CORRESPONDENCE Enzyme Electrode for Amygdalin SIR: The use of biological materials in membrane electrodes has resulted in the development of new potentiometric sensors such as the valinomycin-based potassium electrode (2-3) and an enzyme electrode for urea ( 4 ) . We now report on a novel electrode, responsive to amygdalin, which is the first successful example of a potentiometric enzyme electrode utilizing a nonglass membrane. The electrode consists of a crystalline cyanide sensor coated with a layer of the immobilized enzyme p-glucosidase; the electrode functions viu the enzyme-catalyzed hydrolysis of amygdalin to yield glucose, benzaldehyde, and cyanide ion at the electrode surface. The cyanide ion, produced in stoichiometric proportion to the concentration of amygdalin in the sample solution, gives rise to the potentiometric response of the electrode system. The electrode system is prepared by coating the sensing element of the solid-state cyanide electrode (Orion Research, Inc.) with a film of @-glucosidaseenzyme (Sigma Chemical; Emulsin type, from almonds) immobilized in acrylamide gel (0.05-gram enzyme per I. ml of polymer) according to the method of Guilbault and Das (5)and mechanically fixing the immobilized enzyme layer in place with cellophane film secured to the electrode by an “0”-ring. When this electrode system is exposed to aqueous sample solutions of amygdalin (Sigma Chemical, Grade l), the immobilized enzyme catalyzes the hydrolysis of amygdalin at the electrode surface according to HzO
CsHsCHCN -+2CsH120s
I
enzyme
+ CeHSCHO + HCN
To ensure complete dissociation of the HCN produced, measurements were made at pH 12.7 (Beckman 22326 Buffer) but could be successfully carried out at pH values as low as 8 with some loss in sensitivity. The potentiometric measurements were made with the enzyme electrode us. a saturated calomel reference electrode in a thermostated vessel held at 25 “C 0.1. In solutions of pH 12.7, the enzyme electrode responded to 5 X lO-7M to amygdalin concentration over the 5 X range with best reproducibility and stability in the to 5 x 10WM region. Since not all of the cyanide produced reaches the crystal surface, the response of the electrode system is less than Nernstian; typically, potential changes of 20 mV per concentration decade of amygdalin were observed (Figure 1). When exposed to rapid changes in amygdalin concentration, the electrode system reached potentiometric equilibrium in 1
*
(1) M. S . Frant and J. W. Ross, Jr., Science, 167, 987 (1970). (2) G. A. Rechnitz, ANAL.CHEM., 41 (12), 109A (1969). (3) G. A. Rechnitz and M. S . Mohan, Science, 168, 1460 (1970). (4) G. G. Guilbault and J. G. Montalvo, J . Amer. Chem. SOC.,92, 2533 (1970). ( 5 ) G. G. Guilbault and J. Das, Anal. Biochem., 33,341 (1970).
3
5
7
-La3 Canc.
Figure 1. Calibration curve of enzyme electrode as a function of amygdalin concentration Full and open circles denote separate runs. pH = 12.7; E us. SCE
minute or less. When properly prepared and stored between measurements, the enzyme electrode can be preserved for several weeks. Ultimately, the overall lifetime of the electrode system is limited by the dissolution of the cyanide-sensing crystal membrane which is claimed (6) to have a working life of 200 hours under continuous use. While the full procedural details remain yet to be optimized, amygdalin and other natural products which produce cyanide by &glucosidase catalyzed decomposition, can be determined rapidly and conveniently with this electrode. Moreover, the electrode described represents a new variation of potentiometric enzyme electrodes; the principle involved may be more generally applicable through the use of other enzymes and sensing elements. G. A. RECHNITZ RAMONLLENADO Department of Chemistry State University of New York Buffalo, N. Y.14214 RECEIVED for review September 25, 1970. Accepted November 16, 1970. Supported by grants from the National Institutes of Health and the Office of Saline Water. G. A. Rechnitz is an Alfred P. Sloan Fellow, 1966-72. ( 6 ) Instruction Manual, Model 94-06 Electrode, Orion Research, Inc., 11 Blackstone St., Cambridge, Mass., p 12.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971
283
