Gel-tool Sensor Positioned by Optical Tweezers for Local pH Measurement in a Microchip

2007 IEEE International Conference on Robotics and Automation, Roma, Italy, 10-14 April 200

Laser trapping microchip for biotechnological applications: design and development

This work presents a novel approach towards integrated dual-beam optical trapping achieved using planar lightwave circuit (PLC) technology. Three fabrication technologies sol-gel, photolithography and reactive ion etching were combined to fabricate a Laser Trapping Microchip (LTM) allowing one-dimensional manipulation of transparent micrometer-size spherical objects. Detailed steps of the LTM development are described, beginning with a theoretical approach and numerical simulations through the design and synthesis of a suitable photopatternable sol-gel material, culminating in the fabrication process and experimental confirmation of the trapping properties of the device. The proof of concept of this unique device was achieved by demonstrating its optical trapping abilities using micrometer size polystyrene beads with diameters in the range between 4 pm and 10 pm and the refractive index of 1 59. The LTM device possesses many advantages over currently existing dual-beam laser trapping systems such as small overall dimensions (~15 x 30 x 0 5 mm), low power optical power consumption (<15mW), improved stability of the optical trap due to precise alignment of the optical paths and a relatively easy fabrication process. For these reasons there are many potential applications of the LTM device in biotechnology, microfluidics and other sciences making it an attractive device for commercial use

Genetic Analysis and Cell Manipulation on Microfluidic Surfaces

Personalized cancer medicine is a cancer care paradigm in which diagnostic and therapeutic strategies are customized for individual patients. Microsystems that are created by Micro-Electro-Mechanical Systems (MEMS) technology and integrate various diagnostic and therapeutic methods on a single chip hold great potential to enable personalized cancer medicine. Toward ultimate realization of such microsystems, this thesis focuses on developing critical functional building blocks that perform genetic variation identification (single-nucleotide polymorphism (SNP) genotyping) and specific, efficient and flexible cell manipulation on microfluidic surfaces. For the identification of genetic variations, we first present a bead-based approach to detect single-base mutations by performing single-base extension (SBE) of SNP specific primers on solid surfaces. Successful genotyping of the SNP on exon 1 of HBB gene demonstrates the potential of the device for simple, rapid, and accurate detection of SNPs. In addition, a multi-step solution-based approach, which integrates SBE with mass-tagged dideoxynucleotides and solid-phase purification of extension products, is also presented. Rapid, accurate and simultaneous detection of 4 loci on a synthetic template demonstrates the capability of multiplex genotyping with reduced consumption of samples and reagents. For cell manipulation, we first present a microfluidic device for cell purification with surface-immobilized aptamers, exploiting the strong temperature dependence of the affinity binding between aptamers and cells. Further, we demonstrate the feasibility of using aptamers to specifically separate target cells from a heterogeneous solution and employing environmental changes to retrieve purified cells. Moreover, spatially specific capture and selective temperature-mediated release of cells on design-specified areas is presented, which demonstrates the ability to establish cell arrays on pre-defined regions and to collect only specifically selected cell groups for downstream analysis. We also investigate tunable microfluidic trapping of cells by exploiting the large compliance of elastomers to create an array of cell-trapping microstructures, whose dimensions can be mechanically modulated by inducing uniform strain via the application of external force. Cell trapping under different strain modulations has been studied, and capture of a predetermined number of cells, from single cells to multiple cells, has been achieved. In addition, to address the lack of aptamers for targets of interest, which is a major hindrance to aptamer-based cell manipulation, we present a microfluidic device for synthetically isolating cell-targeting aptamers from a randomized single-strand DNA (ssDNA) library, integrating cell culturing with affinity selection and amplification of cell-binding ssDNA. Multi-round aptamer isolation on a single chip has also been realized by using pressure-driven flow. Finally, some perspectives on future work are presented, and strategies and notable issues are discussed for further development of MEMS/microfluidics-based devices for personalized cancer medicine

Microrobotique pour composants micrométriques : les challenges pour leur manipulation et leur assemblage.

