This dissertation presents the design and implementation of a 16-channel sinusoidal generator to stimulate microfluidic devices that use electrokinetic forces to manipulate particles. The generator has both, independent frequency and independent amplitude control for each channel. The stimulation system is based upon a CMOS application specific (ASIC) device developed using 0.35¦Ìm technology. Several generator techniques were compared based on frequency range, total harmonic distortion (THD), and on-chip area. The best alternative for the microfluidic applications is based in a triangle-to-sine converter and presents a frequency range of 8kHz to 21MHz, an output voltage range of 0V to 3.1VPP, and a maximum THD of 5.11%. The fabricated device, has a foot- print of 1560¦Ìm¡Á2030¦Ìm. The amplitude of the outputs is extended using an interface card, achieving voltages of 0V to 15VPP. The generator functionality was tested by performing an experimental set-up with particle trapping. The set-up consisted of a micromachined channel with embedded electrodes configured as two electrical ports located at different positions along the channel. By choosing specific amplitude and frequency values from the generator, different particles suspended in a fluid were simultaneously trapped at different ports. The multichannel stimulator presented here can be used in many microfluidic experiments and devices where particle trapping, separation and characterization is desired. / This dissertation presents the design and implementation of a 16-channel sinusoidal generator to stimulate microfluidic devices that use electrokinetic forces to manipulate particles. The generator has both, independent frequency and independent amplitude control for each channel. The stimulation system is based upon a CMOS application specific (ASIC) device developed using 0.35¦Ìm technology. Several generator techniques were compared based on frequency range, total harmonic distortion (THD), and on-chip area. The best alternative for the microfluidic applications is based in a triangle-to-sine converter and presents a frequency range of 8kHz to 21MHz, an output voltage range of 0V to 3.1VPP, and a maximum THD of 5.11%. The fabricated device, has a foot- print of 1560¦Ìm¡Á2030¦Ìm. The amplitude of the outputs is extended using an interface card, achieving voltages of 0V to 15VPP. The generator functionality was tested by performing an experimental set-up with particle trapping. The set-up consisted of a micromachined channel with embedded electrodes configured as two electrical ports located at different positions along the channel. By choosing specific amplitude and frequency values from the generator, different particles suspended in a fluid were simultaneously trapped at different ports. The multichannel stimulator presented here can be used in many microfluidic experiments and devices where particle trapping, separation and characterization is desired.

Le phénomène d’interférométrie par réinjection optique, ou effet self-mixing dans un laser permet de concevoir des capteurs non-invasifs, auto-alignés, ne nécessitant que peu d’éléments optiques et simples à implémenter. Ce type de capteur permet de mesurer avec la précision propre à l’interférométrie laser le déplacement, la vitesse ou la position de cibles dite coopératives (cibles réfléchissantes ou fortement diffusantes). Dans cette étude, ce type de capteurs est appliqué à la mesure de profil d’écoulement des fluides dans des microcanaux. Le faible coût et la polyvalence des capteurs à réinjection optique sont d’un grand intérêt dans l’industrie biomédicale et chimique, ainsi que pour la recherche en mécanique des fluides. Dans un premier temps, et en se basant sur les études réalisées dans des macro-canaux, nous avons proposé un modèle d’interferométrie par réinjection optique dans une diode laser lorsque la cible est constitué de particules en mouvement, en suspension dans un liquide. A partir de ce modèle, nous avons étudié expérimentalement l’impact du volume de mesure ainsi que du type de particules (taille et concentration) sur le signal mesuré. Nous avons ensuite proposé des méthodes de traitement du signal permettant de calculer le calcul du débit du fluide, ainsi que sous certaines conditions identifiées, la vitesse locale en tout point d’un microcanal. Ces études préliminaires nous ont permis de reconstruire le profil d’écoulement de différents liquides dans des canaux de 320µm de diamètre. Enfin, nous avons comparé les performances du capteur développé dans cette thèse avec un capteur basé sur la technique du Dual-Slit, technique déjà validée pour la microfluidique, en mesurant le profil d’écoulement dans un canal à section rectangulaire de 100x20µm. / The phenomenon of optical feedback interferometry (OFI) or self-mixing effect in a laser is used to design non-invasive and self-aligned sensors, requiring only few optical elements and simple to implement. This type of sensor is used to measure the displacement, velocity or position of cooperative targets (reflective or strongly scattering targets). In this study, this phenomenom is applied to the measurement of fluid flow profile in microchannels. The low cost and versatility of optical feedback sensors are of great interest in biomedical and chemical industry as well as research in fluid mechanics. Based on studies in macro-channels, we proposed first a theoretical model of OFI in a laser diode when the target consists of moving particles suspended in a liquid. Based on this model, we then studied experimentally the impact of the sensor’s sensing volume and the type of particles (size and concentration) on the OFI signal. We then proposed signal processing methods for calculating the fluid flow rate, as well as the local velocity at any point in a microchannel. These preliminary studies allowed us to reconstruct the flow profile of different liquids flowing in a circular channel of 320μm diameter. Finally, we compared the performance of the sensor developed in this thesis with a sensor based on the Dual-Slit technique, which has been already validated for microchannels, by measuring the flow profile in a rectangular shaped channel (100x20µm).

