Abstract

Cortisol is a member of the glucocorticoid hormone family and a key metabolic regulator. Increased intracellular cortisol levels have been implicated in type 2 diabetes, obesity, and metabolic syndrome. Cortisol is an important bio-marker of stress and its detection is also important in sports medicine. However, rapid methods for sensitive detection of cortisol are limited. Functionalized gold nanowires were used to enhance the sensitivity and selectivity of cortisol detection. Gold nanowires are used to improve the electron transfer between the electrodes. Moreover, the large surface to volume ratio, small diffusion time and high electrical conductivity and their aligned nature will enhance the sensitivity and detection limit of the biosensor several fold. The biosensor was fabricated using, aligned gold (Au) nanowires to behave as the working electrode, platinum deposited on a silicon chip to function as the counter electrode, and silver/silver chloride as reference electrode. The gold nanowires were coupled with cortisol antibodies using covalent linkage chemistry and a fixed amount of 3α-hydroxysteroid dehydrogenase was introduced into the reaction cell during each measurement to convert (reduce) ketosteroid into hydroxyl steroid. Furthermore, the micro-fluidic, micro-fluid part of the sensor was fabricated using micro-electro-mechanical system (MEMS) technology to have better control on liquid flow over Au nanowires to minimize the signal to noise ratio. The biosensor was characterized using SEM, AFM and FTIR technique. The response curve of the biosensor was found to be linear in the range of 10–80 μM of cortisol. Moreover, the presence of hydrocortisone is sensitively detected in the range of 5–30 μM. It is concluded that the functionalized gold nanowires with micro-fluidic device using enzyme fragment complementation technology can provide an easy and sensitive assay for cortisol detection in serum and other biological fluids.

Keywords: Nanowire; Cortisol; Steroid; Covalent linkage; Microfluid device

Article Outline

1. Introduction

2. Materials and methods

2.1. Chemicals

2.2. Apparatus

2.3. Fabrication of the electrochemical biosensor

2.3.1. Preparation of Au nanowires/working electrode

2.3.2. Fabrication of micro-fluidic chip

2.3.3. Integration of Au nanowires into the micro-fluidic chip

2.3.4. Surface functionalization and characterization of Au nanowires with cortisol antibodies

2.4. Electrochemical measurements

2.5. Assay procedure

3. Results and discussion

3.1. SEM investigations of functionalized gold nanowires

3.2. AFM analysis of the functionalized Au (gold) nanowires

3.3. Electrochemical response studies of the cortisol detection

3.4. Calibration plot for determination of cortisol

3.5. Biosensor stability analysis

3.6. Interferences studies

4. Conclusions

Acknowledgements

References