National audienceLa miniaturisation de nombreux produits manufacturés est une réalité et ce processus s'accentue. Ceci conduit la communauté scientifique à proposer des systèmes de production permettant de fabriquer des systèmes hybrides, c'est-à-dire dont les composants proviennent de plusieurs processus de fabrication ou de microfabrication, et ayant des structures 3D complexes afin d'intégrer plusieurs fonctions dans des volumes les plus réduits possibles. Le micro-assemblage réalisé avec des systèmes microrobotiques est une réponse pertinente à ce besoin de produits micromécatroniques. A travers une présentation des principales activités dans ce domaine dans le monde, on peut constater que la mise en oeuvre réelle de systèmes pour manipuler et assembler de façon automatisée (ou en partie) des composants sous-millimétriques reste un véritable challenge si l'on veut cumuler des propriétés de haute précision, de fiabilité, de productivité et de flexibilité. D'autre part, l'intérêt de groupes industriels Européens et l'émergence de start-ups montrent que l'assemblage de composants de taille sousmillimétrique est un enjeu sociétal. En terme de prospective, le passage de la barrière dimensionnelle des 10

Workshop on "Robotic assembly of 3D MEMS".

Proceedings of a workshop proposed in IEEE IROS'2007.The increase of MEMS' functionalities often requires the integration of various technologies used for mechanical, optical and electronic subsystems in order to achieve a unique system. These different technologies have usually process incompatibilities and the whole microsystem can not be obtained monolithically and then requires microassembly steps. Microassembly of MEMS based on micrometric components is one of the most promising approaches to achieve high-performance MEMS. Moreover, microassembly also permits to develop suitable MEMS packaging as well as 3D components although microfabrication technologies are usually able to create 2D and "2.5D" components. The study of microassembly methods is consequently a high stake for MEMS technologies growth. Two approaches are currently developped for microassembly: self-assembly and robotic microassembly. In the first one, the assembly is highly parallel but the efficiency and the flexibility still stay low. The robotic approach has the potential to reach precise and reliable assembly with high flexibility. The proposed workshop focuses on this second approach and will take a bearing of the corresponding microrobotic issues. Beyond the microfabrication technologies, performing MEMS microassembly requires, micromanipulation strategies, microworld dynamics and attachment technologies. The design and the fabrication of the microrobot end-effectors as well as the assembled micro-parts require the use of microfabrication technologies. Moreover new micromanipulation strategies are necessary to handle and position micro-parts with sufficiently high accuracy during assembly. The dynamic behaviour of micrometric objects has also to be studied and controlled. Finally, after positioning the micro-part, attachment technologies are necessary

Miniaturized single-cell analyses for biomedical applications

Numerous diseases affecting living beings have as a cause a modification of gene expression resulting in anomalous expression of proteins in up- or down-regulated fashion, with altered functions or expression of proteins that are not present in the cell in a normal situation. Among the well-known examples is cancer, where a series of DNA damages turn cells out of control of the rest of the body, from which they do not receive or follow control signals. To have a better understanding of the mechanism of these diseases, in particular the genetic diversity existing in a single affected tissue, it is necessary to be able to perform single-cell analyses that unveil the subpopulations of diseased cells leading to adequate medical treatment. In the course of this thesis, we report on the development of single-cell analysis methods which are of interest for medical applications. The first part focuses on the investigation of the viscoelastic properties of cell membranes by observing the back-relaxation of plasma membrane nanotubes which have been pulled out of a cell by an optical tweezer. Applied to the investigation of the viscoelastic properties of individual tumor cells taken from patients, we could show that this method can distinguish the state of progress of skin melanoma. In the second part, we used beads comprising a chemically modified surface to capture specifically one or several proteins inside single-cells. After extraction out of the cell, the affinity bead is transferred in a microfluidic stream of a fluorescently labeled antibody to detect and quantify the protein(s) of interest. The extraction and detection procedures occur inside a microfluidic platform to allow future automatization of the process. The last chapter focuses on the use of cell-derived extracellular vesicles (EVs) as diagnostic and therapeutic agents. With this goal in mind, we explore the potential of EVs as carriers to transfer genetic material into cells. To demonstrate the feasibility of this approach, we encapsulate EVs inside a giant unilamellar vesicle and release their cargo in a time- and space-controlled manner. This method could have therapeutic applications using a patient's self-EVs for gene therapy

Highly efficient selection, enumeration, enrichment, and molecular profiling of low-abundance biological cells

After brief overviews of low-abundance cell selection techniques in chapter 1 and circulating tumor cells in chapter 2, this dissertation initially focuses on the development of aptamer incorporated high-throughput microfluidic techniques to select rare circulation prostate cancer cells (LNCaP) directly from whole blood with subsequent quantification of these rare cells using a non-labeling approach. Then, I extended the technology to environmental samples in an effort around time, sensitivity, and portability of traditional groundwater assessment. As a model bio- pathogen, E. coli O157:H7 was chosen due to its toxicity and its adverse impact on recreational waters. Low-abundance (\u3c100 cells mL-1) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip that its capture beds were covalently decorated with E.coli O157:H7 specific polyclonal antibodies. The selected cells were enumerated using RT-qPCR technique. Finally, I have integrated HTMSU with electrokinetic enrichment microfluidic unit for performance of single recombinant low-abundance CTC cell-based assay. A series of analytical processes were carried out, including immunoaffinity selection of rare CTCs, quantification of selected cells via conductivity impedance and electrophoretic enrichment of selected cells for PCR/LDR/CE interrogation for detection of low-abundance point mutations in genomic DNA