Live single-cell imaging has emerged as an advanced single-cell study tool for approaching a quantitative understanding of many biological questions in recent years. In previous cell studies using bulk cell measurements, the population averages can miss the information from cell to cell variability and mask the underlying signaling networks and mechanisms. Currently, some single cell analysis methods, including but not limited to, live single-cell imaging experiments that built around a fluorescent imaging setup and microfluidic devices enable the measurement and analysis of cell dynamics and responses of single cells across a population and across time. Furthermore, by changing the cells’ environmental conditions in well controlled ways, e.g. balanced steady growth, or temporal pulses, live single-cell imaging can record the cellular behaviors corresponding to these changes in exquisite details. An important question of current interest in both developmental, stem cell and cancer biology is the question of epigenetic differentiation. Continuous long-term live single-cell observations offer insights into the molecular control of cell fate. However, maintaining the imaged cells in a healthy state remains a major challenge. One of our aims in this work was to develop a semi-automated single-cell live imaging and analysis platform to obtain dynamic information of the cellular processes. An imaging incubator that controls and regulates the environmental conditions of the imaged cells also had to be designed and tested. In this thesis, I address the key design considerations of developing a single-cell live imaging platform and demonstrate the capability of this technology through three case studies. To test the design and fabrication of microfluidic devices and micro-valves in imaging malaria infected red blood cells (iRBCs), I recorded the flow of iRBCs through microfluidic channels and constrictions in Chapter 3. Our results illustrate the behaviors of iRBCs with different flow rates and the potential to offer dynamic control in studying the infection probability of iRBCs by implementing the micro-valve system. In order to develop a more adaptable live cell imaging platform, we further developed our semi-automated imaging software and in house built imaging incubator to explore the link between proliferation and differentiation of CD4+ T cells in Chapter 4. By using cells expressing an IL-13-GFP reporter, we distinguished between differentiating and non-differentiating CD4+ T cell population and demonstrated a positive association between cycling differentiation of CD4+ T cells. In Chapter 5, we incorporated the FUCCI cell reporter system in our single cell live imaging system to reveal the effect of different media conditions on the cell cycle progression and cell fate choices of mouse embryonic stem (mES) cells. By improving different factors such as longer pre-incubation time before imaging and exchanging media during the experiments, we maintained a healthy state of mES cells during live cell imaging for extended periods. We observed significant differences in time between divisions of mES cells cultured in 2i +LIF and serum + LIF media, and also small but significant differences in durations of sub-cell cycle phases (G1,G1/S,S/G2/M) between the two media conditions. We further applied this imaging setup to study the behaviors of differentiating mES cells in vitro, and observed lengthening of the G1 phase for both 2i-LIF and serum-LIF cells in agreement with literature. Overall, our semi-automated single cell imaging platform not only offers adjustable intervals between fluorescent imaging, but also provides a constant temperature and gas feeding devices that allows the cells to proliferate for extended microscope imaging. Commercially produced incubators that fit onto the microscope stage and satisfied all requirements in restriction of the cell movement, gas feeding, temperature regulation and optical accessibility are not easily available. Thus, there exists a significant potential for our imaging setup to provide a versatile and adaptable live cell imaging platform for both academia and industrial researchers.

