Dynamic Actuation of Magnetic Beads for Immunoassays on-chip

This thesis was carried out in the frame of the European project DetectHIV aiming the development of a new biosensor platform for the highly sensitive detection of the HIV capsid protein p24. We explore the implementation of a magnetic bead-based lab-on-a-chip system, offering significant advantages compared to conventional systems, mainly through the possibility of controlled manipulation of the magnetic carriers on-chip. In particular, microfluidic immunoassays using functionalized magnetic beads raise increasing interest. In this thesis, we present a microsystem for the magnetic manipulation of superparamagnetic beads on-chip. A highly confined and dynamically actuated plug of biochemically functionalized beads is formed in a microchannel. This plug extends over the channel cross-section, thus allowing efficient analyte capture from the flow. Subsequent immobilization of the plug for incubation modifies the colloidal state (agglutination test). Dynamic actuation of beads is enabled by superposing a static magnetic field and a time-varying magnetic field. The latter field is highly focused and concentrated across the microchannel by means of soft magnetic microtips. A new method for the fabrication of rigid monolithic SU-8 microchannels allows control and ready mechanical integration of the microtips with the microfluidic structure. A protocol for performing magnetic bead-based immuno-agglutination assays on-chip using our system was developed. A simple detection method based on the swelling of the released plug after agglutination is presented. We demonstrate the feasibility of on-chip agglutination tests by means of a streptavidin/biotinylated-bovine serum albumin (bBSA) model assay. A detection limit of about 200 pg/mL (≈3 pM) was obtained. Furthermore, the potential of the magnetic actuation method was emphasized by implementing a heterogeneous immunoassay with a dendritic amplification mechanism. Dendritic amplification aims to increase the detection sensitivity. Amplification of the detection signal is achieved by alternating exposure of the beads to a flow of fluorescently labeled streptavidin molecules and biotin conjugated anti-streptavidin, respectively. The magnetic system developed in the frame of this thesis was integrated in the final biosensor platform of the DetectHIV project. This platform comprises an integrated chip cartridge with an optical detection module. This design is outlined in the last part of the thesis.

Microelectronics Meets Biology

Emile Dupont

Microfluidics plays a key role in the design of automated platforms for realizing biological assays in a miniaturized format. Several advantages are offered by a microfluidic system, namely, low sample and reagent consumption (typically a few microliters), the possibility to mass-produce and to integrate several analysis modules enabling portable automated applications, fast analysis and low cost. Furthermore, the combination of advanced Complementary Metal-Oxide-Semiconductor (CMOS) technology and microfluidics can result in flexible and easy-to-use hybrid systems that hold large potential for application in an analytical laboratory or at the point-of-care. In this Thesis, two different monolithic silicon CMOS chips were designed, fabricated and tested, demonstrating state-of-the-art performance utilising a novel fluorescence detection principle. They allowed the detection of monoclonal antibodies in a solution or the precise counting of specifically labelled fluorescent cells in a mixed-cell sample. The proposed detection method does not require the utilization of a fluorescence camera and filter set. The first part of this Thesis presents different methods, fabrication processes and materials for microfluidic cartridges that can be integrated in a hybrid way with silicon CMOS chips. Glass and SU-8 capillaries are tested, a novel material (NOA 63) is proposed for fabricating a natively hydrophilic microfluidic cartridge, and the fabrication of custom-designed polydimethyl(-siloxane) (PDMS) cartridges for easy integration on top of a CMOS chip is introduced. The second part of this Thesis presents the combination of microfluidics with a monolithic and fully-integrated CMOS chip for the manipulation and detection of fluorescent magnetic beads, inside a PDMS microchannel that is loosely positioned on top of the chip. Magnetic manipulation is done by current actuation of microcoils on the chip; detection is achieved using single photon avalanche diodes (SPADs), located in the centre of each microcoil, that count the fluorescent photons originating from a single labeled magnetic bead. This approach permits in principle microscope-less fluorescence detection with high sensitivity. Using sandwich immunoassays on the beads' surfaces, the selective detection of the cancer biomarker 5D10 monoclonal antibody (mAb) from a non-purified hybridoma cell-culture medium is demonstrated. In the third part, a CMOS chip comprising a matrix of SPADs is designed, fabricated and tested. The recognition of single fluorescently-labelled cells from the breast cancer cell line MCF-7 in a test solution of mixed MCF-7 and non-fluorescent Jurkat cells is demonstrated, without the need of a bulky and expensive fluorescent microscope setup. Moreover, due to the use of a SPAD matrix spanning the complete width of the microchannel, the system does not require any cell focusing mechanism, needed when using a single detector. This allowed the use of a very simple, easily replicable and disposable plastic microfluidic cartridge.

