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Agricultural and Environmental Chemistry

Lignosulfonate: a Convenient FRET Platform for the Construction of Ratiometric Fluorescence pH-Sensing Probe Yuyuan Xue, Zechen Wan, Xinping Ouyang, and Xueqing Qiu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05286 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Journal of Agricultural and Food Chemistry

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Lignosulfonate: a Convenient FRET Platform for the Construction of Ratiometric

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Fluorescence pH-Sensing Probe

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Yuyuan Xue,†, ‡, ‖ Zechen Wan,† Xinping Ouyang,†,* Xueqing Qiu,†, ‡, *

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† School of Chemistry and Chemical Engineering, South China University of Technology,

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Guangzhou, China.

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‡ State Key Laboratory of Pulp and Paper Engineering, South China University of Technology,

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Guangzhou, China.

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‖ College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan, China.

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* Corresponding authors:

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Prof. Xinping Ouyang

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School of Chemistry and Chemical Engineering

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South China University of Technology

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Wu Shan Road, Guangzhou 510640, China

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E-mail: [email protected], Tel.: +86-020-87114033.

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Prof. Xueqing Qiu

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School of Chemistry and Chemical Engineering

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South China University of Technology

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Wu Shan Road, Guangzhou 510640, China

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E-mail: [email protected]

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Abstract

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Lignin is a kind of natural fluorescent polymer material. However, the application

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based on the fluorescent property of lignin was rarely reported. Herein, a non-covalent

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lignin-based Fluorescence Resonance Energy Transfer (FRET) system was readily

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constructed by physical blending method with spirolactam Rhodamine B (SRhB) and

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lignosulfonate (LS) as the acceptor and donor groups, respectively. The FRET behavior,

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self-assembly and energy transfer mechanism of SRhB/LS composite were systematically

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studied. It was demonstrated that LS could be used as a convenient aptamer as energy

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donor to construct water-soluble ratiometric sensors because of its inherent property of

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intra-micelle energy transfer cascades. Our results not only presented a facile and general

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strategy for producing lignin-based functional material, but also provided a fundamental

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understanding about lignin fluorescent to promote the functional and high-valued

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applications of lignin fluorescence characteristic.

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Keywords: lignin, ratiometric sensor, FRET, intra-micelle energy transfer cascade


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INTRODUCTION

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Lignin is one of the most abundant components in plants. Annually, more than 70

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million tons of industrial lignin is obtained from the pulp mill.1 More and more efforts are

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devoting to the high-valued utilization of lignin.2-8 As a natural and environmental-

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friendly fluorophore from plant cell walls, lignin can exhibit blue-green fluorescence

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under UV excitation, with a broad peak in the general range of 350-650 nm.9-11 The

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fluorescent property of lignin has been widely applied, focusing on the cell wall

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imaging,12 exploration of lignin distribution in woods13 and the original fluorophores of

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lignin.9-10 However, the fluorescent functional material based on lignin was rarely

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reported. Most reported lignin-based fluorescent materials were only focused on the role

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of lignin as an abundant and nontoxic carrier.14 Nevertheless, comparing with other

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structured biomass materials, the disordered and complex structure of lignin limited the

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attractiveness as polymer carrier materials.15-17 Thus, exploring the real functional

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application of lignin fluorescence not only opens up a valuable pathway for the utilization

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of industrial lignin recovered from pulping spent liquor, but also brings in a new area for

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high value-added applications of lignin-based products.

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Fluorescence detection/imaging has been attracting continuous attention to varieties of

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fields ranging from chemical analysis to intracellular probing and bio-imaging owing to

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their distinguished advantages in terms of lossless detection, high sensitivity and low-cost

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instrument.18-20 To date, a large number of fluorescence probes, especially ratiometric

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probes, which can avoid external interference from environmental factors by the self-

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calibration of two emission bands, have been constructed based on the mechanism of

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Forster resonance energy transfer (FRET).21-22 Spirolactam Rhodamine B (SRhB) is a 3 ACS Paragon Plus Environment


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classical fluorescent probe and has been widely exploited in design of intensity-based and

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ratiometric probes, resulting from the significant changes in the emission profiles of

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rhodamine.17, 23 In our previous work, a primary research of ratiometric fluorescent probe

