BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 18: Biosensors Last time: engineering intracellular delivery Drug targeting Today: biosensor device classes Detection methods Overview of biosensor technology Classes of biosensor devices External analysis/detection o Large instruments o Objectives null Maximum sensitivity null Highest throughput o Samples probed null Biochemical null Cell populations null Intracellular (single cells) Field detection o Usually simpler, need to be more robust In vivo detection o Usually catheter/needle-based for minimal invasiveness, with detector outside body Classes of sensor mechanisms 1 Capture-based null Binding of labeled analyte to capture reagent null Binding of unlabeled analyte to capture reagent triggers detectable signal Catalysis 2 null Enzymatic reaction generates a detectable product o Change in proton concentration, release of O 2 , NH 3 , CO 2 o Release of metals, halides o Ion/electron transfer o Change in optical properties (e.g. production of a colored product) Cell-based null Single-cell-based o Binding of analyte to cell surface receptor triggers detectable signal null Tissue-based biosensors 3 o Binding of analyte to one cell type triggers cell-cell interactions and signaling cascades that can be detected Lecture 18 – Biosensors 1 of 6 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Capture-based Binding of labeled analyte to capture reagent Binding of unlabeled analyte to capture reagent triggers detectable signal Applications null Microanalysis o Small sample sizes, high throughput null Toxicology and drug testing o Testing drug safety Cell-based Single-cell: Binding to cell surface receptor triggers signal (Griffith and Naughton, 2002) Tissue-based: Binding to multiple cells triggers cell-cell interacdtions o Screening libraries of candidate drug compounds null Toxin and pathogen detection Detection Elements Optical o Concept null Capture analyte and detect binding by optical tag or binding-sensitive optical phenomenon o Capture null Surface-immobilized capture molecules ? E.g. single-stranded DNA (DNA), antibodies (target antigens) Lecture 18 – Biosensors 2 of 6 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 identification based on x-y Capture agent uniquely marked for identification (Lehmann, 2002) Optical bar-coding location of capture agent ? Detection surface can either be planar or composed of capture particles null Planar surface: null Identification based on x-y location of tag null Particle-based detection: null Faster kinetics of binding due to reduced distances to be traveled by analyte null Identification based on particle-specific labeling (challenge) ? Commercial technology example of planar detection surface: gene chips Lecture 18 – Biosensors 3 of 6 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 QuickTime? and a Graphics decompressor are needed to see this picture. o Composition of arrays: null Oligonucleotides ? Each ‘spot’ composed of ~40 oligos 25 base pairs long and a matching control with one central base changed o Need different permutations for each gene to account for redundancy in short probe sequences ? Must know gene sequence to prepare appropriate oligos null CDNA-sized fragments ? Usually produced by PCR ? Long fragments where each fragment uniquely identifies a gene ? Can pack all 6000 yeast genes onto a 1.28 cm x 1.28 cm glass slide ? Random cDNA clones can be used o Application null Label mRNA from cell sample, apply to chip and allow to hybridize null Scan chip for bound fluorescence o Gene chips can detect mRNA present at < 1 molecule in 100,000 (equivalent to detecting one transcript per 20 yeast cells) o Entire yeast genome can be put on a chip Lecture 18 – Biosensors 4 of 6 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 (Johnston, 1998) 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 18 – Biosensors 5 of 6 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 18 – Biosensors 6 of 6