Ionic Liquid-Based Dye for Highly Sensitive Optical Sensing Based on FRET and Ion Extraction System Using Plasticized PVC Membrane
Authors: Mizuta Tatsumi , Sueyoshi Kenji , Endo Tatsuro , Hisamoto Hideaki 
Published: May 2020

Introduction

We have been using a dye-doped plasticized poly(vinyl chloride) (PVC) membrane for various optical sensor applications such as ion-sensing [1], bioassay [2] and immunoassay [3]. PVC membranes are mainly composed of 30-33wt% PVC, 60-66wt% plasticizer and a rest of components are functional molecules like a lipophilic dye and ionophore. The PVC membrane responds by color change based on bulk extraction or interfacial reaction. In order to improve sensitivity, increasing the membrane thickness and dye concentration in the PVC membrane is necessary. However, increase in the membrane thickness causes a slower response, and increase in dye concentration causes dye precipitation due to the low solubility of dye. To solve these problems, we have developed ionic liquid-based dye (IL-dye), which was an ionic liquid composed of a pH indicator dye, as the plasticizer. The PVC membrane sensor using IL-dye allowed to contain unusually high concentration of dye and the absorbance sensitivity was significantly improved with much thinner membrane, compared with that of conventional one [4]. On the other hand, direct fluorescence measurements for sensitive analysis using this membrane is generally difficult due to the concentration quenching.
Recently, luminescent solid or liquid materials using Förster resonance energy transfer (FRET) from large amounts of FRET donor molecules to small amounts of FRET acceptor molecules were reported, where efficient FRET behavior and highly bright acceptor fluorescence were observed [5]. Thus, by combining this technique with PVC membrane sensors based on IL-dyes, demonstration of highly-sensitive fluorescence-based analysis compared with that of conventional PVC membrane-based optical sensors is expected.
Here, we developed a highly-sensitive FRET-based ion measurement system using highly-fluorescent IL-dye that we developed recently (Figure 1). A pyrene-based ionic liquid, [P66614][HP-SO3] (Figure 2(a)), which had the bright fluorescent characteristic originated from pyrene eximer, was used as the FRET donor and plasticizer, and fluorescein derivative, [P66614][12-DCF] (Figure 2(b)), was chosen as the FRET acceptor. To maximize the sensitivity of FRET-based sensing, donor and acceptor ratio was optimized. Then, FRET-based anion sensing scheme, coextraction of proton and anion, was demonstrated as a proof of concept.

Method

Ionic liquid composed of lipophilic phosphonium cation and pyrene or fluorescein derivative anion was synthesized based on ion-exchange reaction using [P66614][Cl] and each anion. Plasticized PVC membrane was prepared based on spin-coating (1600 rpm, 20 s) on the poly(ethylene terephthalate) (PET) film using the THF cocktail containing 10 wt% PVC and 90 wt% [P66614][HP-SO3], where 0-5 mol% of [P66614][12-DCF] was doped. Then, the fluorescence spectrum (λex = 348 nm) and excitation spectrum (λem = 560 nm) of each PVC membrane was measured to investigate the FRET behavior and optimal acceptor concentration. Finally, as the demonstration of ion sensing, FRET response toward different pH solutions (50 mM phosphate buffer containing 100 mM NaCl) was evaluated.

Results and Discussion

The molar ratio of phosphonium and dye in synthesized [P66614][HP-SO3] was characterized as 1 : 1 (mol/mol) by 1H NMR measurement. The fluorescence spectra of the PVC membrane containing acceptor showed FRET efficiency of close to 100% at 0.5 mol% of acceptor. From the fluorescence and excitation spectra, the amplification factor (AF) of acceptor fluorescence was calculated by equation (1) where Iex
D and Iex
A are fluorescence intensities at excitation wavelengths of donor and acceptor in the excitation spectrum of the PVC membrane without acceptor, Iex
D-FRET and Iex
A-FRET are those of the PVC membrane with acceptor, Iem
D and Iem
D-FRET are fluorescence intensities at the donor emission wavelength in the fluorescence spectra of the PVC membrane without or with acceptor, respectively[5]. In this case, AF was larger when smaller amounts of acceptor were doped. Especially, the largest AF (approximately 6) was obtained in the 0.01 mol% acceptor condition. That means the fluorescence intensity per an acceptor molecule was amplified 6-fold. On the other hand, fluorescence intensity (IA) of acceptor calculated by equation (2) was the largest at the 0.5 mol% acceptor condition. Thus, 0.5 mol% acceptor condition was determined to be optimal donor and acceptor ratio (Figure 3). In this experiment, IA was compared with that of conventional PVC membrane prepared with 2-nitrophenyl octyl ether (NPOE) as the plasticizer under the same membrane thickness condition (200 nm). As the result, FRET-based fluorescence of PVC membrane was larger than that of conventional one in all cases of acceptor concentration. Especially in 0.5 mol% acceptor condition, 12-fold fluorescence intensity was observed (Figure 3). That is supposed the change of fluorescence intensity between fluorescence on and off state is expanded to 12-fold when the chemical structure change of acceptor is caused by responding to analytes. Finally, proposed PVC membrane was applied for ion measurement in aqueous solutions. In fluorescence spectra under the several pH conditions, the obvious changes of spectra, which was obtained by highly-efficient FRET system, were successfully observed under the constant chloride concentration. In this case, spectral change was caused by coextraction of proton and chloride anion, and that result means the present FRET-based system can detect the very small amounts of acceptor molecules that change their molecular structure. Thus, FRET-based system proposed in this study is expected to improve the sensitivity of various PVC membrane-based optical sensors for ions, biomolecules and proteins.

References

[1] H. Hisamoto et al., Anal. Chem., 2004, 76, 3222.
[2] H. Hisamoto et al., Anal. Chim. Acta., 2006, 556, 164.
[3] S. Funano et al., Analyst, 2015, 140, 1459.
[4] T. Mizuta et al., Sens. Actuators B, 2018, 258, 1125.
[5] N. Melnychuk et al., J. Am. Chem. Soc., 2018, 140, 10856.

Figure 1