BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Lecture 20: Cell-and tissue-based biosensors Last tir 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.Eng27,697-711(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 DIC/ura over Calcium signalling Potential for single molecule sensitivity TIme relative to Ca+ signal (ml retinal neurons triggered by PMrG PE fuorescence single photons In T/APC Interface ifactory neurons detect single odorant molecules T cell recognition of foreign peptide(shown at night 5 um -Cellular machinery maintains physiological status of receptors involved in detection 30%100 Complex @valuation@f 6 aaen 03040506070 Error! (Irvine et al. 2002) 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 Lecture 20-Biosensors 1 of 8
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 biosensors1-6 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 peptides7 Error! (Irvine et al. 2002) Calcium signaling ¥Potential for singlemolecule 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 ÔevaluationÕ 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 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 Transducers(Haruyama 2003) secondary Biomolecule secretion Light emission Gene expression 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 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 Lecture 20-Biosensors 2 of 8
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 • Neurons2,8 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 Cardiomyocytes 1. changes in reative activity patterrs Neuronal cels synaptically aclive (e. g. nerve) agents 2. changes in newark signalling csaslatiors on channel blockers and toxins 3. paRoxysmal responses due to pathologcal membrane UMd casette network activity S6006NMIAIRN MIlluillulull # Activty fall 几Amm团 newark te 2 p trimethylolpropane phesphate (seizure-inducing compaund) Tenger e a. 20013 0204060B0100120140160 Glutamate. DFP Gross et al. 1997) FIGURE 7. Culture of eva ation, (Upper pane) Phase contrast image or embry m the same site. As shown from Pancrazio et al. 1999)the utility of neur ablation o spontaneus mrig, shlstrating Is cultured on microelectrode arrays for ection of toxic compounds Microphysiometer 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 Lecture 20-Biosensors 3 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 20 – Biosensors 3 of 8 (Gross et al. 1997) (Pancrazio et al. 1999) Microphysiometer9-11 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 Current Adherent Cells Potential Monitor Effects on proton release rate 自自 Silicon FReceptor-ligand binding oxynitride Metabolic drugs/poisons LEDS General cell stress COnnell et al. 1992 Pancrazio et al. 1999 Detecting antigens using T cells and a microphysiometer MCCs 130FC4R3.9TCells MBPI-I4)M4 poptie 是m (McConnell et al. 1995 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 o Receptors, channels, and enzymes maintained in a physiologically-relevant state by the machinery of the o May provide alternatives to animal testing in the future Lecture 20-Biosensors 4 of 8
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 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 (materiat ot pnM Stre desired panem Removal of stream I PDMS Str (Park and shuler, 2003) Dispense of Removal of Fsgs rm lcr ochametis s af the prar fow pamernnat. te ite n Cal pahterInthgraphy, (b) microcontact printing (e) microfluidic patterning 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 Lecture 20-Biosensors 5 of 8
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 sensing12 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 closepacked alkyl chain structures on a surface
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 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 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 Ncohthclene Dihydrodiol GSH Conjugate 悯R,250xide1s2R-0 Richly fused Perfused enzymatic Poor 4. Transferase Perfused Liver tx’y? Figure 1. Reaction scheme for naphthalene and its products. Dihydrodiol GSH Conjugate Quick and shuler 1999 Lecture 20-Biosensors 6 of 8
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 naphthalene13 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 Gas Exchange ID DO o4 a Other Tissues Out Models retention of chemical in blood and interstitial fluid Figure 4.(a)Microscopic CCA system with four chambers. The dimensions(w x I x d of the chambers are: lung 2 mm x 2 mm x 20 um; liver 3.5 mm x 4.6 mm x 20 um; other tissue 0.4 m x 100am:fat0.42mm×50.6mm×100m Cells are cultured as monolayers on the silicon surfaces modified by adsorption of polylysine and collagen(b) ( Park and Shuler 2003 In vivo detection Biofouling typically limits lifetime of in vivo measurements to 1-2 days o Loss of vasculature Lecture 20-Biosensors 7 of 8
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 References Stenger, D Aet 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 DeBusschere, B D. Kovacs, G. T A Portable cell-based biosensor system using integrated CMOS cell cartridges. Biosensors Bioelectronics 16, 543-556 (2001) ilchrist, K. H. et al. General purpose field-portable cell-based biosensor platform. Biosensors Bioelectronics 16,557-564(2001) Makohliso, S. A. et al. Surface characterization of a biochip prototype for cell-based biosensor applications Langmuir15,2940-2946(1999) Gray, S A et al. Design and demonstration of an automated cell-based biosensor. Biosensors Bioelectronics 16,535542(2001) Irvine, D. J, Purbhoo, M. A, Krogsgaard, M.& Davis, M. M. Direct observation of ligand recognition by T cells Nature419,8459(2002) Pancrazio, J J et al. Portable cell-based biosensor system for toxin detection. sensors and Actuators B- Chemica/53,179185(1998) 9. McConnell, H M et al. The cytosensor microphysiometer: biological applications of silicon technology. Science 257,1906-12(192 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 ll molecules. Proc Nat/ Acad SciUSA 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. Biotechnology Progress 15, 540-555 (1999) Lecture 20-Biosensors 8 of 8
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 cellcartridges. 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 BChemical 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 cellbased 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. Biotechnology Progress 15, 540-555 (1999)
