Study of methodologies for detecting bilirubin by electrochemical, UV,fluorescence and chemiluminescence techniques and their applicationfor CE determination of bilirubin and arsenic anions in biofluid

Abstract

Capillary-based analytical methodologies were developed to meet the need for metabolite determination in two major areas. The first area is the determination of free bilirubin in sera for the management of jaundiced neonates under critical conditions. Three sensitive detection techniques were investigated, Quantum dots (QD) mediated fluorescence, Chemiluminescence (CL) and Microelectrode detection. Four different types of QDs were synthesized for the direct bilirubin determination. The CAH-capped CdTe QDs were selected as it shows the best performance compared to organic dyes and other QDs. Its optimized preparation conditions are: refluxing solution containing Cd/Te/CAH (1:0.5:2.4 w/w) for 4 hours at 100 °C. From Transmission Electron Microscope characterization, nano-size QDs with an uniform size distribution, high luminescence and good stability were obtained. The optimized detection conditions were: incubation of bilirubin with CAH-capped CdTe QDs (5 10-6 mol/L) in water at pH=5.6 and 20 oC for 8 min prior to spectrofluorometric determination (λex=473 nm and λem=580 nm). A linear working range from 0.043-0.86 μg/mL with 0.9943 correlation coefficient and 2 ng/mL detection limit (LOD, S/N=3) were achieved. Results from nFIA-CL indicate a quick response within seconds though a poorer LOD (S/N=3) of 15 μg/mL for the direct bilirubin determination.

The third technique investigated used an enzyme microelectrode and it was found to be able to couple with capillary electrophoresis (CE) in frontal analysis (FA) for the determination of free bilirubin in serum samples. Making use of the micron size of the carbon-fiber electrode, a new MCNTs (Multi-wall Carbon Nanotubes) modified CFMEs (Carbon fiber microelectrodes) was fabricated within a microchip-CE device with three guided channels to enable electrodes alignment. Method to immobilize bilirubin oxidase (BOD) onto the CFMEs surface by the carbodiimide chemistry achieved the highest detection sensitivity. Under optimized conditions (sample introduced by hydrodynamic injection at △H (20 cm), and a running/detection buffer (10 mM phosphate) at pH 7.4, working potential for amperometric detection at +0.8 V), a linear working range between 1-40 μg/mL and a detection limit (S/N=3) at 0.15 μg/mL for free bilirubin was achieved.

The second area for metabolite determination was developing a new analytical method for the management of APL (acute promyelocytic leukemia) patients under arsenic treatment, a drug required continued monitoring. The analytical requirements include a high detection sensitivity and the capability to provide timely results for multiple drug residues. Using a 20 mM phosphate as the running buffer and 0.05mM CTAH (Cetyl-trimethyl-ammonium hydroxide) as an additive for EOF reversal, co-EOF (co-electroosmotic flow) stacking was established to enhance up to 200 times of the detection limit for arsenite. Satisfactory baseline separation for arsenite, arsenate, MMA (Methylarsonic acid) and DMA (Dimethylarsinic acid) was achieved with linear working ranges (correlation coefficients > 0.999) from 1-50 μg/mL for arsenate and DMA, 0.5-50 μg/mL for MMA as well as 0.1-50 μg/mL for arsenite. Detection limits (S/N=3, n=3) achievable for arsenate, arsenite, MMA and DMA were found to be 0.41 μg/mL, 0.01 μg/mL, 0.04 μg/mL and 0.32 μg/mL respectively at levels meeting the requirement for APL patient urine monitoring.