Real-Time Determination of Picomolar Free Cu(II) in Seawater Using a Fluorescence-Based Fiber Optic Biosensor Hui-Hui Zeng, ² Richard B. Thompson,* ,²,‡ Badri P. Maliwal, ²,‡ Gary R. Fones, §,| James W. Moffett, § and Carol A. Fierke ⊥ Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 21201, Center for Fluorescence Spectroscopy, University of Maryland, 725 West Lombard Street, Baltimore, Maryland 21201, Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, and Departments of Chemistry and Biochemistry, University of Michigan, Ann Arbor, Michigan 48109-1055 We report real-time, in situ determination of free copper ion at picomolar levels in seawater using a fluorescence- based fiber optic biosensor. The sensor transducer is a protein molecule, site-specifically labeled with a fluoro- phore 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 a typical measurement requiring 3 0 s. The sensor demonstrates a detection limit of 0.1 pM free Cu(II) in a seawater 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. Determination of trace metal ions such as Cu(II) in natural waters remains an important task in marine chemistry and environmental monitoring. The importance of Cu(II) as a pollutant is well established and is a source of worldwide concern. Existing methods used for trace metal determination in seawater are sensitive, selective, and accurate. These methods include stripping voltammetry, inductively coupled plasma-mass spectrometry, graphite furnace atomic absorption spectroscopy, atomic emission spectroscopy, flow injection analysis, and ion-selective electrodes. 1-3 Except for the last two methods, however, these methods require collection and processing of the sample prior to analysis, steps that often require more time and labor than the analysis itself. The labor-intensive nature of these methods and the need to process samples immediately to avoid deterioration of stored samples makes it difficult to make measurements frequently over periods of days without multiple operators working in shifts. Particularly on shipboard, numbers of operators may be limited. Moreover, in view of the well-known risk of contamination during the sampling process, it is desirable to avoid sampling altogether. In marine chemistry, it is often useful to be able to determine analytes such as metal ions rapidly and continuously (or quasi- continuously). Certainly the temporal variation of analyte levels is a key parameter in understanding their sources, sinks, and transport kinetics. In addition, it is typically important to be able to correlate the chemical properties of the ocean with physical properties such as temperature, depth, density, or current velocity. The slowness of retrieval of samples from any significant depth (tens of minutes for depths in the 100-m range) makes it difficult to perform temporal correlation of physical measurements with the analytical determinations for any but the slowest processes. For all these reasons, it is desirable to use a sensor: an instrument capable of frequent, real-time measurements, ideally made in situ. We (and others) have pursued the development of such sensors. Some workers have described flow injection analyzers using a stream of seawater collected from overside that provide continuous readouts, but at the current state of the art, they are limited to shallow depths in the water column unless they are self-contained. The need for rapid determination would seem to require that no separation process such as chromatography or electrophoresis take place; in short, there must be a recognition molecule or transducer that interacts with the analyte to produce a signal that can be related to its presence or concentration. For some time, many workers have been developing metal ion sensors where the transducing molecule is of biological origin and may be termed biosensors. 4,5 In our case, the transducing molecules are variants of the enzyme human carbonic anhydrase II. This protein binds certain divalent cations in its active site with high affinity and specificity; in vivo Zn( II) is found there and is required for catalysis. The carbonic anhydrase binding site also binds Cu(II) * To whom correspondence should be addressed: (phone) (410) 706-7142; (fax) (410) 706-7122; (e-mail) rthompso@ umaryland.edu. † University of Maryland School of Medicine. ‡ University of Maryland. § Woods Hole Oceanographic Institution. | Current address: School of Ocean and Earth Science, Southampton Oceanography Centre SO14 3ZH United Kingdom. ⊥ University of Michigan. (1) Bruland, K. W. Limnol. Oceanogr. 1989 , 34, 269-285. (2) Bruland, K. W.; Rue, E. L.; Donat, J. R.; Skrabal, S. A.; Moffat, J. W. Anal. Chim. Acta 1999 , 405, 99-113. (3) Belli, S. L.; Zirino, A. Anal. Chem. 1993 , 65, 2583-2589. (4) Thompson, R. B.; Walt, D. R. Naval Res. Rev. 1994 , 46, 19-29. (5) Wolfbeis, O. S., Ed. Fiber Optic Chemical Sensors and Biosensors; CRC Press: Boca Raton, FL, 1991. Anal. Chem. 2003, 75, 6807-6812 10.1021/ac0345401 CCC: $25.00 © 2003 American Chemical Society Analytical Chemistry, Vol. 75, No. 24, December 15, 2003 6807 Published on Web 11/13/2003