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was reported via chemical grafting SRhB to lignosulfonate (LS), a water-soluble and

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fluorescent polymer material.24 It was found that LS could serve as not only the water-

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soluble carrier but also the energy donor group in FRET system. Nevertheless, multi-step

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synthetic process resulted in the disadvantage of low reproducibility, complexity and high

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cost. Generally, the constant distance between donor and acceptor groups was considered

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to be the most important factor to ensure the stable FRET efficiency.23 Except for the

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covalent link, recently, intense researches have been directed toward the exploitation of

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non-covalent strategy to construct FRET systems without any synthetic effort, such as

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entrapment, self-assembly, nanoprecipitation, adsorption and emulsion methods25-28. It is

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well-known that LS has natural hydrophilic and hydrophobic parts in its three-

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dimensional network structure, which could act as the natural scaffolds for hydrophobic

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nanomaterial.29-30 Therefore, non-covalent strategy was an operable method to construct

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the lignin/organic functional materials composite.

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Here, a facile approach to construct lignin-based FRET system (SRhB/LS) was

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presented by the self-assembly of LS with SRhB nanoparticles (SRhB NPs) via

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electrostatic interactions, without complex and time-consuming synthetic effort.

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Compared with our previous work,24 the preparation of SRhB/LS composite was greatly

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simplified. Moreover, based on the convenient and controllable synthetic strategy, the

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energy transfer mechanism and effect of LS content on the fluorescent response of

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SRhB/LS were also systematically studied in the first time. It was found that LS could be 4 ACS Paragon Plus Environment

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used as a convenient FRET platform for the construction of water-soluble ratiometric

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sensors, resulting from its inherent intra-micelle energy transfer.



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Materials and methods

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Materials

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LS was obtained as a byproduct of sulfite pulping processes from pines. The raw

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material was further purified by dialysis with the molecular weight cut-off of 1000 Da.

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Physicochemistry parameters of LS showed in Table 1. Gel Permeation Chromatography

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(GPC) analysis presented that the molecular weight (Mw) and polydispersion index (PDI)

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of LS were 3×104 Da and 2.118, respectively. Elemental analysis showed that the

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contents of C%, N%, S%, H% and sulfonation were 45.58, 0.420, 4.749, 4.314 and 1.48

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mmol/g, respectively. Sodium dihydrogen phosphate, sodium hydrogen phosphate, citric

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acid, trisodium citrate, sodium polystyrene sulfonate (PSSNa), 35% mass fraction of poly

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dimethyl diallyl ammonium chloride aqueous solution (PDAC) were of analytical grade.

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SRhB was synthesized according to the literature.24 Buffer solutions as follows: citric

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acid buffer saline for pH 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, and 6.2;

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phosphate buffer saline for pH 6.4, 6.6, 6.8, 7.0, 7.2, and 7.4.

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Table 1 Molecular weight distribution, elemental analysis and sulfonation degree of LS GPC analysis

Elemental analysis

Sample Mw (Da)

LS

3.0×104

PDI

Sulfonation degreea (mmol/g)

C%

N%

S%

H%

45.58

0.420

4.749

4.314

2.11 8

100

a:

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Preparation of SRhB/LS composite

1.480

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10 mL water was injected into 0.5 mL of SRhB alcohol solution (3 mg/mL) at once.

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After stirring for 5 min at 200 rpm, the residual alcohol was removed under vacuum

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rotary evaporation at room temperature. SRhB NPs stock solution was obtained with the 6 ACS Paragon Plus Environment

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concentration of 0.15 mg/mL. The composite of SRhB/LS was obtained by the physical

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blending of LS and SRhB NPs aqueous solutions. Then, 10 mL SRhB NPs stock solution

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was gradually added into 0.3 mL LS water solution (100 mg/mL). According to our

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previous work, the mass ratio of SRhB NPs:LS was chose as 1:20.24 After stirring for 10

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min at 1000 rpm, SRhB/LS aqueous solution was obtained. The powder of SRhB/LS was

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obtained by lyophilization. Corresponding schematic diagram for the synthetic method of

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SRhB/LS was showed in Fig. 1.

