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Cell-based biosensors (CBB) exploit the naturally evolved sensitivity of cells in order to detect a broad range of biochemical agents as a cellular response. This cellular response is detected by a secondary transducer and converted into an electronic form suitable for analysis. In comparison to other biosensor types, CBBs offer the advantage of a functional assay without requiring foreknowledge of the chemical structure. This advantage would be a powerful tool in potential field applications such as toxin detection and environmental monitoring if portable CBB systems were available. The development of portable CBBs has been limited due to many issues including preparation of the sample, maintenance of the biological environment, incorporation of the appropriate sensors, development of ìportable-friendlyî sensing algorithms, and integration of electronics for data collection and analysis.

This dissertation reports on the development of a portable cell-based biosensor recording platform that addresses several of these portability issues with the fabrication of an integrated silicon / polydimethylsiloxane cell-cartridge. The cell-cartridge contains a CMOS silicon die that incorporates digitally controlled multiplexers, a temperature control system, microelectrode electrophysiology sensors, and analog signal buffering. Additionally, the cell-cartridge supports two separate cell populations in two 10 Ïl sealed chambers that have independent fluidic channels for sample injection. The sealed chambers were designed to be sufficiently gas-permeable to eliminate the need for a continuous perfusion system, even at high cell densities. A handheld, microcontroller-based electronics system capable of monitoring the action potential activity within the cell-cartridges was also developed.

HL-1 cardiomyocytes were observed to adhere, proliferate, and grow to confluency inside the cell-cartridge chambers. Extracellular action potentials were recorded up to one week after plating at which point the cells became overconfluent and pulsatile activity was no longer apparent. Shifts in temperature were observed to cause shifts in the electrophysiological activity of the cardiomyocyte syncytia of both chambers, while the two chambers responded differentially to the flow of a control medium versus the flow of a biochemical agent. To demonstrate portability, the cell-cartridges and handheld electronics system were used to successfully record action potential activity from cardiomyocytes outside of the laboratory under realistic application conditions.

In addition to the work on a portable CBB recording platform, new sensing algorithms suitable for portable implementation were investigated. One of the primary obstacles in the practical implementation of CBBs is the interpretation of the output signal of the cell since multiple biochemical pathways can lead to the same cellular response. Signal interpretation has many constraints in a portable system with its limited resources, and the CBB algorithm used needs to generate high-confidence results with the minimal number of experiments. A new approach presented in this work used improved primary transducers (genetically-engineered cells) in a differential measurement to achieve a specific functional classification of biological agents without requiring a large number of experiments. The ability to use this technique on microelectrode arrays and monitor a single parameter such as beat rate makes it suitable for a portable system. The approach reduces signal processing complexity, minimizes the number of required experiments, and is compatible with low-power and compact microelectrode sensors. This approach retains the advantages of a functional assay while allowing specificity of agent recognition, and importantly, required only a limited number of experiments to achieve a highly significant response. The cell-cartridge, with its two independent chambers on the same sensing substrates and separate fluid paths, is a very appropriate sensing platform for this technique.

 

DeBusschere, B. D., ìPortable Cell-based Biosensors,î Ph.D. Thesis, Stanford University, CA, 2002.