Chemiluminescent Determinations of Clinical Analytes Using Microporous Membrane Flow Cells
Malavolti, Nathan Lee
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Permalink
https://hdl.handle.net/2142/70331
Description
Title
Chemiluminescent Determinations of Clinical Analytes Using Microporous Membrane Flow Cells
Author(s)
Malavolti, Nathan Lee
Issue Date
1986
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Abstract
The use of various chemiluminescence (CL) reactions as detection techniques for clinically important analytes has been explored in more depth over the last several years. This thesis project presented the use of microporous membrane flow cells for the chemiluminescent determinations of some of these species using flow injection analysis as an advantageous alternative to more classical methods of detection. The compounds studied were glucose, glactose, cholesterol (in both the free and esterified forms), creatinine, and creatine. Due to the lack of specificity of the CL system, an oxidase enzyme was used to convert the substrate (in the first three systems, the analyte of interest) into an equivalent amount of hydrogen peroxide, which in turn was quantitated by the luminol CL reaction. This oxidase enzyme was forced through a microporous membrane under pressure into an analyte stream that was flowing approximately three orders of magnitude faster. This approach had significant implications such as minimal enzyme consumption and negligible sample contamination by the enzyme, in addition to the fact that the enzymatic step occurred at its optimal pH simultaneously with the CL detection step at more basic pH.
The membrane CL analyzer was, for the most part, successful for those species mentioned. The oxidase enzyme requirement per assay was generally 0.01-0.1 I.U., with the amount of time needed for one determination ranging one to four miniutes. The detection limits observed for these compounds were in the range of 1-5 nanomoles (or in other words, 2-20 (mu)M for injected volumes of 0.25-1.0 mL); the precision that was recorded was usually 2-3% RSD, which was comparable to that reported by many other FIA systems. Blood sera was assayed for free and total cholesterol by standard addition using this system, after pretreatment with the Somogyi-Nelson procedure. Also, an active splitter configuration was designed for the simultaneous determination of glucose and galactose. Difficulties were encountered when attempting to extend the application of this configuration to the simultaneous determination of creatinine and creatine in aqueous standards, as well as when assaying urine and blood samples for these two species.
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