Microfluidic Immunoassays Based on Self-Assembled Magnetic Bead Patterns and Time-Resolved Luminescence Detection

Venkataragavalu Sivagnanam
EPFL, 2010
Thèse EPFL

Résumé

Microfluidic bio-assays have emerged as the most privileged solutions and provide the basis for the realization of miniaturized bio-analytical systems and clinical diagnostic devices that are portable, user-friendly and cost-effective (Lab-on-a-chip). Two important steps that are implemented in a microfluidic bio-assay are: (a) the immobilization and/or patterning of target-specific bio-molecules on the surface of a microfluidic channel, for selectively capturing bio-targets like antigens or pathogens, followed by (b) sensitive detection of the bio-targets. In this thesis, we demonstrate microfluidic bio-assays based on novel methods for generating protein-patterns and on sensitive detection of the bio-targets. First, we introduce a simple and fast method for creating protein micropatterns both on a bare substrate and in-situ inside a microfluidic channel, in a matter of minutes, through electrostatic self-assembly of pre-functionalized magnetic beads. A lift-off patterned positively-charged aminosilane layer is used as the template for immobilizing the protein-coated negatively-charged beads. The number and arrangement of the beads can be well-controlled by altering the silane template design. Subsequently, we use patterned beads as assay substrates for performing on-chip bioassays. We demonstrate highly-sensitive full on-chip sandwich immunoassays for single and multi-analyte detection using beads as assay substrate. We successfully explored the possibility to lower the detection limit of immunoassays by concentrating the target antigens on a very small number of patterned beads. We also present the application of bead patterns as a platform for immuno-separation, culture and analysis of target (cancer) cells. Finally, we demonstrate a rapid on-chip immuno-histo-chemical assay on breast cancer tissues. We use luminescent lanthanide probes in place of conventional fluorescent probes, as labels for detection antibodies, for sensitive detection and quantification of biomarkers. Thanks to the time resolved microscopy and luminescent probes, the background noise due to the autofluorescence of the samples (i.e. tissue, cells) and microfluidic chips is successfully eliminated resulting in an improved signal-to-noise ratio when compared with the fluorescent microscopy results. Our assay results fully agree with the clinical analyses outcome, and this opens perspectives for a fully-integrated cancer detection platform for bedside diagnostics.

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https://infoscience.epfl.ch/record/144092?ln=fr

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Venkataragavalu Sivagnanam
EPFL, 2010
Thèse EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/144092?ln=fr

À propos de ce résultat

Concepts associés (20)

Concepts associés

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Publications associées (3)

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Optical trapping allows manipulating very small objects – varying from Angstöms to micron size particles – without physical contact by taking advantage of laser light. This technique, relying on light momentum transfer, allows manipulating biological matter (such as cells, organelles, vesicles or chemically functionalized artificial particles, etc.) and offers promising potentialities for research in biotechnology and biochemistry. Individually confining a large number of microscopic objects opens new ways for the downscaling of analysis tools for drug screening, particles sorting and the assessment of statistical data. In particular, the combination of optical trapping with microfluidics greatly enlarges the possibilities offered by both techniques. This PhD work takes place in a research aiming at developing novel bio-analytical instrumentation relying on large arrays of optical traps compatible with microfluidic systems. The dissertation focuses on the use of micro-optics for generating extended matrices of three-dimensional optical traps capable of capturing a large number of particles within microfluidic environments. The stability of optical traps is first studied with special emphasis on the conditions present in microfluidic flows, both from a theoretical and an experimental point of view, and trapping forces achievable on biological cells are investigated. A multiple optical trapping system relying on microlens arrays, adapted to work on commercial microscopes, is shown to be capable of generating arrays of more than 500 three-dimensional traps. Simultaneously trapping in multiple planes is also evidenced using matrices of microlenses, thanks to a self-imaging effect of the array of traps. Furthermore, possibilities offered by multiple optical trapping within microfluidic environments are explored. Polystyrene spheres as well as biological particles, such as native vesicles, can be trapped in arrays, manipulated inside microfluidic devices and analyzed in parallel through fluorescence microscopy while extremely small quantities of chemical reagents are flown past the array. Finally, for the sake of system miniaturization and further extension of the number of traps, multiple three-dimensional optical trapping based on arrays of miniaturized high numerical aperture parabolic mirrors is proposed, allowing the optics necessary for optical trapping, fluorescence excitation and collection to be integrated at the level of a microfluidic chip. Such miniaturized mirror matrices did not even exist before this thesis; their fabrication and characterization are detailed in this dissertation.

