Development of microelectrode arrays for real-time monitoring of alkaline phosphatase release from cells in clinical analysis

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Balbaied, Thanih
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University College Cork
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The alkaline phosphatase (ALP) biomarker is a membrane-bound enzyme widely distributed in the tissues of living organisms, which can instigate particular chemical processes, such as hydrolyses, to provide inorganic phosphate in the human body. ALP activity is routinely used as an indicator in point-of-care applications for some diseases. However, there is mystery about its biological role in terms of regulation during pathogenesis, due to the limitations of real-time monitoring detection tools. The current methods, although sensitive, are costly processes, are time consuming and are based on optical detection, which is hard to miniaturize. There is thus a demand for a reliable technique that addresses these issues and therefore enhances ALP detection in living systems. Our approach in the research is to develop an electrochemical method that will pave the way for performing direct, cost effective and simple monitoring of the ALP biomarker from release in cells for the purposes of clinical analysis. The thesis discusses a novel strategy that integrates cell culture and electrochemical techniques to develop a real-time detection method for monitoring ALP release from cells using an electric cell-substrate impedance sensing (ECIS) system. The integration of these techniques can enhance the detection of biological targets based on electrochemical changes in electrode interfaces. Our integrative approach took advantage of microelectrode arrays technology, facilitating the yield of recordings, signal shape, and signal-to-noise ratio. For the purposes of the research, electrochemical impedance devices had 40 electrodes distributed in 8 wells that facilitated continuous visualization of cell adhesion, spreading, proliferation, and detachment. Amperometric sensors used three-electrode systems to monitor cellular signal transduction under a monolayer of cell culture conditions. The amperometric sensor application offers solutions that can be used in screening cellular ALP expression detected by electroactive species. This helps to avoid radioactivity, antibodies and mRNA tools that are presently used in clinics. The experimental optimization of the target sample was performed by using resazurin assay. The standard method for ALP detection was used for optimizing the conditions of the enzymatic assay during ALP release. Balb/c 3T3, A549 and MCF-7 cells displayed an optimal initial density of 40×103 cell/mL, whereas the Ht20 cells displayed an optimal density of 80×103 cell/mL. This was compatible with the hemocytometer and microscope images. The kinetic enzyme assay of ALP release based on the absorbance analysis showed the optimum activity at 30 minutes incubation time and at 6 mM pNPP concentration. Electrochemical analysis allowed the highest activity after 10 minutes incubation with concentration of pAPP of 9.6 mM. Activity of ALP during the proposed differentiation strategy showed a significant difference in the values of electrochemistry compared to the values of absorbance. The analysis produced findings that were compatible with capillary electrophoresis, which was used as a comparative study. The frequencies required for the target monolayers were optimized on the ECIS system, illustrating a linear relationship between concentration of cells and impedance magnitude. Cellular behaviour was investigated during ALP release, which corresponded to the resazurin-based assay. Real-time ALP release monitoring was proven during differentiation of the colon cancer cell line. A concentration of sodium butyrate of 0.5mM and 1 mM allowed impedance of 550 Ω and 400 Ω compared to the control and to 600 Ω for untreated cells. This indicated a correlation between the cell response and the concentration of sodium butyrate, which suggested that more investigation was necessary to determine the LOD of the developed methodology. Relevant findings were also produced during an investigation of cell morphology and cells viability. This was also proven by amperometry in order to obtain sufficient real-time monitoring of ALP under cellular conditions. In conclusion, this PhD thesis presents and describes the real-time monitoring for the detection of the ALP biomarker in living cells, with suitable detection results. It contributes to the development of microfluidic testing that would facilitate work on small scale te chniques.
Absorbance , Cell culture , Electrochemistry , Capillary electrophoresis , Alkaline phosphatase
Balbaied, T. A. 2020. Development of microelectrode arrays for real-time monitoring of alkaline phosphatase release from cells in clinical analysis. PhD Thesis, University College Cork.
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