Electrokinetics for sample preparation of biological molecules in biological samples using microfluidic systems (original) (raw)
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Monoclonal antibody (mAb) technology is one of the most important scientific achievements in the twentieth century. However, the traditional mAbs screening encounters a bottleneck for the high-throughput process. Here we integrate the immunoassay methods on the digital microfluidic platform for the mAbs analysis that potentially holds high throughput. In our prototype devices, the nitrocellulose dots spotted on the Teflon surface let the isotyping antibodies be bound on the chip stably. Furthermore, the droplet of antibodies mixture and washing buffer were manipulated by EWOD (electrowetting-on-cielectric) force. The preliminary results show that the isotyping analysis was executed on the digital microfluidic platform.
Toward Integrated Molecular Diagnostic System ($i$ MDx): Principles and Applications
IEEE Transactions on Biomedical Engineering, 2014
Integrated molecular diagnostic systems (iMDx), which are automated, sensitive, specific, userfriendly, robust, rapid, easy-to-use, and portable, can revolutionize future medicine. This review will first focus on the components of sample extraction, preservation, and filtration necessary for all point-of-care devices to include for practical use. Subsequently, we will look for low-powered and precise methods for both sample amplification and signal transduction, going in-depth to the details behind their principles. The final field of total device integration and its application to the clinical field will also be addressed to discuss the practicality for future patient care. We envision that microfluidic systems hold the potential to breakthrough the number of problems brought into the field of medical diagnosis today.
Multimodal ligands are synthetic molecules comprising multiple types of interactions that have been increasingly used for the capture of different biopharmaceutical compounds within complex biological mixtures. For monoclonal antibodies (mAbs) in particular, these ligands have shown the possibility of direct capture from cell culture supernatants in native conditions, as well as enhanced selectivity and affinity compared to traditional single-mode ligands. However, performing the capture of a target mAb using multimodal chromatography comes with the need for extensive optimization of the operating conditions, due to the multitude of interactions that can be promoted in parallel. In this work, a high-throughput microfluidic platform was developed for the optimization of chromatographic conditions regarding the capture of an anti-interleukin 8 mAb, using a multimodal ligand (2-benzamido-4-mercaptobutanoic acid), under a wide range of buffer pH and conductivities. The interaction of the ligand with the fluorescently labeled target mAb was also analyzed with respect to the individual contribution of the hydrophobic (phenyl) and electrostatic (carboxyl) moieties using fluorescence microscopy. The results were further validated at the macroscale using prepacked columns in standard chromatography assays, and recovery yield values of 94.6% ± 5.2% and 97.7% ± 1.5% were obtained under optimal conditions for the miniaturized and conventional approaches, respectively. In summary, this study highlights that a microfluidic-based approach is a powerful analytical tool to expedite the optimization process while using reduced reagent volumes (<50 μL), less resin (∼70 nL), and delivering results in less than 1 min per assay condition.
Though only my name appears on the cover of this dissertation, many people have contributed and extended their valuable assistance to its production. I owe my gratitude to all those people who have made this dissertation possible and because of whom my graduate experience has been one that I will cherish forever. My deepest gratitude is to my advisor, Dr. James F. Rusling for his patience, motivation, enthusiasm, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor for my PhD program. Jim taught me how to question thoughts and express ideas. I am grateful to him for holding me to a high research standard and enforcing strict validations for each research result, and thus teaching me how to do research. His patience and support helped me overcome many crisis situations and finish this dissertation. His insightful comments and constructive criticisms at different stages of my research were thought-provoking and they helped me focus my ideas. I am also thankful to him for reading my reports, commenting on my views and helping me understand and enrich my ideas. I am deeply grateful to him for the long discussions that helped me sort out the technical details of my work. I hope that one day I would become a good mentor as Jim has been to me. Besides my advisor, I would like to thank the rest of my thesis committee: Prof.
Sample preparation: the weak link in microfluidics-based biodetection
Biomedical Microdevices, 2008
As a broad generalization, clinicians and laboratory personnel who use microfluidics-based automated or semi-automated instrumentation to perform biomedical assays on real-world samples are more pleased with the state of the assays than they are with the state of the frontend sample preparation. The end-to-end procedure requires one to collect, manipulate, prepare, and analyze the sample. The appeal of microfluidics for this procedure is partly based on its combination of small size and its ability to process very small liquid volumes, thus minimizing the use of possibly expensive reagents. However, real-world samples are often large and incompatible with the input port and the μm-scale channels of a microfluidic device, and very small liquid volumes can be inappropriate in analyzing low concentrations of target analytes. It can be a worthy challenge to take a raw sample, introduce it into a microfluidics-based system, and perform the sample preparation, which may include separation and concentration of the target analytes, so that one can benefit from the reagentconserving small volumes and obtain the correct answer when finally implementing the assay of interest.
2016
Though only my name appears on the cover of this dissertation, many people have contributed and extended their valuable assistance to its production. I owe my gratitude to all those people who have made this dissertation possible and because of whom my graduate experience has been one that I will cherish forever. My deepest gratitude is to my advisor, Dr. James F. Rusling for his patience, motivation, enthusiasm, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor for my PhD program. Jim taught me how to question thoughts and express ideas. I am grateful to him for holding me to a high research standard and enforcing strict validations for each research result, and thus teaching me how to do research. His patience and support helped me overcome many crisis situations and finish this dissertation. His insightful comments and constructive criticisms at different stages of my research were thought-provoking and they helped me focus my ideas. I am also thankful to him for reading my reports, commenting on my views and helping me understand and enrich my ideas. I am deeply grateful to him for the long discussions that helped me sort out the technical details of my work. I hope that one day I would become a good mentor as Jim has been to me. Besides my advisor, I would like to thank the rest of my thesis committee: Prof.
Digital microfluidic assay for protein detection
Proceedings of the National Academy of Sciences, 2014
Global studies of the human proteome have revealed a plethora of putative protein biomarkers. However, their application for early disease detection remains at a standstill without suitable methods to realize their utility in the clinical setting. There thus continues to be tremendous interest in developing new technology for sensitive protein detection that is both low in cost and carries a small footprint to be able to be used at the point of care. The current gold standard method for protein biomarker detection is the ELISA, which measures protein abundance using bulky fluorescent scanners that lack portability. Here, we present a digital microfluidic platform for protein biomarker detection that is low in cost compared with standard optical detection methods, without any compromise in sensitivity. This platform furthermore makes use of simple electronics, enabling its translation into a portable handheld device, and has been developed in a manner that can easily be adapted to assay different types of proteomic biomarkers. We demonstrate its utility in quantifying not only protein abundance, but also activity. Interleukin-6 abundance could be assayed from concentrations as low as 50 pM (an order of magnitude lower than that detectable by a comparable laboratory designed ELISA) using less than 5 μL of sample, and Abelson tyrosine kinase activity was detectable in samples containing 100 pM of kinase.
Biosensors and Bioelectronics, 2009
Requirements for a point-of-care device are an easy and robust read-out and -above all -a simple handling. We integrated an established robust electrical read-out for DNA-chips into a microfluidic device, thereby creating an automated analysis system that combines the necessary steps for a chip-based analysis. It is based on the electrical detection of biotin-labeled DNA in a gap between two microstructured electrodes on the surface of a DNA-chip. The biotin serves as binding molecule for streptavidin-conjugated horseradish peroxidase. A following enzyme-induced silver deposition bridges the gap by a conductive layer. The miniaturized chip gives the possibility to realize a durable system suitable for point-of-care applications.