abstract: Synthetic biology is a novel method that reengineers functional parts of natural genes of interest to build new biomolecular devices able to express as designed. There is increasing interest in synthetic biology due to wide potential applications in various fields such as clinics and fuel production. However, there are still many challenges in synthetic biology. For example, many natural biological processes are poorly understood, and these could be more thoroughly studied through model synthetic gene networks. Additionally, since synthetic biology applications may have numerous design constraints, more inducer systems should be developed to satisfy different requirements for genetic design. This thesis covers two topics. First, I attempt to generate stochastic resonance (SR) in a biological system. Synthetic bistable systems were chosen because the inducer range in which they exhibit bistability can satisfy one of the three requirements of SR: a weak periodic force is unable to make the transition between states happen. I synthesized several different bistable systems, including toggle switches and self-activators, to select systems matching another requirement: the system has a clear threshold between the two energy states. Their bistability was verified and characterized. At the same time, I attempted to figure out the third requirement for SR – an effective noise serving as the stochastic force – through one of the most widespread toggles, the mutual inhibition toggle, in both yeast and E. coli. A mathematic model for SR was written and adjusted. Secondly, I began work on designing a new genetic system capable of responding to pulsed magnetic fields. The operators responding to pulsed magnetic stimuli in the rpoH promoter were extracted and reorganized. Different versions of the rpoH promoter were generated and tested, and their varying responsiveness to magnetic fields was recorded. In order to improve efficiency and produce better operators, a directed evolution method was applied with the help of a CRISPR-dCas9 nicking system. The best performing promoters thus far show a five-fold difference in gene expression between trials with and without the magnetic field. / Dissertation/Thesis / Masters Thesis Bioengineering 2016

abstract: Microfluidics has shown great potential in rapid isolation, sorting, and concentration of bioparticles upon its discovery. Over the past decades, significant improvements have been made in device fabrication techniques and microfluidic methodologies. As a result, considerable microfluidic-based isolation and concentration techniques have been developed, particularly for rapid pathogen detection. Among all microfluidic techniques, dielectrophoresis (DEP) is one of the most effective and efficient techniques to quickly isolate and separate polarizable particles under inhomogeneous electric field. To date, extensive studies have demonstrated that DEP devices are able to precisely manipulate cells ranging from over 10 μm (mammalian cells) down to about 1 μm (small bacteria). However, very limited DEP studies on manipulating submicron bioparticles, such as viruses, have been reported. In this dissertation, rapid capture and concentration of two different and representative types of virus particles (Sindbis virus and bacteriophage M13) with gradient insulator-based DEP (g-iDEP) has been demonstrated. Sindbis virus has a near-spherical shape with a diameter ~68 nm, while bacteriophage M13 has a filamentous shape with a length ~900 nm and a diameter ~6 nm. Under specific g-iDEP experimental conditions, the concentration of Sindbis virus can be increased two to six times within only a few seconds, using easily accessible voltages as low as 70 V. A similar phenomenon is also observed with bacteriophage M13. Meanwhile, their different DEP behavior predicts the potential of separating viruses with carefully designed microchannels and choices of experimental condition. DEP-based microfluidics also shows great potential in manipulating blood samples, specifically rapid separations of blood cells and proteins. To investigate the ability of g-iDEP device in blood sample manipulation, some proofs of principle work was accomplished including separating two cardiac disease-related proteins (myoglobin and heart-type fatty acid binding protein) and red blood cells (RBCs). Consistent separation was observed, showing retention of RBCs and passage of the two spiked protein biomarkers. The numerical concentration of RBCs was reduced (~70 percent after one minute) with the purified proteins available for detection or further processing. This study explores and extends the use of the device from differentiating similar particles to acting as a sample pretreatment step. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2017

Cette thèse s’inscrit dans le développement d’un Dispositif Médical d’Injection (DMI) automatisé dans lequel est intégrée une micropompe en technologie silicium. Le cœur de cette micropompe est constitué d’une membrane actionnée permettant de déplacer un volume de liquide à travers des canaux micro-fluidiques. Deux types de membranes actionnées ont été étudiés : une membrane à actionnement bimétallique intégré et une membrane à actionnement piézoélectrique externe. Des simulations FEM ont permis d’affiner les modèles théoriques existants et de mieux rendre compte des effets non linéaires qui régissent le fonctionnement de ces membranes. Une méthode d’optimisation spécialement mise en place a permis de calculer des configurations géométriques optimales en fonction des plages de fonctionnement visées. Des membranes ont ensuite été fabriquées en salle blanche. Leurs caractéristiques mécaniques ont été mesurées et comparées aux prédictions des simulations FEM. / This thesis fits into the development of an automated Injection Medical Device (IMD) in which is integrated a micropump in silicon technology. The heart of this micropump is constituted by an actuated membrane which moves a volume of liquid through microfluidic channels. Two types of actuated membranes were studied: a membrane with an integrated bimetallic actuator and a membrane with an external piezoelectric actuator. FEM simulations enabled the refinement of existing theoretical models and a better representation of nonlinear effects that govern the mechanics of such membranes. An optimization method specially putted in place enabled the computation of optimal geometric configurations according to the targeted functioning ranges. Some membranes were then fabricated in cleanroom. Their mechanical characteristics were measured and compared to predictions of FEM simulations.

