BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 19: Biosensors (continued) Last time: biosensor device classes Gene array biosensors Today: detection methods Detection Elements o Readout ? Macroscopic fluorescence, diffraction, or interference ? what ? Optical bar-coding 4 ? Example: quantum dot-loaded microsphere capture agents 5 ? QDs show size-dependent luminescence ? Narrow emission bands from a common excitation wavelength ? Stable against photobleaching ? Approach: ? Load polymer microspheres with different amounts of several colors of QDs to obtain a unique fluorescence signature ? 6 colors at 10 possible intensities allows for > 10 6 possible ‘codes’ ? Capture molecule on surface of beads grabs labeled analyte (Han et al, 2001) Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Excitation of bar-code and target fluorochrome by same wavelength Microscope-based spectrophotometer for detection of emission spectra from individual beads ? Optical absorption (colorimetric) ? what ? Surface plasmon resonance and SPR arrays ? Developed commercially later 1980’s (Cooper 2002) ? Typically, receptor is immobilized and free ligand is passed over sensor chip ? Both ways possible, small ligands simply interfere with binding if immobilized (Cooper 2002) Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Biacore sensor chips Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 (Cooper 2002) ? Optical fiber-based ? Single cell analysis optical fiber probes 6 Advantages/disadvantages ? Pros o Fast measurements o Sensitive ? Cons o Cannot perform detection on turbid solutions Electrochemical Electrochemical readouts 7 o Conductometric o Measure changes in the conductance of the biological component arising between a pair of metal electrodes due to e.g. metabolism o Potentiometric o Measure electrical potential difference between a sample and reference electrode o Monitor the accumulation of charge at zero current created by selective binding at the electrode surface Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o Electrode may be selective for certain ions or gases ? E.g. F-, I-, CN-, Na+, K+, Ca2+, H+, NH4+ ? CO2, NH3 o Amperometric o Measure current generated by electrochemical oxidation or reduction of electroactive species at a constant applied potential Electrochemical detection: ISFET - ion-sensitive field-effect transistors Common pH-modifying enzymatic reactions: (Mulchandani and Rogers, 1998) Advantages/disadvantages ? Pros o Fast measurements o Sensitive ? Low detection limits typically ~ 10 -9 M o Ability to perform measurements on turbid/opaque solutions ? Cons o PH-sensing mechanisms require weakly buffered or non-buffered solutions Calorimetric Calorimetric readouts o Measurement of heat generated by an enzymatic reaction o Typically utilize thermistors to transform heat into an electrical signal Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Calorimetric detection: QuickTime? and a TIFF (LZW) decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. http://www.sbu.ac.uk/biology/enztech/calorimetric.html Schematic diagram of a calorimetric biosensor. The sample stream (a) passes through the outer insulated box (b) to the heat exchanger (c) within an aluminium block (d). From there, it flows past the reference thermistor (e) and into the packed bed bioreactor (f, 1ml volume), containing the biocatalyst, where the reaction occurs. The change in temperature is determined by the thermistor (g) and the solution passed to waste (h). External electronics (l) determines the difference in the resistance, and hence temperature, between the thermistors. Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Piezoelectric 8,9 ? Based on quartz crystal microbalance detectionetection o Crystal is oscillated at a defined frequency by an oscillating applied voltage ? Shear deformation induced as dipoles in crystal seek to align with direction of electric field field ? Deformation typically 10-100 nm for AT-cut crystals operating in freq. range of 1-10 MHz MHz o Binding of analyte to surface changes mass of crystal and alter oscillation frequencyo cy ? Figure below from : www-bond.chem.monash.edu.au/theses/ Graeme%20Snook/Chapter1.pdf Piezoelectric detection: Quartz crystal microbalance Analyte solution http://www.q-sense.com/main.qcmd_tech.html Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Piezoelectric detection: Quartz crystal microbalance Detecting HIV virions: (Cooper et al. 2001) Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 SPR Arrays 10 ? External analysis/detection ? Optical method Cell- and tissue-based biosensors (Stenger 2001, Gross 1997) 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 (Irvine 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 ? 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 ? 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 Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 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) ? Cardiomyocytes ? Generate electric signals in a substance-specific and concentration-dependent manner ? Signals generated can be monitored by microelectrodes o Microphysiometer 11,12 ? 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 ? EXAMPLE OF HARDING MCCONNELL’S WORK WITH T CELLS 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 May provide alternatives to animal testing in the future ? Cons o Issues of maintaining cell viability and reproducibility in measurements 13 Patterning cells for sensing ? Techniques used: o Photolithography o Microcontact printing (soft lithography) o Microfluidic patterning o Membrane lift-off Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 (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 Tissue analogs ? 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 14 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 Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 (Quick and Shuler 1999) Models retention of chemical in blood and interstitial fluid (Park and Shuler 2003) Lecture 19 – Biosensors BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 In vivo detection ? Biofouling typically limits lifetime of in vivo measurements to 1-2 days o Inflammation o Fibrosis o Loss of vasculature References 1. Shah, J. & Wilkins, E. Electrochemical biosensors for detection of biological warfare agents. Electroanalysis 15, 157-167 (2003). 2. Chaplin, M. & Bucke, C. Enzyme Technology (Cambridge Univ Press, New York, 1990). 3. Griffith, L. G. & Naughton, G. Tissue engineering--current challenges and expanding opportunities. Science 295, 1009-14 (2002). 4. Lehmann, V. Biosensors: Barcoded molecules. Nat Mater 1, 12-3 (2002). 5. Han, M., Gao, X., Su, J. Z. & Nie, S. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat Biotechnol 19, 631-5 (2001). 6. Vo-Dinh, T., Alarie, J. P., Cullum, B. M. & Griffin, G. D. Antibody-based nanoprobe for measurement of a fluorescent analyte in a single cell. Nat Biotechnol 18, 764-7 (2000). 7. Mulchandani, A. & Rogers, K. R. Enzyme and Microbial Sensors (Humana Press, New York, 1998). 8. Cooper, M. A. et al. Direct and sensitive detection of a human virus by rupture event scanning. Nat Biotechnol 19, 833-7 (2001). 9. Saphire, E. O. & Parren, P. W. Listening for viral infection. Nat Biotechnol 19, 823-4 (2001). 10. Cooper, M. A. Optical biosensors in drug discovery. Nat Rev Drug Discov 1, 515-28 (2002). 11. McConnell, H. M. et al. The cytosensor microphysiometer: biological applications of silicon technology. Science 257, 1906-12 (1992). 12. 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). 13. Park, T. H. & Shuler, M. L. Integration of cell culture and microfabrication technology. Biotechnology Progress 19, 243-253 (2003). 14. 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 19 – Biosensors