Polymer Microsystems for the Enrichment of Circulating Tumor Cells and their Clinical Demonstration

Cancer research is centered on the discovery of new biomarkers that could unlock the obscurities behind the mechanisms that cause cancer or those associated with its spread (i.e., metastatic disease). Circulating tumor cells (CTCs) have emerged as attractive biomarkers for the management of many cancer-related diseases due primarily to the ease of securing them from a simple blood draw. However, their rarity (~1 CTC per mL of whole blood) makes enrichment analytically challenging. Microfluidic systems are viewed as exquisite platforms for the clinical analysis of CTCs due to their ability to be used in an automated fashion, minimizing sample loss and contamination. This has formed the basis of the reported research, which focused on the development of microfluidic systems for CTC analysis. The system reported herein consisted of a modular design and targeted the analysis of CTCs using pancreatic ductal adenocarcinoma (PDAC) as the model disease for determining the utility of the system. The system was composed of 3 functional modules; (i) a thermoplastic CTC selection module consisting of high aspect ratio (30 µm x 150 µm) channels; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for phenotype identification of selected CTCs. The system could exhaustively process 7.5 mL of blood in \u3c45 min with CTC recoveries \u3e90% directly from whole blood. In addition, significantly reduced assay turnaround times (8 h to 1.5 h) was demonstrated. We also show the ability to detect KRAS gene mutations from CTCs enriched by the microfluidic system. As a proof-of-concept, the ability to identify KRAS point mutations using a PCR/LDR/CE assay from as low as 10 CTCs enriched by the integrated microfluidic system was demonstrated. Finally, the clinical utility of the polymer-based microfluidic device for the analysis of circulating multiple myeloma cells (CMMCs) was demonstrated as well. Parameters such as translational velocity and recovery of CMMCs were optimized and found to be 1.1 mm/s and 71%, respectively. Also demonstrated was on-chip immunophenotyping and clonal testing of CMMCs, which has been reported to be prognostically significant. Further, a pilot study involving 26 patients was performed using the polymer microfluidic device with the aim of correlating the number of CMMCs with disease activity. An average of 347 CMMCs/mL of whole blood was recovered from blood volumes of approximately 0.5 mL

In Situ Preconcentration by AC Electrokinetics for Rapid and Sensitive Nanoparticle Detection

Reducing cost and time is a major concern in clinical diagnostics. Current molecular diagnostics are multi-step processes that usually take at least several hours or even days to complete multiple reagents delivery, incubations and several washing processes. This highly labor-intensive work and lack of automation could result in reduced reliability and low efficiency. The Laboratory-on-a-chip (LOC), taking advantage of the merger and development of microfluidics and biosensor technology, has shown promise towards a solution for performing analytical tests in a self-contained and compact unit, enabling earlier and decentralized testing. However, challenges are to integrate the fluid regulatory elements on a single platform and to detect target analytes with high sensitivity and selectivity. The goal of this research work is to develop an AC electrokinetic (ACEK) flow through concentrator for in-situ concentration of biomolecules and develop a comprehensive understanding of effects of ACEK flow on the biomolecule transport (in-situ concentration) and their impact on electronic biosensing mechanism and performance, achieving automation and miniaturization. ACEK is a new and promising technique to manipulate micro/bio-fluids and particles. It has many advantages over other techniques for its low applied voltage, portability and compatibility for integration into lab-on-a-chip devices. Numerical study on preconcentration system design in this work has provided an optimization rule for various biosensor designs using ACEK technique. And the microfluidic immunoassay lab-chip designed based on ACET effect has showed promising prospect for accelerated diagnostics. With optimized design of channel geometry, electrode patterns, and properly selected operation condition (ac frequency and voltage), the preconcentration system greatly reduced the reaction time to several minutes instead of several hours, and improved sensitivity of the assay. With the design of immunoassay lab-chip, one can quantitatively study the effect of ACET micropumping and mixing on molecular level binding. Improved sensors with single-chip form factor as a general platform could have a significant impact on a wide-range of biochemical detection and disease diagnostics including pathogen/virus detection, whole blood analysis, immune-screening, gene expression, as well as home land security