Lin Li,' Mark A. Arnold,'* and Jonathan S. Dordick2 'Department of Chemistry, *Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa 52242 Received August 24, 1992/Accepted December 29, 1992
A kinetic model that accurately describes intensity vs. time reaction profiles for thechemiluminescence reaction between luminol and hydrogen peroxide, as catalyzed by horseradish perioxdase, is derived and evaluated. A set of three differential equations is derived and solved to provide intensity time information for the first 200 seconds of the reaction. The model accurately predicts intensity-time profiles when literature values are used for all but one of the reaction rateconstants. Furthermore, the model predicts a nonlinear curve for plots of light intensity versus the initial hydrogen peroxide concentration. Experimental data confirm that such plots are nonlinear. Finally, a linear double-reciprocal plot is predicted by the model and the experimental data verify this relationship. 0 1993 John Wiley & Sons, Inc. Key words: luminol chemiluminescence peroxidasehydrogen peroxide
Chemiluminescence (CL) is a phenomenon that provides for rapid and sensitive analytical measurements. Light generated by biocatalytic approaches offers high selectivity in combination with reasonable quantum yields at near neutral pH levels. Peroxidases are known to catalyze light generation upon the oxidation of luminol in the presence of hydrogen peroxide. Thisis particularly useful in coupled reactions with oxidases to measure a wide variety of clinically, biomedically, and chemically important compounds.1 2 5 11,13
7 7 7
state 3-aminophthalate (3-AP) is the light emitting species that forms directly from LO*H-. Because 3-AP can be generated by a number of different, and simultaneous reaction steps, the observed generation of light at a giventemperature depends upon several system variables including the concentrations of enzyme, luminol, hydrogen peroxide, and oxygen, as well as the pH of the solution. The complexity of this reaction mechanism and its analytical potential motivate us to develop a mathematical model that describes the time-dependent generation of light as a function of system variables. Such a model may be expanded toinclude oxidase-catalyzed reactions, thereby providing a means to better understand and control these important analytical systems. In this study, we present a kinetic model that describes the relationship between the CL intensity and concentrations of HRP, luminol, and hydrogen peroxide. Our ultimate objective is to develop an optical method for the continuous, in situ detection of analytes ofphysiological significance. The reaction conditions maintained in this work have been selected with such applications in mind. For example, the solution pH has been restricted to the physiological value of 7.5.
MATERIALS AND METHODS Materials
The mechanism of CL catalyzed by horseradish peroxiLuminol, hydrogen peroxide (30%), HRP (type 11, E.C. dase (HRP) has been studied extensively over the pasttwo 188.8.131.52, 220 units/mg, pH 6.0, 20°C) and superoxide decades. The individual reaction steps and their kinetics dismutase (E.C. 184.108.40.206, 3250 units/mg, pH 7.8, 25°C) that comprise the catalytic mechanism have been identified were obtained from Sigma Chemical Co. (St. Louis, MO). (see Scheme I) and fall into three distinct c a t e g ~ r i e s . ~ ~ , The water used for all solutions was type-Ireagent grade '~ Initially, luminol (LH2) is oxidized by the peroxidatic obtained from a Milli-Q Reagent Water System (Millipore, pathway of HRP to generate a luminol radical (LH.) (steps Bedford, MA). Hydrogen peroxide solutions were standard1 through 3). This is followed by a series of nonenzymatic ized by titration with thio~ulfate.~ reactions involving luminol radical, oxygen, and H202...