by hexokinase and glucose-6-phosphate dehydrogenase, with the concomitant reduction of NADP(+) to NADPH. A fluorescence microscopy setup was used to monitor the different processes (fluid flow and enzymatic reaction) in the microfluidic chip. A two-dimensional finite element model was applied to understand the different aspects of design and to improve the performance of the device without extensive prototyping. To our knowledge this is the first work to exploit numerical simulation for understanding a multisubstrate double-enzyme on-chip assay. The assay is very complex to implement in electrokinetically driven continuous system due to the involvement of many species, which has different transport velocity. With the help of numerical simulation, the design parameters, flow rate, enzyme concentration, and reactor length, were optimized. The results from the simulation were in close agreement with the experimental results. A linear relation exists for glucose concentrations from 0.01 to 0.10 g l(-1). The reaction time and the amount of enzymes required were drastically reduced compared to off-chip microplate analysis.

Evaluation and optimization of high-throughput enzymatic assays for fast l-ascorbic acid quantification in fruit and vegetables.

In this paper, we compare and evaluate the applicability of three UV-VIS absorbance based assays for high-throughput quantification of ascorbic acid in horticultural products. All the methods involve the use of a common enzyme (ascorbate oxidase) in combination with a different indicator molecule. The three methods were retrieved from literature: a direct oxidase-method, an OPDA coupled oxidase-method and a PMS-method, which is commercially available. The analysis in high-throughput context involved the analysis in microplates in combination with the use of an automated liquid handling system. We checked (i) the performance factors of the selected methods on standard solutions, (ii) the applicability of the defined methods in high-throughput context, and, (iii) the accuracy of the methods on real samples using HPLC as a reference technique. The OPDA-method was found to be the most appropriate method for the quantification of ascorbic acid in high-throughput context with a linear range between 7.0 and 950 mgL(-1) and excellent correlation parameters (slopes close to 1, intercepts close to 0, R(2)>0.91) with the reference technique when real samples were analyzed. Finally, this method was optimized for assay cost and assay time. Hereto the enzymatic reaction was mathematically described using a model for enzyme kinetics, which was then used to calculate the optimal concentrations of ascorbate oxidase and OPDA. As a result of the modeling the amount of enzyme in the assay could be reduced with a factor 2.5 without affecting significantly the reaction time. In a last step the optimal concentrations were used for a successful validation with the HPLC-reference technique.

Computational fluid dynamics model for optimal flow injection analysis biosensor design

This paper presents the optimization of a flow injection analysis (FIA) biosensor with respect to its design and operational parameters such as flow cell geometry, microfluidic channel dimensions, and flow rate. Since it is time consuming and costly to investigate the effect of each factor on the biosensor performance by building it, computational fluid dynamics (CFD) theory is presented as a great tool for finding optimal parameter values. This modeling approach has a high potential in the design of high accuracy FIA-biosensors, regardless of the chosen enzyme substrate system. As an example the optimal design for a glucose/glucose oxidase FIA biosensor is calculated with the CFD theory

High throughput flavour profiling of fruit

Publisher Summary For the analysis of the flavor of fruit and vegetables that require minimal sample preparation, there is a need for high-throughput techniques that are easy to operate at the lowest possible cost. The shorter commercial life cycle of fruit and vegetables and the increasing importance of flavor have necessitated the development of new high-throughput techniques for flavor analysis. This chapter describes the biology of aroma and taste perception by humans as this knowledge has inspired the development of biomimetic sensors, such as electronic noses and tongues. It introduces high-throughput spectroscopic techniques for measuring taste components, with an emphasis on Near-Infrared (NIR) spectroscopy. It also discusses the principle and applications of electronic tongues and describes new developments in high-throughput aroma profiling based on mass spectrometry and electronic noses. Biomimetic sensors use sensor arrays that generate complex signals when exposed to a headspace or immersed in juices. These signals are then analyzed by means of chemometric techniques and related to either sensory attributes of the fruit or vegetable or to individual flavor components. Electronic noses and tongues and spectroscopic techniques, such as (near) infrared spectroscopy require less sample preparation than traditional techniques and are faster. Some of them, in particular NIR spectroscopy, for measuring soluble solids content, are non-destructive and have been mounted on grading lines.

Model-based design and optimization of a multiplexed microfluidic biochip for multi-analyte detection

Multiple enzyme assays were systematically performed for simultaneous quantification of sucrose, D-glucose and D-fructose in a single microchannel reactor. The assay was based on optical detection of the reaction product, NADH, formed through a cascade of enzymatic reactions. For design optimization of the system, a model was developed for the microfluidic flow, enzyme kinetics and mass transfer of the different species involved in the analysis. The performance of the device was then optimized using the reduced form of these models in terms of process conditions (reagents volume and flow rate) thereby facilitating biosensor development. The proposed multiplexed device increases throughput and improves user-friendliness compared to equivalent microtiter plate assays. All sugars were quantified within 2.5 min in the optimized microchip based continuous system whereas it took at least two hours in standard microtiter plate analysis. Parallelization can further improve the throughput. In addition, the amount of reagents consumed reduced drastically. For example, the amount of Hexokinase used to detect glucose in 96 well microtiter plates with a total volume of 200 muL was 21.7 ng whereas in the designed microfluidic chip it only required 4.1 ng.