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Fig. 1 The schematic diagram for the synthetic method of SRhB/LS Preparation of SRhB/LS probe aqueous solutions

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30 mg of SRhB/LS powder was dissolved in 10 mL deionized water. Then, 50.0 μL of

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SRhB/LS aqueous solution was added into the flask containing 1.95×103 μL different

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buffer solutions (pH from 4.0 to 7.4). After the mixtures were stirred for 30 min at 1000

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rpm, the fluorescent spectra were recorded.

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Preparation of LS film on gold-coated QCM-D crystals sensor

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The gold-coated substrate was cleaned sequentially under sonication with detergent,

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deionized water and acetone, and then dried at room temperature overnight, followed by a

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plasma treatment. 3 wt% PDAC aqueous solution was introduced to adsorb on gold film

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at a flow rate of 0.15 mL/min until the baseline stabilized at 25 oC. Then, deionized water

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was injected (0.15 mL/min) until an equilibrium state was reached. Subsequently, 1 g/L 7 ACS Paragon Plus Environment


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LS was injected into the QCM-D flow module (0.15 mL/min). LS film on gold-coated

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QCM-D crystals sensor was obtained after the unstable adsorption of LS was removed by

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deionized water. 20.2 Hz LS film was prepared by the assistance of PDAC on gold-

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coated substrate.25

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In situ 1H-NMR measurement

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10 mL D2O was injected into 0.5 mL of SRhB acetone-d6 solution (3 mg/mL) at once.

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After stirring for 5 min at 200 rpm, the residual acetone-d6 was removed under vacuum

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rotary evaporation at room temperature. Then, the 1H-NMR spectra of SRhB NPs in D2O

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was recorded with different content of LS.

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Characterization

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The molecular weight of LS was detected using Waters 1515 Isocratic HPLP

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pump/Waters 2487 Gel Permeation Chromatography (Waters Co., America). Elemental

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analyses were measured using an Elementar Vario EL cube (Elementar Co., Germany).

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The preparation of SRhB/LS were detailedly characterized by UV-vis, Zeta potential,

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DLS, SEM, TEM and 1H-NMR, respectively. The UV-spectra were acquired in a UV-

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2450 spectrometer (Shimadzu Co., Japan). Dynamic light scattering (DLS) and Zeta

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potential experiments were performed at 25 °C on a Malvern Zetasizer 2000 instrument

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(Malvern Co., England) and were repeated at least ten times and the average values were

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obtained. SEM/TEM images of the morphology were obtained using a HITACHI H-7650

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transmission electron microscopy (HITACHI Co., Japan) with an accelerating voltage of

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10 kV. 1H-NMR spectra was recorded on an Advance Digital 400 MHz NMR

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spectrometer (Bruker Co., Germany).


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The pH-response of SRhB/LS probes were explored by steady-state and transient

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fluorescence techniques. The fluorescence spectra were recorded at 25 °C on a HITACHI

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F-4600 luminoscope spectrometer (HITACHI Co., Japan). The fluorescent lifetime was

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recorded by a Quantaurus-Tau C11367-11 instrument (Hamamatsu Photonics Co., Japan).



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RESULTS AND DISCUSSION

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Formation and characterization of SRhB/LS

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SRhB was insoluble in pure water. In order to realize the fabrication of SRhB/LS in

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aqueous phase, the stable dispersion of SRhB NPs was prepared through the solvent-

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exchange method. The characterizations of SRhB NPs and SRhB/LS were presented Fig.

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2. Along with the change of solvent environment of SRhB ethanol solution, the red-shift

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of absorption peak at 313 nm and absorption tail at long wavelength (350-500 nm) were

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observed, indicating the formation of SRhB NPs (Fig. 2A). The nano-dispersion of SRhB

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could be stabilized by electrostatic repulsion and the Zeta potential of SRhB NPs was +15

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mV at pH 7 (Fig. 2B). The size of SRhB NPs was found to be 40 and 90 nm by DLS, and

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the signal of large size distribution might mainly result from the aggregates of

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monodisperse SRhB NPs (Fig. 2C).

