Development of an insitu quantitative measurement system for stress hormones : towards open microfluidic system

Abdulsattar, Jwan Oday

Abstract

Personalized health diagnostic and monitoring has gained serious attention in recent years and one area of interest is the analysis of hormones which indicate increased stress levels. Cortisol (known also as hydrocortisone) and cortisone are steroid hormone (also known as stress hormones) that plays an important role in the regulation of many physiological processes such as glucose levels, blood pressure, and carbohydrate metabolism and they are considered as a potent biomarker for post-traumatic stress. The determination of stress hormones represents a challenge because their secretion follows a circadian rhythm (all day cycle) and their secretion are dependent on environmental and behavioral triggers. As a result, there is a need to develop a system for cheaply and rapidly monitoring their levels using approaches such as lab-on-a-chip (LOC) which combine high selectivity and sensitivity to provide valuable health informatics, not just in human but in animals such as fish being bred in a fish farm.
Selectivity in these devices can be achieved utilising an immunoassay approach taking advantage of the lock and key mechanism that is related to the antibody-antigen interaction. In this work, a new immunoassay method was developed to measure the stress hormones which involved the reproducible immobilization of cortisol and cortisone antibodies onto a tin-doped indium oxide (ITO) electrode. This was achieved by modifying the electrode in a two process step; the deposition of a nitro group onto the ITO electrode followed by the reduction of the nitro group to amino group using cyclic voltammetry. This approach enables a good orientation of the antibody on the surface. The antibodies were then immobilized using an EDC/Sulfo-NHS linkage. To enable electrochemical detection the antibodies were tagged with ferrocene to give a redox tag. When square wave voltammetry was utilised the method gave good limits of detection (LOD) of 1.03 pg ml-1 for cortisol and 0.68 pg ml-1 for cortisone.
The methodology was carried out using biological sample including Zebrafish whole- body sample and artificial saliva and reliable results were obtained without the need for complex extraction procedure.
The results of the analysis suggest that the proposed method has promise for the routine detection of stress hormones, which gives a good reason to detect cortisol and cortisone based on a chemiluminescence immunoassay. The antibodies were immobilised using the electrochemical method but then chemiluminescence detection was selected due to its high sensitivity and the simple instrumentation required. A static system was first constructed using a micropipette to add the chemiluminescence reagents with the use of a CCD camera and image J software to capture the chemiluminescence. To achieve chemiluminescence detection the ferrocene tag on the antibodies was first oxidised and then this acted as a catalyst for luminol and hydrogen peroxide chemiluminescence reaction. Optimum conditions were investigated and 20 mM luminol and 10 mM hydrogen peroxide were used with a 200 seconds exposure to the camera and an incubation time of 30 minutes. Using this approach limits of detection were obtained of 0.47 pg ml-1 and 0.34 pg ml-1 also R2 0.9912 and 0.9902 for cortisol and cortisone respectively. The method was also applied to Zebrafish and artificial saliva without analyte extraction and good results were obtained.
Once the successful chemiluminescence immunoassays had been developed it could be incorporated into in situ measurement devices. In this work, an open microfluidic approach was investigated to overcome the problems of blockages, high back pressure and air bubbles seen in closed microfluidic systems. A superhydrophobic ITO electrode substrate was prepared depending on the lotus leaf effect of extreme water repellence by the deposition of dichlorodimethylsilane (DCDMS) onto the substrate, followed by dip coating the hydrophobised ITO electrode into fumed silica nanoparticle suspensions to increase the hydrophobicity. Superhydrophilic patterns were then produced using a mask and a UV/ozone lamp. The wetting properties were investigated in detail using a drop shape analysis system. Optimum conditions for the formation of a homogeneous coating were established giving the following results; fumed silica suspension concentration 4%, dip coating velocity 3.18 cm min-1, and sonication time of 10 minutes. The results obtained from fluorescence microscopy showed the capability of fluid to flow along the superhydrophilic pattern acting as an open microfluidic channel.
Finally, the open microfluidic approach was combined with the immobilisation procedure. Although further work will be needed to optimise the system, chemiluminescence detection was achieved when the chemiluminescence reagents were passed through the open microfluidic channels over the immobilised antibodies.
To conclude an electrochemical immobilization platform has been exploited to reproducibly immobilize the antibodies and develop a quantitative novel chemiluminescence assay for stress hormone analysis in combination with an open microfluidic device.

Citation

Abdulsattar, J. O. (2017). Development of an insitu quantitative measurement system for stress hormones : towards open microfluidic system. (Thesis). University of Hull. Retrieved from https://hull-repository.worktribe.com/output/4224572

• Thesis Type: Thesis
• Deposit Date: Jan 5, 2023
• Publicly Available Date: Feb 24, 2023
• Keywords: Chemistry
• Public URL: https://hull-repository.worktribe.com/output/4224572
• Additional Information: Department of Chemistry, The University of Hull
• Award Date: Aug 1, 2017