EPFL2010

Separation and Focusing of Magnetic Beads for Agglutination Tests

Rana Afshar Ghasemlouy

Functional magnetic micro- and nanoparticles are used in bioanalytical applications as solid carriers for capture, transport and detection of biomolecules or magnetically labeled cells. Colloidal suspensions of such particles provide a large specific surface for chemical binding and therefore allow highly efficient interactions with target molecules in a sample solution. Controlled actuation and manipulation of these mobile substrates in the microfluidic format offers interesting new opportunities for on-chip bioassays with previously unmatched properties. Separation of functional magnetic particles or magnetically labeled entities is therefore a key feature for bioanalytical or biomedical applications and also an important component of lab-on-a-chip devices for biological applications. In this thesis we present two novel integrated microfluidic magnetic bead manipulation devices. The first system consists of dosing of magnetic particles, controlled release and subsequent magnetophoretic size separation with high resolution. On-chip integrated soft-magnetic microtips with different shapes provide the magnetic driving force for the bead manipulation. The system is designed to meet the requirements of specific bioassays, in particular of on-chip agglutination assays for the detection of rare analytes, in which the latter can be quantified via the counting of the particle doublets. In a second approach, magneto-microfluidic three-dimensional (3D) focusing of microparticles has been developed. In this system, magnetic microparticles from a dense plug are released into a single streamline with longitudinal inter-particle spacing. Plug formation is induced by a high-gradient magnetic field generated at the sidewall of a microchannel by a micromachined magnetic tip that is connected to an electromagnet. Controlled release of the microparticles is achieved using an exponential damping protocol of the magnetic retention force in the presence of an applied flow. Carefully balancing the relative strengths of the drag force imposed by the flow and the magnetic retention force moreover allows in-flow size separation of the microparticles. Adding subsequently a lateral sheath flow microchannel focuses the microparticles into a single stream situated within 0plusmn; 5 µm from the channel center axis. Our system for 3D focusing and in-flow separation of magnetic microparticles has been used for performing an immuno-agglutination assay on-chip. 3D focusing was of the basis of reliable in-flow counting of singlets and agglutinated doublets. We demonstrated the potential of the agglutination assay in a microfluidic format using a streptavidin/biotinylated-bovine serum albumin (bBSA) model system. A bBSA detection limit of about 400 pg/mL (6 pM) is achieved.

EPFL2011

Manipulation of magnetic microparticles in liquid phases for on-chip biomedical analysis methods

Ulrike Lehmann

Magnetic microparticles and their application in bioanalytical microfluidic systems have been steadily gaining interest in recent years. This progress is fueled by the comparatively large and long range magnetic forces that can be obtained independently of the fluidic flow pattern. This thesis work presents new approaches for using magnetic microparticles in Lab-on-a-Chip systems. The first approach deals with the design of a magnetic droplet manipulation system and the second combines magnetic particle actuation with integrated optical detection. The applicability of both systems for miniaturized bioanalysis will be shown, demonstrating the potential of magnetic particle based Lab-on-a-Chip systems. The magnetic droplet manipulation system tackles the handling of small liquid volumes, which is an important task in miniaturized analytical systems. The careful adjustment of hydrophilic/hydrophobic surface properties and interfacial tensions leads to the design of a system, where small droplets are manipulated in a controllable fashion. The system's setup permits the direct implementation of bioanalytical protocols and two different procedures are in consequence examined. Based on a commercial laboratory kit, a platform for the on-chip extraction and purification of DNA will be designed. The miniaturized setup allows the user to capture and clean the DNA obtained from a raw cell sample containing as little as 10 cells, which is several orders of magnitude lower than known for macroscopic systems. A similar performance is observed for the colorimetric antibody detection further-on evaluated in the droplet manipulation system, where the small sample volumes permit a significant reduction of the reaction times. With the possibility of concentrating the biomolecules of interest on the particle surface, a sensitive and fast immunosorbent assay can be devised. A further miniaturization is examined in a CMOS system, which combines magnetic actuation and optical detection. The small dimensions of the actuation system allow the manipulation of single magnetic microparticles and the integration of Single Photon Avalanche Diodes (SPADs) enables their optical detection. An innovative detection algorithm permits hereby to distinguish the particles in size and, in combination with a velocity measurement, to evaluate the magnetic properties of the detected particles. In consequence, bioanalysis on a single magnetic particle using fluorescent measurements can be performed, as is shown by preliminary experiments.

EPFL2008