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Fig. 2 (A) UV-vis spectra and (B) Zeta potential of SRhB NPs; (C) Size distributions of

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SRhB NPs, LS and SRhB/LS; SEM and TEM images of (D) SRhB/LS and (E+F) SRhB

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NPs


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The surface of SRhB NPs was positively charged, while LS is a natural anionic

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surfactant. Therefore, SRhB NPs would be inevitably adsorbed on LS micelles by the

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electrostatic attraction in the mixture. DLS provided evidences for the success of

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SRhB/LS formation (Fig. 2C). Compared with SRhB NPs, the size distribution of

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SRhB/LS increased and broadened, finding at 80 and 370 nm, respectively. The

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expanding size distribution was mainly ascribed to the electrostatic attraction of SRhB

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NPs onto LS micelle, which was found to be 90 and 800 nm, respectively, in the aqueous

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solution. In addition, after the adsorption of SRhB NPs, the size of LS was also relatively

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compressed, resulting from the electrostatic shielding effect between the opposite electric

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charge of LS and SRhB NPs.

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SEM measurement illustrated the morphology of SRhB/LS powder (Fig. 2D). Quasi-

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spherical shape of SRhB/LS nanoparticles were observed and the size was about 100 nm,

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which was consistent with the DLS results. Although SRhB NPs could be stabilized by

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electrostatic repulsion in the preparation process, the re-dispersibility of SRhB NPs

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disappeared after the drying process, because of the aggregation between SRhB NPs (Fig.

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2E and 2F). After encapsulated with LS, an efficient dispersant, the aggregation effect of

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SRhB NPs was significantly inhibited, and the powder of SRhB/LS would be easily

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disperse in aqueous solution. The enhanced re-dispersibility of SRhB was benefit for the

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application in water environment detection.

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FRET behavior of SRhB/LS composite

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Compared with our previous work,24 the preparation of SRhB/LS composite was

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greatly simplified. Except for the successful formation, the FRET behavior of SRhB/LS

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was then studied (Fig. 3). The fluorescent pH-response of SRhB/LS in various pH buffer 11 ACS Paragon Plus Environment


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solutions between 4.0 and 7.4 was recorded (Fig. 3A and 3B). LS exhibit blue-green

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fluorescence centered at 400 nm under 350 nm excitation wavelength. When the pH

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value was higher than 7, the fluorescence of SRhB/LS was mainly from LS, and SRhB

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was non-fluorescent. Upon the gradual decrease of pH, a new band centered at 581 nm

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from SRhB was formed, while the fluorescent intensity at 400 nm was gradually

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decreased. The dual-emission fluorescent intensity ratio of I581/I400 increased linearly with

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pH in the range of 4.4-6.6 and the R2 was 0.9935. When the concentration of SRhB/LS in

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the buffer solutions was different, the linear relationships between pH values and

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fluorescent intensities at 400 and 581 nm, respectively, were both broken (Fig. 3C).

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However, the fluorescent intensity ratio of I581/I400 could calibrate the interference. Thus,

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the ratiometric detection of SRhB/LS to pH was achieved based on the dual-emission

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from LS and SRhB fluorescence. The ratiometric detection might be achieved by the

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energy transfer between LS and SRhB.23-24

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Fig. 3 (A) Fluorescent pH-response spectra and (B) corresponding intensity changes of

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SRhB/LS; (C) fluorescent pH-response curve of SRhB/LS with different concentration;

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(D) Photobleaching curves of SRhB/LS and SRhB NPs 12 ACS Paragon Plus Environment

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The energy transfer behavior between LS and SRhB was further confirmed in the

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photobleaching experiment (Fig. 3D). To obtain a believable result and avoid excessive

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response time24, the buffer solution with pH 5.4, which was close to the pKa value of

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SRhB probe, was selected. Under continuous UV-350 nm irradiation, the emission

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intensity of SRhB/LS was decreased, and it dropped 30% and 10% at 400 and 581 nm,

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respectively. In order to understand the phenomenon above, the photobleaching of

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individual LS and SRhB was investigated. The emission intensity of LS was decreased

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under the same irradiation condition (Fig. S1), however, no emission intensity change

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was observed in the individual SRhB solution. It revealed that the photobleaching of

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SRhB/LS at 581 nm had no connection with the destruction of SRhB fluorophore but was

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mainly due to the photobleaching of LS. For the FRET system, photobleaching of energy

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donor would result in a decreasing intensity of energy acceptor groups, indicating the

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FRET behavior between LS and SRhB.31

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Self-Assembly and Adsorption behavior between LS and SRhB NPs

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The ratiometric pH-response of SRhB/LS revealed that the FRET efficiency between

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LS and SRhB was stable in the pH detection. As the ‘spectroscopic ruler’, FRET was

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very sensitive to the distance between donor and acceptor fluorophores. To in-depth

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understand the FRET behavior in SRhB/LS composite, 1H NMR, Zeta potential and

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QCM-D were carried out to determine the self-assembly and adsorption behavior

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between LS and SRhB NPs (Fig. 4 and 5).

