1740
Anal. Chem. 1085, 57, 1740-1743
device did not vary with the temperature within experimental error. CONCLUSION The correlation between this method of ozone determination and UV photometry is 1.01 f 0.02. The transient response time ( b 2 / D )for the device, estimated from the membrane thickness and diffusion coefficient of ozone in the membrane (4,8), is of the order of 1.0 s. This short response time makes the device sensitive to rapid fluctuations in ambient ozone concentrations. The sampling rate can be calculated from the relationship (14)
O3collected (pg) = Co,
X sampling rate X time
where Co, is the ozone concentration. By use of results from a typical experiment, the number of moles of DBA that have reacted equals the number of moles of ozone trapped. Converting this to microgram and the concentration of ozone from parts per million to micrograms, at 25 “C and 760 torr we found a sampling rate of 180 mL/min. Registry No. DBA, 23663-77-6; Os,10028-15-6. LITERATURE CITED (1) Reiszner, K. D.; West, P. W. Environ. Sci. Techno/. 1973, 7, 526-532.
Neims, L. H. Ph.D. dlssertation. Louisiana State University, 1976. Matthews, T. G.; Hawthorne, A. R.; Howell, T. C.; Metcalfe, C. E.; Gammage, R. B. Environ. I n t . 1982, 8, 143-151. Rubin, R. J. NBSIR 80-1975. National Bureau of Standards: Washington, DC, 1980. General Electric Co. “General Electric Permselective Membranes”; Membrane Products Operation, Medical Systems Business Operations: Schenectady. NY, 1983. Kearns, D. R. J. Am. Chem. SOC. 1989, 91, 6554-6563. Turro, N. J. “Modern Molecular Photochemistry”; Benjamin-Cummings: Menlo Park, CA, 1978; p 278. Crank, J.; Park, G. S. “Diffusion in Polymers”; Academic Press: New York, 1968. Mukaiyama, T.; Sato, T. Hanna, J. Chem. Lett. 1973, 1041-1044. Hodgeson, J. A.; Surgi, M. R. I n “The Air Quality Criterla Document for Ozone and Other Photochemical Oxidants”; U S . Environmental Protection Agency: Research Triangle Park, NC, 1984. Benson. F. B.; Henderson, J. J.; Caidwell, D. E. ”Indoor-Outdoor Air Pollution Relationships. A Literature Review”; U.S. Environmental Protection Agency: Research Triangle Park, NC, 1972; Pubi. No. AP-112. Yasuda, H.; Clark, H. 0.; Stannett, V. I n ”Encyciopedia of Polymer Science and Technology”; Mark, H. G., Gaylord, N. G., Eds.; Interscience: New York, 1968; Voi. 9. Pryor, W. A.; Collard, R. S. J. Environ. Sci. Health Par? A 1981, A 16, 73-86. Cardoff, B.; Hodgeson, J. A. Anal. Chem. 1983, 55, 2083-2085.
RECEIVED for review December 17,1984. Accepted March 15, 1985. The authors are indebted to Louisiana State University, Department of Chemistry, for their financial support and permission to publish this work.
High-Sensitivity Enzyme Thermistor Determination of L-Lactate by Substrate Recycling Frieder Scheller,’ Nils Siegbahn, Bengt Danielsson,* and Klaus Mosbach
Pure and Applied Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S-22100 Lund, Sweden
Substrate recycling was used to increase the sensltivlty of enzyme probes by several orders of magnitude. One system studled was L-lactate determination wlth lactate oxidase coimmoblilzed with catalase and lactate dehydrogenase. I n the presence of sufficient amounts of NAD(H) and oxygen, Llactate was repeatedly oxidized to pyruvate by lactate oxidase and the pyruvate formed was subsequently reduced to L-lactate by lactate dehydrogenase durlng the passage through the enzyme column. An enzyme thermistor, wlth a normal sensltlvity for L-lactate of 10 pM, was used to follow the recycling reactlons. An enhancement of the sensitivity by up to 1000-fold was noted, which lowered the detection lImN to an amount less than 5 pmol of L-lactate. We have also made some prelimlnary studles on glucose oxldase colmmoblilzed with catalase and glucose dehydrogenase.
The concept of enzymic recycling of an analyte in order to get amplified signal output has been successfully applied to a number of systems including the determination of pyridine dinucleotides ( I , 2). The basis for the latter amplification is two coupled reactions, one oxidizing (enzyme 1)and the other reducing (enzyme 2) the coenzyme according to Scheme 1. ‘ O n leave f r o m Akademie der Wissenschaften der DDR, Zent r a l i n s t i t u t fur Molekularbiologie, R o b e r t Rossle Strasse 10, DDR1115 Berlin-Buch, GDR. 0003-2700/85/0357-1740$01.50/0
Scheme I enzyme I
NAD+
NADH t Ht
u enzyme 2
C
The number of cycles that occur during a given time will reflect the initial coenzyme concentration ( I ) . Effective coenzyme recycling can also be obtained by combining the enzymic reduction of NAD+ with chemical oxidation of the formed NADH using, e.g., immobilized alcohol dehydrogenase and phenazine methosulfate (3). In analogy to coenzyme recycling, substantial amplificationof the substrate signal can also be obtained. The substrate is recycled by choosing the enzymes such that the product of the first enzyme reaction is in turn the substrate for the second enzyme which will then convert it back to the original substrate. In connection with biosensor assays, coenzyme (NAD(H)) recycling has been utilized previously in enzyme electrode units for the determination of glutamate and pyruvate, respectively (4). The principle of signal amplificationas described above, however, has to our knowledge first been utilized in a biosensor unit, i.e., enzyme electrode, in the determination of glucose employing coimmobilized glucose oxidase (GOD) and glucose dehydrogenase (GDH) (5,6). More recently, lactate oxidase 0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985 1741 BrdgelAmplifier
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