Este trabalho descreve o desenvolvimento de um biossensor contendo transdução condutométrica sem contato (C4D, capacitively coupled contactless conductivity detection) e ácido fólico (FA) como biorreceptor em microchip, uma nova alternativa que poderá ser utilizada na determinação do biomarcador tumoral FR-α. Essa espécie exibe interações com FA altamente específicas, com constantes de formação da ordem de 109-1010. Os dispositivos microfluídicos, os quais consistiram de uma lâmina de vidro (integrando os eletrodos), dielétrico (contendo a fase biossensora) e substrato de poli(dimetilsiloxano) (PDMS, incorporando os microcanais), foram fabricados utilizando-se processos de fotolitografia e deposição de filmes finos em fase vapor. Objetivando melhorias nos níveis de detecção da C4D, estudos de sensibilidade com base em parâmetros da curva analítica foram conduzidos alterando-se a natureza do dielétrico e a configuração dos eletrodos. Posteriormente, estudos de caracterização foram realizados para as superfícies modificadas com os intermediários de imobilização; condições reacionais distintas (reagente, concentração, solvente e tempo) foram consideradas. As técnicas de microscopia eletrônica de varredura e espectroscopia de fotoelétrons excitados por raios-X foram usadas, respectivamente, a fim de se verificar a possível formação de aglomerados e permitir determinações qualitativas e quantitativas sobre as composições químicas das superfícies. Como resultado dos experimentos de sensibilidade e caracterização de superfície, adotamos os parâmetros seguintes para os ensaios de interações biomoleculares posteriores: filme de SiO2 como dielétrico, eletrodos seletivos à C4D com formato retangular e orientação antiparalela e monocamadas automontadas do reagente 3-aminopropil(trietoxisilano) como intermediário de imobilização de FA. As duas etapas finais do trabalho foram: otimização do tempo de funcionalização com FA (3, 5 e 7 h) e caracterização da fase biossensora, realizada a partir de medidas de C4D e microscopia de força atômica (AFM). Para o primeiro caso, os microchips foram aplicados a um padrão de anticorpo monoclonal específico a FA (α-FA). Os ensaios biomoleculares indicaram uma adsorção efetiva de FA junto à superfície de SiO2 silanizada, sem a ocorrência (ao menos em níveis significativos) de impedimentos estéricos de sua espécie bioativa. Dentre os tempos de funcionalização investigados, 3 h foi aquele que resultou em uma maior sensibilidade do método. Em termos da etapa de caracterização eletroquímica da fase biossensora, seus resultados mostraram haver correlação entre a resposta analítica e as interações FA/α-FA. Em adição, conforme indicaram as medidas de AFM, não houve alterações drásticas na morfologia do substrato (SiO2) em função dos processos de modificação química de superfície. Por fim, o uso da C4D como uma técnica de transdução em biossensores mostrou-se uma alternativa promissora para a análise do biomarcador tumoral FR-α. Dentre outros aspectos, essa plataforma analítica requer uma instrumentação simples, barata e portátil, não apresenta inconvenientes relacionados ao contato eletrodo/solução, dispensa o uso de mediadores redox e permite a determinação simultânea de multianalitos. Neste ínterim, alterações no transdutor devem ser implementadas visando um aumento na sensibilidade do método, o qual representa seu fator limitante principal. / This work describes the development of a biosensor containing capacitively coupled contactless conductivity transduction (C4D) and folic acid (FA) as bioreceptor in microchip, a new alternative that can be used in FR-α tumor biomarker analysis. FR-α exhibits highly specific interactions with FA, showing formation constants of the order of 109-1010. The microfluidic devices consisted of a glass layer (integrating the electrodes), dielectric (containing the biosensor phase), and poly(dimetilsiloxane) substrate (PDMS, incorporating microchannel). The microfabrication stage evolved photolithography processes, metal adsorption via sputtering, and plasma-enhanced vapor film deposition. In order to improve detection levels of C4D, sensitivity studies were conducted by changing the dielectric nature and electrode configuration. Through flow analysis with given electrolyte standards, the limits of detection and quantification were calculated based on analytical curve parameters. Subsequently, researches were performed to characterize the modified surfaces with immobilization intermediate considering reaction conditions distinct (reagent, concentration, solvent, and