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The assembly behavior of SRhB/LS composite was established by 1H NMR. The 1H

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NMR spectra of SRhB/LS was dependent on the LS content (Fig. 4). All the proton

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signals of SRhB became more and more broad as the content of LS increased, indicating 13 ACS Paragon Plus Environment


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the decreasing degree of freedom of SRhB. When the mass ratio of SRhB:LS was 1:5, the

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signals of SRhB even disappeared. With the continue addition of LS, the signals at 3.5

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and 7.0 ppm from methoxyl and aromatic proton of LS, respectively, were observed (Fig.

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4 and S2). These results suggested the gradual formation of SRhB/LS composite because

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of the increasing aggregation effect of SRhB NPs. Meanwhile, the magnetic shielding

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effect of LS on SRhB was weak and nearly no chemical-shift change of SRhB was

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observed in the in-situ 1H-NMR experiment. it was suggested that SRhB NPs was mainly

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adsorbed onto the surface but not the inside of LS micelle.

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Fig. 4 In situ 1H-NMR spectra of SRhB NPs with different content of LS

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QCM-D measurement was a sensitive and effective sensor to explore the adsorbing

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mass of between LS and SRhB NPs. Changes in the resonance frequency (Δf3) was

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primarily related to mass uptake or release at the sensor surface. The adsorbing mass of

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SRhB NPs on LS film was very sensitive to the ionic strength (Fig. 5A). The maximum

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adsorption capacity of SRhB NPs on LS film increased to 46.3, 72.72, 78.38 and 112.11

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Hz, which were 2.16, 3.60, 3.88 and 5.55 times mass of LS film, under the NaCl

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concentration of 0, 10-4, 10-3 and 10-2 mol/L, respectively. When the concentration of 14 ACS Paragon Plus Environment

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NaCl was 10-1 mol/L, the adsorption equilibrium of SRhB NPs on LS film was still not

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reached after 1000 s, but the adsorption capacity was significantly increased, compared

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with that in the NaCl concentration of 10-2 mol/L. This phenomenon might be connected

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with the change of electrostatic effect. LS has the anti-static shielding capability under

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relatively low ionic strength, due to the special micelle structure.32 However, the

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shielding effect on the surface charge of SRhB NPs would become more and more strong

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as the ionic strength increases. Thus, the mass ratio of SRhB NPs and LS at isoelectric

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point was increased along with the increase ionic strength.

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Fig. 5 Change in frequency (Δf3) as a function of time during the adsorption of SRhB NPs

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on LS film under different (A) ionic strength and (B) pH; (C) Zeta potentials of SRhB

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NPs as a function of LS content in different pH aqueous solution

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SRhB/LS was used as pH probe in neutral and weakly acidic solution. The adsorbing

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mass of SRhB NPs on LS film in different pH solution was further explored by QCM-D

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(Fig. 5B). To reduce the interference of ionic strength at different pH, SRhB NPs solution 15 ACS Paragon Plus Environment


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with the NaCl concentration of 10-2 mol/L was used. Upon the gradual decrease of

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solution pH, the maximum adsorption capacity of SRhB NPs on LS film decreased. This

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phenomenon was related to the pH-dependent Zeta potential of SRhB NPs. The surface

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changes of SRhB NPs increased as the pH change from 6.98 to 4.08 (Fig. 2B), resulting

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in the decrease mass ratio of SRhB NPs and LS at isoelectric point. The QCM-D results

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above further revealed that electrostatic interaction was the main driving force to realize

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the constant distance between SRhB NPs and LS micelle.

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The isoelectric point of SRhB and LS was determined by Zeta potential (Fig. 5C).

