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<p>This thesis demonstrates a new way to achieve sparse biological sample detection, which uses magnetic bead manipulation on a digital microfluidic device. Sparse sample detection was made possible through two steps: sparse sample capture and fluorescent signal detection. For the first step, the immunological reaction between antibody and antigen enables the binding between target cells and antibody-­‐‑ coated magnetic beads, hence achieving sample capture. For the second step, fluorescent detection is achieved via fluorescent signal measurement and magnetic bead manipulation. In those two steps, a total of three functions need to work together, namely magnetic beads manipulation, fluorescent signal measurement and immunological binding. The first function is magnetic bead manipulation, and it uses the structure of current-­‐‑carrying wires embedded in the actuation electrode of an electrowetting-­‐‑on-­‐‑dielectric (EWD) device. The current wire structure serves as a microelectromagnet, which is capable of segregating and separating magnetic beads. The device can achieve high segregation efficiency when the wire spacing is 50µμm, and it is also capable of separating two kinds of magnetic beads within a 65µμm distance. The device ensures that the magnetic bead manipulation and the EWD function can be operated simultaneously without introducing additional steps in the fabrication process. Half circle shaped current wires were designed in later devices to concentrate magnetic beads in order to increase the SNR of sample detection. The second function is immunological binding. Immunological reaction kits were selected in order to ensure the compatibility of target cells, magnetic bead function and EWD function. The magnetic bead choice ensures the binding efficiency and survivability of target cells. The magnetic bead selection and binding mechanism used in this work can be applied to a wide variety of samples with a simple switch of the type of antibody. The last function is fluorescent measurement. Fluorescent measurement of sparse samples is made possible of using fluorescent stains and a method to increase SNR. The improved SNR is achieved by target cell concentration and reduced sensing area. Theoretical limitations of the entire sparse sample detection system is as low as 1 Colony Forming Unit/mL (CFU/mL).</p> / Dissertation

Magnetic biosensors detect biological targets through sensing the stray field of magnetic beads which label the targets. Commonly, magnetic biosensors employ the “sandwich” method to immobilize biological targets, i.e., the targets are sandwiched between a bio-functionalized sensor surface and bio-functionalized magnetic beads. This method has been used very successfully in different application, but its execution requires a rather elaborate procedure including several washing and incubation steps. This dissertation investigates a new magnetic biosensor concept, which enables a simple and effective detection of biological targets. The biosensor takes advantage of the size difference between bare magnetic beads and compounds of magnetic beads and biological targets. First, the detection of super-paramagnetic beads via magnetic tunnel junction (MTJ) sensors is implemented. Frequency modulation is used to enhance the signal-to-noise ratio, enabling the detection of a single magnetic bead. Second, the concept of the magnetic biosensor is investigated theoretically. The biosensor consists of an MTJ sensor, which detects the stray field of magnetic beads inside of a trap on top of the MTJ. A microwire between the trap and the MTJ is used to attract magnetic beads to the trapping well by applying a current to it. The MTJ sensor’s output depends on the number of beads inside the trap. If biological targets are in the sample solution, the beads will form bead compounds consisting of beads linked to the biological targets. Since bead compounds are larger than bare beads, the number of beads inside the trapping well will depend on the presence of biological targets. Hence, the output of the MTJ sensor will depend on the biological targets. The dependences of sensor signals on the sizes of the MTJ sensor, magnetic beads and biological targets are studied to find the optimum constellations for the detection of specific biological targets. The optimization is demonstrated for the detection of E. coli, and similar optimization processes can be performed for the detection of other biological targets. Third, we demonstrate the new magnetic biosensor concept using a mechanical trap capable of detecting nucleic acids via the size difference between bare magnetic beads and bead compounds. The bead compounds are formed through linking nonmagnetic beads of 1 µm in diameter and magnetic beads of 2.8 µm in diameter by the target nucleic acids. The purpose of the nonmagnetic beads is to increase the size of the compounds, since the nucleic acid is very small compared to the magnetic beads. Alternatively, smaller magnetic beads could be used but their detection would be more challenging. Finally, an enhanced version of the magnetic biosensor concept is developed using an electromagnetic trap for the detection of E. coli. The trap is formed by a current-carrying microwire that attracts magnetic beads into a virtual sensing space. As in the case of the mechanical trap, the sensor signal depends on the number of beads inside of the sensing space. The distance which magnetic beads can be detected from by the MTJ sensor defines the sensing space. The results showed that the output signal depends on the concentration of E. coli in the sample solution and that individual E. coli bacterium inside the sensing space could be detected using super-paramagnetic beads that are 2.8 µm in diameter. In summary, this dissertation investigates a new magnetic biosensor concept, which detects biological targets via the size difference between bare magnetic beads and compounds of magnetic beads and biological targets. The new method is extremely simple and enables the detection of biological targets in two simple steps and within a short time. The concept is demonstrated for the detection of nucleic acid and E. coli.

