This proposal involves the development and evaluation of new measurement and data-processing approaches for fast clinical determinations. Approaches emphasized in the proposal are stopped-flow mixing, nonlinear kinetic data-processing methods, and use of imaging detectors for multiwavelength spectroscopy. Stopped-flow mixing methods will provide clinical chemists with access to reactions with half-lives as short as 20 ms, which is well beyond the time frame accessible to any existing clinical instrumentation. Some typical problems that will make effective use of the fast-mixing capability are quantitation of albumin, reserve bilirubin binding capacity of infant sera, heart and muscle subunits of lactate dehydrogenase, and oxy- and methemoglobin. Nonlinear kinetic data-processing offers advantages of reduced dependence on experimental variables, improved linear range for enzyme substrates and for immunoassay procedures, and simultaneous multicomponent determinations. Typical problems to which these methods will be applied include those mentioned above as well as simultaneous quantitation of amino acids and muscle and brain subunits of creatine kinase. Multiwavelength spectral data, including first and second derivative spectroscopy, will be used to obtain degrees of selectivity and procedures of multicomponent determinations that are not possible with single-wavelength measurements. Some typical problems that will be used to evaluate multiwavelength approaches are reserve bilirubin binding capacity of infant sera and simultaneous quantitation of oxy-, met-, and carboxy-hemoglobin in mixtures. In addition, the nonlinear kinetic methods and multiwavelength data will be evaluated for their potential use for internal error detection.
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