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With the addition of LS, the Zeta potential of SRhB NPs sharply decreased from positive

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to negative. The isoelectric point was observed when the mass ratios of SRhB NPs:LS

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was 1:0.2 at pH 7.0. In addition, the isoelectric point only increased to 1:0.4, even the pH

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value at 3.8. Thus, the LS content in the composite was much greater than the amount to

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neutralize positive charges of SRhB NPs in both neutral and acidic solutions.

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Energy transfer mechanism of SRhB/LS composite

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Except for distance, FRET was also sensitive to the spectra overlap of donor emission

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and acceptor absorption. LS belongs to multiple fluorophores system and different kinds

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of fluorophores randomly distribute in the LS micelle. The adsorption site of SRhB onto

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LS micelle was dynamic and random in the preparation. However, despite the donor

281

fluorophore was thereby uncertain for SRhB in different adsorption sites, the random

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combination has no effect on the FRET efficiency in SRhB/LS system. To gain further

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insights on the energy transfer mechanism of SRhB/LS composite, the real donor

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fluorophore of LS, which would be decayed when the FRET occurred, was explored by

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transient fluorescence (Fig. 6).33 FRET process between LS and SRhB occurred only in 16 ACS Paragon Plus Environment

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acid environment. Thus, the fluorescence lifetime of SRhB/LS was measured at pH 4.4

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and 7.4, respectively (Fig. 6A, Table S1 and S2). When the solution pH decreased from

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7.4 to 4.4, the fluorescence lifetime of LS fluorophores at 450, 475, 500 and 525 nm

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decreased from 3.24 to 2.23, from 3.65 to 2.83, from 3.99 to 3.54 and from 3.91 to 3.74,

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respectively. Lifetime decay was observed in the long-wavelength fluorophore of LS

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(>450 nm). The short-wavelength fluorophore of LS (450

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nm), which well overlapped with the absorption spectrum of Rhodamine B. On the other

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hand, another type of energy transfer was occurred at the same time in the LS micelle,

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from the fluorophores with high excitation energy (< 425 nm) to the low excitation

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energy groups (> 450 nm). In addition, the integration of emission energy of LS was

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realized based on the intrinsic intra-micelle energy transfer cascades.

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The effect of LS content on the fluorescent response of SRhB/LS

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The adsorption results revealed that the LS content in SRhB/LS composite, of which

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the mass ratio of SRhB NPs: LS was 1:20, was much greater than the saturated

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adsorption capacity of LS to SRhB NPs. Thus, the effect of LS content on the fluorescent

336

response of SRhB/LS system was further studied (Fig. 7).

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Fig.7 (A) pH-response curves of SRhB NPs; (B) The absorbance and (C) PL intensity of

339

SRhB as a function of surfactants content in the buffer solution of pH 5.4; (D) pH-

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response curves of SRhB/PSSNa; (E) The time variation of absorbance intensity at the 19 ACS Paragon Plus Environment


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567 nm of SRhB/LS in the buffer solution of pH 5.4 with different LS content; (F)

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ratiometric pH-response curves of SRhB/LS with different content of LS

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Actually, individual SRhB NPs was a classical intensity-based fluorescence probe. As

344

presented in Fig. 7A, the fluorescent intensity of SRhB NPs at 581 nm increased linearly

345

with pH values but obvious aggregation-caused quenching (ACQ) phenomenon of SRhB

346

was observed at low pH (4.0-4.6), which was a common problem for rigid fluorophores.

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Except for the achievement of ratiometric fluorescence detection, the ACQ phenomenon

348

of SRhB was passivated in presence of LS. Meanwhile, compared to the pH-response

349

curves of SRhB, the fluorescence intensity of SRhB/LS at 581 nm was increased by about

350

two times, when the pH was 4 (Fig. 3B). The significant emission amplification and

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quenching inhibition for SRhB must be endowed from LS. The effect of LS content on

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the optical spectra of SRhB was systematically explored (Fig. 7B and 7C). Meanwhile,

353

PSSNa, a classical surfactant without visible fluorescence, was also introduced as the

354

reference to verify the role of LS fluorescence. The enhancement of emission intensity

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and absorbance of SRhB was observed with the increase content of LS or PSSNa. When

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the mass ratio of LS or PSSNa:SRhB reached 20:1, the fluorescence intensity of SRhB

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solution at 581 nm was increased by 3.27 and 2.47 times, respectively, indicating that