Two possible ways for increasing the rate of separation for magnetic bead separation has been observed. Increasing NP concentration by 2.5x gave a slight increase in rate of separation while 1.5x and 2.0x concentration increase resulted in a slight decrease in rate of separation. Synthesizing the magnetic beads under the influence of an external magnetic field also showed promising results. In a literature review, several types of magnetic beads and technologies are discussed, and how there is a great future potential for magnetic beads in the isolation of several types of biomolecules. It is concluded that the market for magnetic beads for cell isolation is expanding greatly with many different applications and expects to be worth 14.64 billion USD by 2025.

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This thesis presents some experimental work in the area of manipulation of microparticles. Manipulation of both magnetic and non magnetic beads as well as microorganisms are addressed. The work on magnetic bead manipulation is focused on controlled transport and release, on a micrometer level, of proteins bound to the bead surface. Experimental results for protein transport and release using a method based on magnetization/demagnetization of micron-sized magnetic elements patterned on a modified chip-surface are presented. Special attention has been placed on minimizing bead-surface interactions since sticking problems have shown to be of major importance when protein-coated beads are used. The work with non-magnetic microparticles is focused on the dielectrophoretic manipulation of microorganisms. Preliminary experimental results for trapping and spatial separation of bacteria, yeast and non-magnetic beads are presented. The overall goal was to investigate the use of dielectrophoresis for the separation of sub-populations of bacteria differing in, for example, protein content. This was, however, not possible to demonstrate using our methods.Within the non-magnetic microparticle work, a method for determining the conductivity of bacteria in bulk was also developed. The method is based on the continuous lowering of medium conductivity of a bacterialsuspension while monitoring the medium and suspension conductivities.

Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages. In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.

La dengue et le chikungunya sont deux arboviroses émergentes qui sont transmises à l'homme par la piqûre de moustiques du genre Aedes. Il n'existe ni vaccin ni traitements commercialisés pour ces arboviroses. Il apparaît donc nécessaire de développer de nouvelles stratégies pour isoler les virus circulants et bloquer leur transmission. La compréhension des mécanismes mis en jeu dans les cellules des vecteurs Aedes lors d'une infection par le virus de la dengue (DENV) sont encore très peu étudiés, notamment pour les sérotypes 1 et 3. Par des analyses protéomiques de l'infection d'une lignée cellulaire du moustique Aedes albopictus par ces séroytypes, nous avons démontré qu'en réponse à l'infection, les cellules de moustiques utilisent les mécanismes antioxydants combinés à la production d'énergie pour faire face au virus. Les résultats de notre étude devraient permettre de mieux comprendre l'interaction DENV-vecteur Aedes au niveau cellulaire dans le but de concevoir des stratégies efficaces pour le contrôle du DENV. Nous avons également regroupé dans une revue les connaissances acquises sur les études protéomiques des principaux compartiments des arthropodes vecteurs de maladies humaines. Dans un second volet, nous avons mis en évidence une méthode rapide d'isolement et de concentration des DENV et du chikungunya. Cette technique d'isolement basée sur la capture de virus sur des billes magnétiques enrobées de polymères anioniques permet d'obtenir des particules virales infectieuses. Cette méthode combinée à des approches classiques de détection de virus pourrait non seulement permettre l'identification des échantillons infectés ayant une faible charge virale, mais aussi l'isolement simultanée de particules infectieuses de dengue et de chikungunya à partir d'un seul échantillon. / Dengue (DENV) and Chikungunya (CHIKV) viruses are two emerging arboviruses that are transmitted to humans by the bite of Aedes sp. mosquito vectors. Neither vaccines, nor medical treatments, are commercially available for these infections. It is, therefore, necessary to elaborate novel strategies to isolate the circulating viruses and block their transmission.Our understanding of the molecular mechanisms involved, during the infection of the Aedes vector by dengue virus (DENV), especially serotypes 1 and 3, remains very scant. We, therefore, performed a proteomics analysis of an Aedes albopictus cell line, infected by these two DENV serotypes, and showed that the cells use both anti-oxidant and energy-production mechanisms in the fight against the virus. These results should help to improve our knowledge of the interaction of the DENV virus and the Aedes mosquito vector, at the cellular level, with the aim of designing efficient strategies for the control of this virus. We have, in addition, developed a rapid and sensitive isolation technique, based on viral particle adsorption to magnetic beads coated with an anionic polymer. The use of this technique is of great interest, as it permits the rapid and simultaneous detection and isolation of CHIKV and DENV from samples with reduced viral loads.

The aim of the thesis was the comparison of two DNA microextraction methods with the use of magnetic beads from food of plant origin. Samples had disparate and complex matrices and were either raw (broccoli) or processed (strawberry jam). The first method uses a magnetic separator for the manipulation of magnetic beads and was used as a standart for the comparison. The second method uses a paramagnetic needle, the advantage of which should be the possibility to isolate DNA of higher quality without a significant contamination by polyphenolic compounds or proteins. The former method was validated by statistic analysis of results obtained from both methods. DNA quality was judged by testing the amplificability of isolated DNA via PCR. The amplified products were visualised on an agarose gel with electrophoresis.

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