time). The techniques of scanning electron microscopy and X-ray photoelectron spectroscopy were employed, respectively, aiming to verify the clusters formation and allow qualitative and quantitative determinations about the surfaces chemical composition. From the results of sensitivity experiments and surface characterization, we adopt the following parameters for the biomolecular interactions assays: SiO2 film as dielectric, C4D selective electrodes with rectangular shape and antiparallel orientation, and self-assembled monolayers of 3-aminopropyl(triethoxysilane) as intermediary for immobilization of FA. The two final steps of the work were: optimizing the FA functionalization time (3, 5, and 7 h) and phase biosensor characterization, made from measures of C4D and atomic force microscopy (AFM). For the first case, due to the absence of FR-α standard for purchase, the microchips were applied to FA specific monoclonal antibody (α-FA). The biomolecular assay indicated effective adsorption of FA, without occurrence (at least in significant levels) of steric hindrance of its bioactive specie. Among the investigated times of functionalization, 3 h resulted in a higher sensitivity of the method. In terms of biosensor phase electrochemical characterization stage, their results evidenced correlation between analytical response and FA/α-FA interactions. Additionally, as the AFM measurements showed, drastic changes in the morphology of the substrate (SiO2) with the surface modification processes did not occur. Finally, the use of the C4D as transduction technical in biosensors proved to be a promissory alternative for FR-α tumor biomarker analysis. Among other features, this platform has not drawbacks related to the electrode/solution contact, dispenses the use of redox mediators, allows the simultaneous determination of multianalytes, and employs an instrumentation that is simple, cheap, and portable. Nevertheless, changes in the transducer should be implemented to increase the method sensitivity, which represents its main limiting factor.

Made available in DSpace on 2015-03-26T13:35:12Z (GMT). No. of bitstreams: 1 texto completo.pdf: 4700435 bytes, checksum: bac9eb5b4507aab56d5ee90842c50c1a (MD5) Previous issue date: 2009-07-29 / Emulsions are colloidal systems where one liquid is dispersed in another liquid where the two liquids are immiscible. The control of these dispersions can bring important contributions to science and society since this kind of colloidal system has many and important applications in the food and pharmaceutic area and also in the material science. This work has the main purpose to develop and improve monodisperse emulsions production techniques using simple and low-cost devices. It is shown the production of simple emulsions using devices built from glass capillary tubes. Besides, the drops diameter dependence with the phases flow rate is verified. Solid particles were made from droplets of poly(styrene) dispersed in chloroform, followed by the evaporation of the chloroform, using these devices. Although the glass capillary tube devices show good results, we have made emulsions, for instance, of (80±1) μm with standard deviation of 1,3 μm (σ/dm = 1, 6%), they have limitations concerning the configuration of the tubes. Another kind of device built was made basically from microchannels produced in glass microscope slides using hydrofluoric acid to attack the slide surfaces. The slides were sealed using epoxy glue and the final result were channels with controlled values of depth and width. The dependence of the drops diameter with the continuous and disperse phases flow rate were made to test the devices quality. High quality emulsions were obtained with tipical values for diameter and standar deviation about 100 μm and 2%. Four different channel configurations were tested and the configuration where the channels were built as a cross (one channel carrying the disperse phase and two channels carrying the continuous phase) shown better results then the devices with only two channels (one channel carrying each phase). / Emulsões são sistemas coloidais onde um líquido é disperso em outro líquido imiscível. O controle dessa dispersão pode trazer importantes contribuições para a ciência e para a sociedade, já que este tipo de sistema coloidal apresenta variadas e importantes aplicações nas áreas alimentícia, farmacêutica e de materiais. Este trabalho tem como objetivo principal desenvolver e aperfeiçoar técnicas de produção de emulsões com baixa dispersão de tamanhos utilizando