Exploration of Self-Immolative Cascade Devised Electrochemical Molecular Switches for Activity-Based Biomarker Profiling

• Main Author: Shunmuga Thain Balamurugan Thangaraj
• Other Authors: HUANG, SHENG-TUNG
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/j5r3pw

博士 === 國立臺北科技大學 === 能源與光電材料專班(EOMP) === 107 === Design and development of biosensors to assay critical biomarkers with high sensitivity and selectivity at low cost has become essential in clinical diagnosis. Despite the requirements, developing a distinctively selective, high sensitive detection platform to quantify the trace level biomarkers in a complex biological system remains to be a mammoth task to attain over decades. On this ground, a set of prodrug inspired self-immolative latent electrochemical molecular switches are designed and developed for rapid real-time active profiling of specific biomarkers in physiological samples with no/minimum sampling. A series of four electrochemical probes FCPA, NAS-FC, Leu-FC, and INA-FC were designed to specifically target and quantify three essential biomarkers cysteine (Cys), aminoacylase-1 (ACY-1), leucine aminopeptidase (LAP), and influenza neuraminidase (INA), respectively. The proposed self-immolative electrochemical probe platform involves three key steps starting from 1. Design and synthesis of the electrochemical molecular probes, 2. Proof-of-concept and determine the analytical parameters under lab samples, 3. Examine the real-world utility of the developed assay platforms such, real-time active profiling of specific biomarkers from live cells and complex physiological samples such as whole blood. The functional principle of all three probes was identical and clocked based on a predefined trigger-target specific chemical or biochemical reaction to induce a self-immolative chemical transformation of the linker to eliminate unmasked electrochemical reporter. The probe ferrocene carbamate phenyl acrylate (FCPA) was developed to selectively assay Cys over other biothiols such as homocysteine(Hcy), and glutathione (GSH). As discriminative detection of the three biothiols is necessary to understand the interconnections between the three and their role in various physiological and pathological functions. ACY-1 is a common mammalian enzyme hydrolyze N-acetyl amino acids and have been identified as a potential serum biomarker for liver cirrhosis, liver cancer, small-cell lung cancer, colon cancer, and delayed graft function (DGF) following renal transplantation. The probe FCPA was capable of assay ACY-1 activity employing NAC as transducing substrate. The molecular switch FCPA delivered outstanding analytical performance towards Cys, and ACY-1 detection. The probe was further employed in real time active profiling of cellular Cys production in Escherichia coli W3110 alongside quantification of blood Cys, and ACY-1 spiked in whole blood samples with admirable accuracy and reliability. The probe NAS-FC is designed to be specific for ACY-1 without any transducing substrate. The probe has been synthesized and characterized successfully; however, the electrochemical analysis of the probe with ACY-1 was not successful; as incubation of the probe with ACY-1 does not release the signaling species. The NAS-FC probe was redesigned to rectify the flaws of our previous design. The electrochemical substrate LeuFC has been designed for the activity profiling of LAP. The substrate has delivered outstanding analytical performance for the first-of-its-kind. Further, the real world utility of LeuFC probe has been demonstrated in real-time profiling of cellular LAP activity from liver cancer cells (HepG2 cells). The INA-FC probe was designed for rapid selective quantification of INA, there are three synthetic routes were tried to attain the designed probe for INA detection however, all the proposed synthetic routes are led down in selective installation of two methyl groups to the neuraminic acid core and we are not able to attain the destination compound (INA-FC) in expected duration