Study of fluorescent chemodosimeter platform based on a combination of caged-fluorophore and human serum albumin and development of paper-type sensor based on artificial peroxidase-functionalized nanocarrier

Abstract

Chapter I. A study on fluorescent chemodosimeter platform for the assay of enzymatic activities based on a combination of caged-fluorophore and human serum albumin. Non-specific binding of human serum albumin (HSA), abundant in human serum, to fluorescent probes causes a problematic issue of the non-specific changes in the fluorescence of the probe. There are two drug binding sites in HSA, which have the property of non-specifically binding to various fluorescent probes such as coumarin derivatives and dansylated amino acids. Fluorescent probes bound to HSA, regardless of interaction with the analyte, produce non-specific fluorescence due to the hydrophobic environment inside the HSA. Consequently, the concentration of the target analyte as a biomarker may be overestimated, resulting in an inaccurate diagnosis of the associated disease. Since HSA acts as an interfering agent, it is often difficult to apply a sensor capable of quantitatively detecting an analyte in a buffer to an actual serum sample. In this study, we tried to solve this problem by reversely using non-specific fluorescence, which is inherently problematic. The proposed new sensing strategy uses a turn-on type fluorescent chemodosimeter with a fluorophore having a high binding affinity for HSA. This allows HSA to be directly used as a part of the sensing system for detecting the target biomarker in the serum, instead of acting as an interfering agent affecting the fluorescence signal. The caged-fluorophore is de-caged to the form of a bare fluorophore through irreversible interaction with the analyte. The bare fluorophore then rapidly binds to the HSA, resulting in strong fluorescence. Hence, it is possible to quantitatively detect the analytes in the serum. Moreover, the proposed sensing system does not require additional processes to eliminate the above-mentioned non-specific fluorescence changes. As a proof of concept of the proposed system, butyrylcholinesterase (BChE), which is a biomarker of organophosphate poisoning and its inhibitor has potential as a therapeutic agent for Alzheimer's diseases, was selected as an analyte. Dansyl-sarcosine-choline (DSC), a caged-fluorophore, was developed by combining DS, which is known to generate strong fluorescence by binding to albumin, and choline, a recognition unit of BChE. The assay system for detecting the BChE activity showed excellent linearity over a wide range, low detection limit, and high selectivity over other enzymes containing acetylcholinesterase (AChE; sister enzyme of BChE). In addition, it detects the BChE activity in human serum with only a drop of the serum (10 μL) and DSC in real-time without additional processing. In addition, it not only is utilized to measure the inhibitory efficiency of BChE inhibitors but also has the potential for high-throughput screening for BChE inhibitors. The proposed sensing strategy based on caged-fluorophore and HSA was intended to be applied to the design of a new fluorescent substrate for assay of CP activity. Fluorescent substrates of endopeptidases can be easily designed using various methods, but in the case of CPs, it is difficult to design a fluorescent substrate as the hydrolysis site is located in the front part of the C-terminal amino acid or dipeptide. Fluorescence resonance energy transfer (FRET)-based substrates have been developed for dipeptidyl-CP, but their synthesis is relatively complicated, and their application to mono-CP is severely limited. For this reason, many researchers have used hippuryl-based substrates to analyze CP activity, while enduring various inconveniences such as extraction process. Hence, it is desirable to develop a new strategy that can be adapted for the development of both dipeptidyl- and mono-CP fluorescent substrates. The new fluorescent substrate for CPs was designed by attaching an amino acid or peptide, which acts as the recognition unit for CP, to the C-terminus of DS. As a proof of concept, two dansylated peptides, Dansyl-Sar-Lys-Pro (DS-KP) and Dansyl-Sar-Arg (DS-R), were designed as fluorescent substrates for the assay of angiotensin-converting enzyme (ACE) and carboxypeptidase B (CPB) as dipeptidyl- and mono-CP. The fluorescent substrates can be easily synthesized in the solid-state at a time using the solid peptide synthesis method. The CP assay system was verified to be capable of detecting quantitatively the activities of both dipeptidyl-CP and mono-CP by using the HSA as the signal amplifier. It showed excellent linearity of the detection range for CP activity and applicability as a high-throughput screening method for numerous inhibitor candidates.

Chapter II. Development of long-term stable paper-type glucose sensor based on artificial peroxidase-functionalized, glucose oxidase-loaded pluronic nanocarrier. This study focuses on the development of a smartphone/paper-based sensing system for glucose detection based on nanocarrier with high peroxidase-like activity and long-term stability. Previously developed paper-based glucose sensors using enzyme cascade reactions have problems associated with poor long-term stability due to enzyme denaturation. Therefore, a sensor that retains the high inherent selectivity of glucose oxidase with long-term stability would be desirable. A simple paper-based analytical device (PAD) for the one-pot detection of glucose was developed herein using an artificial peroxidase (Mn2BPMP)-functionalized and glucose oxidase (GOx)-loaded pluronic-based nanocarrier (PNC). Mn2BPMP has high peroxidase-like activity at a physiological pH. In addition, pluronic-based temperature-sensitive nanocarrier system encapsulated GOx with very high loading efficiency and prevent the GOx from denaturation even in a dried state. In solution, Mn2BPMP-PNC showed higher peroxidase-like catalytic efficiency than did Mn2BPMP. In addition, glucose detection via enzyme cascade reaction between GOx and Mn2BPMP in the GOx loaded-Mn2BPMP-PNC was more sensitive than the simple combination of Mn2BPMP and GOx with excellent selectivity. Subsequently, a PAD was fabricated using a laser printer with an assay substance containing GOx-loaded Mn2BPMP-PNC and peroxidase chromogenic substrate. The prepared Mn2BPMP-PNC-based PAD quantitatively measured glucose in human serum ranging from normal levels to those typical for diabetics as well as in buffer by obtaining RGB (red, green, and blue) color values through smartphone readout or the naked eye. Importantly, the present PNC-based PAD maintained the detection efficiency during storage at room temperature for 6 weeks in contrast to the rapid decrease in detection efficiency obtained for PAD containing Mn2BPMP and GOx without PNC.