358

ACQ effect of SRhB was inhibited by the addition of surfactants. This result was also

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consistent with previous correlative researches34-35. It was noted that LS showed a

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stronger ability to enhance the emission of SRhB than that of PSSNa under the same

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addition. Furthermore, the advantage was more obvious at low pH (4.0-4.6) and the ACQ

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effect of SRhB still existed in SRhB/PSSNa system (Fig. 7D). It was proposed that the

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three-dimensional network structure of LS could provide better site isolation for SRhB 20 ACS Paragon Plus Environment

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than that of PSSNa with linear structure36. Meanwhile, FRET also enabled to enhance the

365

optical absorption and fluorescent emission of acceptor groups28. Thus, the strong ability

366

of fluorescence amplification of LS was attributed to the synergistic effect of quenching

367

inhibition and FRET to SRhB. However, the responsive time of SRhB/LS was lengthened

368

with the increase of LS content, suggesting that the good steric effect of LS for SRhB not

369

only inhibited the aggregation but also decreased the sensitivity (Fig. 7E).

370

It was well-known that the FRET efficiency was greatly related to the relative content

371

of acceptor group (SRhB) and donor group (LS) in the composite. The pH-response

372

curves of SRhB/LS composite with different LS contents was recorded (Fig. 7F). When

373

the mass ratios of SRhB:LS ranged from 3:10 to 3:60, good linear relationships between

374

I581/I400 and pH were observed, and the fitted equation and R2 values were presented in

375

Table 2. The pH detection resolution of SRhB/LS could be improved by decreasing the

376

LS content, due to the changes of FRET efficiency between LS and SRhB. In the

377

formation of composite, SRhB NPs homogeneously adsorbed onto LS micelles. Upon the

378

gradual decrease of LS content, the adsorbing mass of SRhB NPs on specific LS micelle

379

must be increased, resulting in the increasement of FRET efficiency.

380

Table 2 The fitting equations and R2 of pH-response curves of SRhB/LSs with different

381

LS content Mass ratio of Fitting equation

R2

SRhB NPs:ELS 3:10

0.9957

3:20

0.9935

3:30

0.9958 21 ACS Paragon Plus Environment


Page 22 of 30

3:40

0.9824

3:50

0.9799

3:60

0.9842

382 383

In summary, a non-covalent lignin-based FRET ratiometric system, in which LS and

384

SRhB were used as the donor and acceptor groups, respectively, was prepared via

385

electrostatic self-assembly method in aqueous solution. The energy transfer between LS

386

and SRhB was non-radiative type (FRET) and the direct donor groups were long-

387

wavelength fluorophores of LS (>450 nm), which well overlapped with the absorption

388

spectrum of Rhodamine B. The emission energy of short-wavelength fluorophore of LS

389

(< 425 nm) indirectly transferred to SRhB via the FRET channel above based on the

390

intra-micelle energy transfer cascades in LS. In addition, the emission energy of LS from

391

multiple fluorophores showed as integration. Meanwhile, the FRET efficiency of

392

SRhB/LS and fluorescent behaviors of SRhB could be facilely adjusted by changing the

393

LS content in the preparation process. This study not only provided a facile, low-cost and

394

sustainable strategy for producing lignin-based functional material, but also promoted the

395

functional

396

Nevertheless, there are also many challenges for the commercialization process of lignin

397

fluorophore owing to its marked heterogeneity and low fluorescence quantum efficiency.

398

More research still needs to be performed on the mechanism of lignin fluorescence to

399

promote the veritable industrial application of lignin fluorescence.

400

Acknowledgment

and

high-valued

applications

of

lignin

401 22 ACS Paragon Plus Environment

fluorescence

characteristic.

Page 23 of 30


402

This work is financially supported by the National Natural Science Foundation of China

403

(No. 21436004, 21576104, 21776108, 21690083) and the Natural Science Foundation of

404

Guangdong, China (2017A030308012).

405

Supporting Information Available

406

Supporting information included the photobleaching of LS, 1H-NMR spectrum of

407

SRhB/LS (the mass ratio of SRhB:LS was 1:20) and additional transient fluorescence

408

data.

409



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Abstract Graphic

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Lignosulfonate: a natural aptamer as energy donor to construct water-soluble ratiometric

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sensors

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