EPFL2008

Chargement

Sandwich immunoassay on a microfluidic chip using patterns of electrostatically self-assembled streptavidin-coated beads

Martinus Gijs, Frédéric Lacharme, Venkataragavalu Sivagnanam, Caroline Vandevyver

In this paper, we demonstrate a bead-based microfluidic immunoassay using self-assembled streptavidin-coated bead micropatterns. The beads are assembled on the microfluidic channel surface using electrostatic forces between the negatively-charged streptavidin-coated beads and the positively-charged aminosilane micropatterns. The immunoassay is performed in the stop-flow mode and uses mouse IgG diluted in phosphate buffered saline (PBS) solution as the model target antigen. The total immunoassay takes around 30 min and is able to detect mouse IgG antigen down to a concentration of 15 ng/mL by using only 560 nl of analyte volume.

Elsevier2009

Chargement

The physiological properties of cells are typically investigated in ensembles yielding averaged data that mask heterogeneities present in any cell population. Single-cell analysis techniques based on microfluidic "lab on a chip" (LOC) devices have recently led to new insights into cellular physiology revealing the biological importance of cell variability. However, we are only at the beginning of understanding the complex nature of cellular constitution and function. Similarly is the study of individual cells in LOC devices a new field requiring further development. The aim of this thesis is to develop novel methods for functional and chemical single-cell analysis in controlled microfluidic environments using optical trapping for cell manipulation. Investigation of cellular function requires experimental techniques allowing us to monitor cell-to-cell variability of the response of a cell population to external stimuli and to sort out interesting cell phenotypes for further investigation. Although flow cytometry is able to sequentially probe and sort thousands of cells per second, dynamic processes cannot be experimentally accessed on single cells due to the sub-second sampling time. Cellular dynamics can be measured by image cytometry of surface-immobilized cells, however, cell sorting is complicated under these conditions. Here, we developed a cytometric tool based on refractive multiple optical tweezers combined with microfluidics and optical microscopy. We demonstrate contact-free immobilization of more than 200 yeast cells into a high-density array of optical traps in a microfluidic chip. The cell array could be moved to specific locations of the chip enabling us to expose in a controlled manner the cells to reagents and to analyze the responses of individual cells in a highly parallel format using fluorescence microscopy. We further established a method to sort single cells within the microfluidic device using an additional steerable optical trap. Observed dose-dependent cytotoxic effects of the trapping laser on the cells' viability imposed an upper limit to the time periods during which optically trapped cells could be studied. For assays involving monitoring of cellular dynamics over several hours or days, we immobilized cell arrays formed by refractive optical tweezers in a hydrogel-matrix confined to a microfluidic chamber of 15 nL volume. We demonstrated multiplexing of this concept by integrating several chambers adjacent to each other in the same microfluidic device. In fluidic contact, each chamber could be exposed to a different reagent. Chemical single-cell analysis deals with identification and quantification of intracellular chemical species. Here, we report an alternative method for chemical cytometry using micro-beads functionalised with capture agents as intracellular affinity probe. Cell internalization of the beads followed by induced endosomal escape leads to binding of the target analyte to the surface of the beads. Using optical trapping with microfluidics, we demonstrate fast extraction of the beads from the cells and detection of bound target analyte under preservation of the single-cell analytics. In contrast to classical chemical cytometry, our method has the potential to reach high analytical sensitivity at simultaneously reasonable throughput.

EPFL2011

Chargement

EPFL2008

EPFL2011