BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 20 – Biosensors 1 of 8 Lecture 20: Cell- and Tissue-based biosensors Last time: detection methods Surface plasmon resonance biosensors Today: cell- and tissue-based sensors Primary transducers and biosensor design with living cells microphysiometer Reading: J.J. Pancrazio et al., ‘Development and application of cell-based biosensors,’ Ann. Biomed. Eng. 27, 697-711 (1999) Cell-based biosensors 1-6 General concepts ? Why cell-based biosensors? o Known ultrasensitivity of cells: ? Olfactory neurons respond to single odorant molecules ? Retinal neurons triggered by single photons ? T cells triggered by single antigenic peptides 7 Error! (Irvine et al. 2002) Calcium signaling ￥Potential for single- molecule sensitivity -retinal neurons triggered by single photons -olfactory neurons detect single odorant molecules -T cell recognition of foreign peptide (shown at right) ￥Cellular machinery maintains physiological status of receptors involved in detection ￥Complex ?evaluation? of agents o Ability to ‘integrate’ cellular or tissue response to compounds ? Detect functionality of compound in addition to its chemical presence ? i.e. tell the difference between a dead and live virus BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 20 – Biosensors 2 of 8 Design of CBBs: ? Cell-based biosensors are based on a primary transducer (the cell) and secondary transducer (device which converts cellular/biochemical response into a detectable signal) o Secondary transducer may be electrical or optical o Example pathways for signal transduction: ? Toxin -> cell stress -> changes in gene expression ? Analyte -> cell metabolism -> changes in extracellular acidification rates Electrical signal Biomolecule secretion Light emission Gene expression Transducers (Haruyama 2003) primary secondary Single-cell arrays Tissue arrays ? Detection of arbitrary targets o Transfect cells with receptors to introduce responsiveness of e.g. neuronal cells to a chosen compound ? Basis of electrical secondary transducers o Electrically-excitable cells ? Example cell types ? Neurons 2,8 o Non-sensory neurons grown in culture outside of normal homeostasis and the insulation of the blood-brain barrier behave in a ‘sensory’ manner (Gross 1997) o Electrical signals play physiological role in control of secretion ? Cardiomyocytes o Electrical signals play physiological role in control of contraction ? Generate electric signals in a substance-specific and concentration-dependent manner ? Signals generated can be monitored by microelectrodes BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 20 – Biosensors 3 of 8 (Gross et al. 1997) (Pancrazio et al. 1999) Microphysiometer 9-11 ? Measures changes in extracellular acidification rate: pH changes associated with alterations in ATP consumption by cells (metabolism) ? Extremely sensitive readout of changes in cell metabolism BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 20 – Biosensors 4 of 8 (McConnell et al. 1992) (Pancrazio et al. 1999) Effects on proton release rate: ￥Receptor-ligand binding ￥Metabolic drugs/poisons ￥General cell stress (McConnell et al. 1995) Detecting antigens using T cells and a microphysiometer: Relative advantages and disadvantages of cell-based sensors ? Pros o Cell-based sensors may utilize the ability of cells to respond to complex mixtures of signals in a unique way o Receptors, channels, and enzymes maintained in a physiologically-relevant state by the machinery of the cell o May provide alternatives to animal testing in the future BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 20 – Biosensors 5 of 8 ? Cons o Issues of maintaining cell viability and reproducibility in measurements o Issues of cell sources ? Often require primary cells in current systems Patterning cells for sensing 12 ? Techniques used: o Photolithography o Microcontact printing (soft lithography) o Microfluidic patterning o Membrane lift-off (Park and Shuler, 2003) soft lithography and self-assembled monolayers ? Techniques based on the formation of gold (or other metal)-thiol bonds and spontaneous assembly of close- packed alkyl chain structures on a surface BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 20 – Biosensors 6 of 8 Tissue-based biosensors ? Any papers out on the liver chip? GRIFFITH LAB In vitro toxicology studies: tissue biosensors ? Shown below is a model of the pharmacology of naphthalene 13 o Tissue distribution and toxic chemistry outlined is a multi-organ, multi-compartment phenomenon ? Potential methodology: Animal-on-a-chip o 2 cm x 2 cm Si chip o designed to have ratio of organ compartment size and liquid residence times physiologically realistic o minimum 10K cells per compartment to facilitate analysis of chemicals and enzyme activity o physiologic hydrodynamic shear stress values (Quick and Shuler 1999) BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 20 – Biosensors 7 of 8 (Park and Shuler 2003) Models retention of chemical in blood and interstitial fluid In vivo detection ? Biofouling typically limits lifetime of in vivo measurements to 1-2 days o Inflammation o Fibrosis o Loss of vasculature BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 20 – Biosensors 8 of 8 References 1. Stenger, D. A. et al. Detection of physiologically active compounds using cell-based biosensors. Trends in Biotechnology 19, 304-309 (2001). 2. Gross, G. W., Harsch, A., Rhoades, B. K. & Gopel, W. Odor, drug and toxin analysis with neuronal networks in vitro: extracellular array recording of network responses. Biosensors and Bioelectronics 12, 373-393 (1997). 3. DeBusschere, B. D. & Kovacs, G. T. A. Portable cell-based biosensor system using integrated CMOS cell- cartridges. Biosensors & Bioelectronics 16, 543-556 (2001). 4. Gilchrist, K. H. et al. General purpose, field-portable cell-based biosensor platform. Biosensors & Bioelectronics 16, 557-564 (2001). 5. Makohliso, S. A. et al. Surface characterization of a biochip prototype for cell-based biosensor applications. Langmuir 15, 2940-2946 (1999). 6. Gray, S. A. et al. Design and demonstration of an automated cell-based biosensor. Biosensors & Bioelectronics 16, 535-542 (2001). 7. Irvine, D. J., Purbhoo, M. A., Krogsgaard, M. & Davis, M. M. Direct observation of ligand recognition by T cells. Nature 419, 845-9 (2002). 8. Pancrazio, J. J. et al. Portable cell-based biosensor system for toxin detection. Sensors and Actuators B- Chemical 53, 179-185 (1998). 9. McConnell, H. M. et al. The cytosensor microphysiometer: biological applications of silicon technology. Science 257, 1906-12 (1992). 10. McConnell, H. M., Wada, H. G., Arimilli, S., Fok, K. S. & Nag, B. Stimulation of T cells by antigen-presenting cells is kinetically controlled by antigenic peptide binding to major histocompatibility complex class II molecules. Proc Natl Acad Sci U S A 92, 2750-4 (1995). 11. Pancrazio, J. J., Whelan, J. P., Borkholder, D. A., Ma, W. & Stenger, D. A. Development and application of cell- based biosensors. Annals of Biomedical Engineering 27, 697-711 (1999). 12. Park, T. H. & Shuler, M. L. Integration of cell culture and microfabrication technology. Biotechnology Progress 19, 243-253 (2003). 13. Quick, D. J. & Shuler, M. L. Use of in vitro data for construction of a physiologically based pharmacokinetic model for naphthalene in rats and mice to probe species differences. 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