1. REAL TIME DETERMINATION OF PICOMOLAR FREE CU(II) IN SEA WATER USING A FLUORESCENCE-BASED FIBER OPTIC BIOSENSOR Huihui Zeng 1, Richard Thompson 1,2, Badri P. Maliwal 1,2, Gary Fones 5, James Moffett 3, and Carol Fierke 4 1 Department of Biochemistry and Molecular Biology, University of Maryland, School of Medicine, Baltimore, MD 21201 2 Center for Fluorescence Spectroscopy, University of Maryland, Baltimore, MD 21201 3 Department of Geochemistry and Biogeochemistry, Woods Hole Oceanographic Institute, Woods Hole, MA 02543 4 Department of Chemistry and Biochemistry, University of Michigan, Ann Arbor, MI 48109 5 Southampton Oceanography center, Southampton, UK Abstract We report real time, in situ determination of free copper ion at picomolar levels in sea water using a fluorescence-based fiber optic biosensor. The sensor transducer is a protein molecule, site-specifically labeled with a fluorophore, that is attached to the distal end of an optical fiber which binds free Cu(II) with high affinity and selectivity. The transducer reports the metal’s concentration as a change in fluorescence intensity or lifetime, using a frequency domain approach. The transducer’s response time is diffusion-limited, with typical measurement requiring thirty seconds. The sensor demonstrates a detection limit of 0.1 picomolar free Cu(II) in a sea water model. Accuracy and precision of the sensor were at least comparable to cathodic ligand exchange/adsorptive cathodic stripping voltammetry. Measurements of tidal flushing of a copper contaminated inlet are shown. References 1.Thompson, R.B. In Topics in Fluorescence Spectroscopy Vol. 2: Principles; Lakowicz, J.R., Ed.; plenum Press: New York, 1991; Vol.2, pp 345-365. 2.Thompson, R.B. et al, Biosensors and Bioelectronics, 1996, 11, 557-564. 3.Thompson, R.B. et al, Analytical Chemistry, 1999, 267, 185-195. 4.Bhatia, S.K. et al, Analytical Chemistry, 1989, 178, 408-413. 5.Szmacinski, H. et al, In Topics in Fluorescence Spectroscopy Vol.4: Probe Design and Chemical Sensing; Lakowicz, J.R., Ed.; Plenum: New York, 1994; Vol.4, pp 295-334. 6.Thompson, R.B. et al, Analytical Chemistry, 1992, 64, 2075-2078. 7.Moffet, J.W. et al, Limnology and Oceanography 1997, 42, 789-799. Figure 6. Schematic of fiber optic phase fluorometer Figure 2. Human carbonic anhydrase II; The Zn(II) ion found in wild type protein is red. Figure 1.Sensitivity and selectivity in Cu sensing in seawater: Total metal ion concentrations in sea water versus affinities of Fura-2( a fluorescent indicator) and variants of carbonic anhydrase. Figure 9. Overview of Eel Pond Figure 4. Principle of phase fluorimetry: Phase, modulation change with lifetime. Figure 5. Sensor calibration curves in cuvette: Fluorescence intensities(●), and phase angles(○) and modulations(□) at 200 MHz for apo-L198C Alexa Fluor 660 as a function of Cu(II)) in a MOPS/NTA buffer. Figure 10. Picture of the Inlet Figure 7. Immobilization procedures for carbonic anhydrase on fiber optic Figure 11. Calibration curves for fiber optic Cu(II) sensor: Fluorescence intensities(●), and phase angles(○) and modulations(□) at 200 MHz for apo-L198C Alexa Fluor 660 immobilized at the distal end of an optical fiber as a function of Cu(II) concentration, with the best two-component fits indicated. Figure 12. Time response of Carbonic anhydrase-based sensor: Time-dependent phase angles of apo-N67C-Oregon Green immobilized on quartz showing response of immobilized carbonic anhydrase to free [Cu]. Figure 13. Time-dependent Fluorescence intensities(●), and phase angles(○) and modulations(□) at 200 MHz for apo-L198C Alexa Fluor 660 immobilized at the distal end of an optical fiber immersed in Eel Pond inlet. Time is given after beginning of ebb tide. Figure 14. Comparison of free Cu(II) determined by CLE/ACSV and by phase fluorimetry with optical fiber in sea water. Figure 3. Principle of fluorescence-based Cu sensing by carbonic anhydrase: Binding of Cu (K D  0.1pM) to fluorescent-labeled apo- carbonic anhydrase results in energy transfer, quenching; Fluorescent label exhibits decreased intensity, lifetime. Figure 8.Total and free Cu levels in Southern Massachusetts Harbors; Eel Pond is at left (From Moffet, et al.)