Development of point-of-care bioassays for renal function marker-creatinine.

摘要

Creatinine is an important diagnostic compound in determining renal and muscular dysfunction. There is an urgent necessity of rapid and simple creatinine assays for point-of-care (POC) applications. In this thesis, novel creatinine lateral-flow bioassays and electrochemical biosensor have been developed.;An enzymatic bar-code lateral-flow creatinine test has been demonstrated. A unique approach that involves both 3,3',5,5'-tetramethylbenzidine (TMB) diffusion and horseradish peroxidase (HRP) kinetics is presented. The hydrophobic TMB tends to form micro-crystals on the hydrophilic polyester pad. The large size of TMB crystals results in their delay in release, which in turn generates a decrease of reaction time on successive lines. Accordingly, a bar-code result in three distinct lines is generated. The assay expresses creatinine concentration at micromole range and is applicable to both urine and serum samples.;Creatinine competitive immunochromatographic (IC) lateral-flow assays using colloidal gold and HRP as signal generator are implemented. The use of HRP as label offers a lower detection limit (3 muM) compared to colloidal gold (88 muM). The HRP-labeled IC assay is further devised into bar-code and one-step format by two unique approaches. The bar-code assay is based on the progressive decrease in the amount of anti-creatinine antibody immobilized on successive lines. The decrease in amount of free creatinine-HRP conjugate bound to antibodies immobilized on nitrocellulose (NC) membrane is therefore more significant in sequence. The one-step IC assay is constructed by incorporating glucose oxidase (GOD) and TMB pads to the IC setup. The delay in TMB release compared to GOD and creatinine-HRP conjugate effectively prevents the simultaneous occurrence of HRP reaction and competitive antigen-antibody binding.;Multi-wall carbon nanotube (MWCNT)-modified sarcosine and creatinine layer-by-layer (LbL) electrochemical biosensors are described. In the presence of ferrocene carboxylic acid (FCA) as a diffusive electron mediator, an enhanced electroanalytical performance at lower working potential (0.37 vs. Ag/AgCl) is achieved.;It is believed that the simple design of our developed creatinine bioassays will provide platforms for portable POC devices in decentralized detection.