Application of flow injection analysis to enzymatic fluorescence kinetic methods Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/dn39x520f

Descriptions

Attribute NameValues
Creator
Abstract or Summary
  • Studies of the use of NADH as the monitored fluorophore for enzymatic fluorescence kinetic methods are discussed. Interference studies for the detection of NADH and the adaptation and optimization of fluorescence kinetic measurements to four serum assays are also described. A microcomputer-controlled flow injection analysis (FIA) system was designed and built to automate fluorescence kinetic assays. The performance of the FIA-fluorometer for kinetic assays was tested. Optimization of the fluorometric reaction-rate instrument was made, resulting in a detection limit of 2 nM for NADH. An interference study of fluorometric measurements of NADH in a serum matrix showed serum proteins and bilirubin to be the major interferents. Assays for ethanol and glucose were adapted to fluorescence kinetic methods which yielded detection limits of 0.3 μM and 0.01 mg/dL (in-cell concentrations), respectively. The analytes of the other two assays were enzymes, lactate dehydrogenase (LDH) and creatine kinase (CK) which showed detection limits of 0.2 and 1 mU/mL (in-cell activities), respectively. The LDH assay was conducted such that the decrease in fluorescence per unit time was measured, while for the other assays the increase in fluorescence due to the formation of NADH was measured. To improve the performance of these fluorometric serum assays, a fluorophore called resorufin was utilized, which could be coupled directly to NADH-producing assays. Coupling the resorufin reaction to the CK assay lowered the CK detection limit to 0.09 mU/mL (in-cell). An FIA system was constructed to provide computer-controlled fluorometric kinetic assays. Operations of the system such as inject ions, flow rates, timing and data acquisition are controlled by soft ware. Optimization of the system and measurements of the performance of the flow cell were conducted. Stopped-flow and continuous-flow modes of kinetic measurements, made the the FIA-fluorometer were thoroughly studied using the ethanol assay. The stopped-flow mode of measurement gave the lowest detection limit for ethanol (0.4 μM, in-cell) and the greatest throughput (75 runs/hr). The CK assay, was adapted to the FIA system and showed a detection limit of 0.2 mU/mL.
Resource Type
Date Available
Date Copyright
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Academic Affiliation
Non-Academic Affiliation
Subject
Rights Statement
Peer Reviewed
Language
Digitization Specifications
  • File scanned at 300 ppi (Monochrome) using ScandAll PRO 1.8.1 on a Fi-6670 in PDF format. CVista PdfCompressor 4.0 was used for pdf compression and textual OCR.
Replaces
Additional Information
  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2013-07-29T16:56:30Z (GMT) No. of bitstreams: 1 ElliottColinB1983.pdf: 4213188 bytes, checksum: 149be4e63cf311e989613803717a3566 (MD5)
  • description.provenance : Approved for entry into archive by Deborah Campbell(deborah.campbell@oregonstate.edu) on 2013-07-29T21:22:24Z (GMT) No. of bitstreams: 1 ElliottColinB1983.pdf: 4213188 bytes, checksum: 149be4e63cf311e989613803717a3566 (MD5)
  • description.provenance : Submitted by Katy Davis (kdscannerosu@gmail.com) on 2013-07-29T16:12:54Z No. of bitstreams: 1 ElliottColinB1983.pdf: 4213188 bytes, checksum: 149be4e63cf311e989613803717a3566 (MD5)
  • description.provenance : Made available in DSpace on 2013-07-29T21:22:24Z (GMT). No. of bitstreams: 1 ElliottColinB1983.pdf: 4213188 bytes, checksum: 149be4e63cf311e989613803717a3566 (MD5) Previous issue date: 1982-11-02

Relationships

In Administrative Set:
Last modified: 08/23/2017

Downloadable Content

Download PDF
Citations:

EndNote | Zotero | Mendeley

Items