dispositivos simples e de baixo custo. Apresenta-se, assim, a técnica de produção de emulsões simples através da utilização de dispositivos construídos a partir de tubos capilares de vidro. Além disso, verifica-se a dependência do diâmetro das gotas das emulsões produzidas com a vazão da fase contínua e dispersa. Utilizando este dispositivo, produzem-se também partículas sólidas a partir da preparação de gotas de poliestireno dissolvido em clorofórmio e posterior evaporação do clorofórmio. Embora esse tipo de dispositivo apresente bons resultados, sendo possível produzir emulsões, por exemplo, com diâmetro médio das gotas igual a (80±1) μm e desvio padrão de 1,3 μm (σ/dm = 1, 6%), ele tem limitações quanto ao número de configurações dos fluxos possíveis de se trabalhar. Outro dispositivo produzido constitui-se basicamente de microcanais produzidos em lâminas de microscópio por ataque controlado de ácido fluorídrico. A vedação superior dos canais é feita utilizando cola epóxi (araldite) tendo-se, assim, canais de largura e profundidades controladas. Para testar a qualidade deste tipo de dispositivo, fez-se também a verificação do diâmetro das gotas de emulsão produzidas com a vazão da fase contínua ou dispersa, tendo sido possível a produção de emulsão com baixa dispersão de tamanhos, produzindo para dada condição, emulsões com diâmetro médio das gotas da ordem de (100±1) μm com desvio padrão típicos de 2%. Foram testadas quatro diferentes configurações de canais, onde as configurações em cruz (um canal contendo a fase dispersa e dois canais com a fase contínua) mostraram melhores resultados que configurações com apenas dois canais (um por fase).

Le cancer constitue un problème majeur de santé publique en France. La nécessité de disposer d’un test capable de détecter très précocement une tumeur, avant même qu’elle ne soit décelable par l’imagerie (et surtout avant les métastases) ne fait pas doute. Pour l’heure, la voie la plus prometteuse pour détecter la maladie demeure la mise au point des tests simples, fiables, rapides et hautement sensibles, reposant sur le dosage de marqueurs génétiques (des biomarqueurs). Nous avons développé une procédure non-invasive, innovante et au moins 1000 fois plus sensible que les méthodes actuelles (0,0005% de séquences mutées détectées parmi un excès de séquences non-mutées), pour le criblage des biomarqueurs spécifiques des cancers. Elle peut facilement être utilisée pour le diagnostic, le pronostique ou la prédiction des procédures de gestion clinique des patients souffrant du cancer colorectal et par après pour les patients souffrant d’autres types de cancer. Cette méthode est basée sur l’utilisation des millions de microgouttelettes en tant qu’autant de bioréacteurs indépendants. Apportant ainsi la possibilité d’analyse chaque molécule d’ADN indépendamment, elle permettra de détecter spécifiquement une minorité de séquences mutantes au sein d’une forte quantité de séquences non mutées et avec un débit important. Nous avons développé cette stratégie pour la détection des mutations de l’oncogène KRAS (responsables des non-réponses aux thérapies ciblées) et nous l’avons validé avec la détection de mutations KRAS dans les échantillons de plasma sanguin et de tumeur des patients atteints d’un cancer colorectal métastatique. Notre méthode trouvera son utilité non seulement dans le domaine du diagnostic précoce des cancers, mais aussi dans cas de la prédiction de la réponse aux traitements ciblés grâce à la détection de biomarqueurs spécifiques. / The aim of this work is to establish novel strategies for the highly sensitive screening of cancer biomarkers in biological samples.To achieve this goal, we developed droplet-based microfluidic dPCR technique. Using a limiting dilution, individual DNA molecules are encapsulated within monodisperse droplets of a water-in-oil emulsion created with a microfluidic device. Fluorescent TaqMan® probes targeting the screened cancer biomarkers allow the detection of mutations. We focused on the mutations in the human KRAS gene for the development of our test. This method is also transposable in a multiplexed format for the parallel detection of multiple mutations in clinical samples.The developed technique allowed the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of un-mutated KRAS genes and enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines. We validated our technique by screening for KRAS mutations in the blood plasma and tumor samples from patients with metastatic colorectal cancer.

No description available.