A pyrylium-based colorimetric and fluorimetric chemosensor for the selective detection of lysine in aqueous environment and real sample

Accepted Manuscript A Pyrylium-Based Colorimetric and Fluorimetric Chemosensor for the Selective Detection of Lysine in Aqueous Environment and Real Sample Xiaomin Qian, Weitao Gong, Furui Wang, Yuan Lin, Guiling Ning PII: DOI: Reference:

S0040-4039(15)00656-5 http://dx.doi.org/10.1016/j.tetlet.2015.04.029 TETL 46168

To appear in:

Tetrahedron Letters

Received Date: Revised Date: Accepted Date:

2 February 2015 30 March 2015 8 April 2015

Please cite this article as: Qian, X., Gong, W., Wang, F., Lin, Y., Ning, G., A Pyrylium-Based Colorimetric and Fluorimetric Chemosensor for the Selective Detection of Lysine in Aqueous Environment and Real Sample, Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.2015.04.029

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Graphical Abstract

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A Pyrylium-Based Colorimetric and Fluorimetric Chemosensor

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for the Selective Detection of Lysine in Aqueous Environment

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and Real Sample

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Xiaomin Qian, Weitao Gong*, Furui Wang, Yuan Lin and Guiling Ning*

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State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Dalian University of

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Technology, Dalian 116024, China.

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Abstract: A new chemosensor based on pyrylium salt has been developed. The

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sensor can selectively fulfil the detection of lysine among 20 common amino

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acids in aqueous environment and exhibit a distinct color change and “turn-on”

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fluorescence. Besides, simple test paper based on this sensor was also presented,

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which elucidated the feasibility of lysine detection in real samples.

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Keywords: lysine, pyrylium, chemosensor, fluorimetric and colorimetric

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1. Introduction

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Amino acids (AA) are pivotal intermediates of primary metabolism in all

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biological cells and effective biomarkers in bioanalytical process.1-4 In this

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family, lysine (Lys) is closely related to the Krebs-Henseleit cycle and

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polyamine synthesis. An appropriate amount of Lys in the diet is essential for

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the metabolic functions and weight gain of animals.5 As the result of

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considerable attention paid to human health, many efforts have been done to 2

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explore new approaches toward Lys detection. Currently, the most common

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analytical

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chromatography

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inevitable drawbacks, such as operational inconvenience, high analysis cost and

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comparatively low test speed restrict its further applications. Hence, the

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development of new detection methods for Lys can be of rather interest.

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procedures

to

(HPLC)

detect and

Lys

are

amperometric

high

performance

methods.

However,

liquid some

Due to the advantages of simplicity, inexpensiveness and sensitivity,

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colorimetric and fluorimetric approach based on synthetic chemosensors

has

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attracted increasing interest during the last decade.6-16 Even though, it is still a

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challenging job to selectively discriminate a specific AA from others owing to

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the similarity in structure and reactivity. In the past few years, sensors for

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cysteine (Cys) and homocysteine (Hcy) have been largely described, utilizing

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the unique nucleophilicity of their thiol group.17-19 On the other hand, the

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examples for the selective sensing of Lys with the interference from histidine

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(His), arginine (Arg) or cysteine (Cys) are comparatively rather common.20-25

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To the best of our knowledge, only one example, exhibiting both distinct color

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change and only moderate fluorescent enhancement upon interaction with Lys

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exclusively, had been reported to date.26 Accordingly, it is still quite necessary

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to develop new Lys chemosensors with high efficiency and selectivity.

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Considering the structural features and chemical reactivity of pyrylium salts

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and their derivatives, they had already been utilized in the field of molecular

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(ion) recognition extensively.27-31 For example, Mouradzadegun’s group 3

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reported a designed organic/inorganic solid receptor based on a triarylpyrylium

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derivative incorporated into γ-alumina which displayed a highly selective

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colorimetric chemodosimeter toward

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Martínez-Máñez et al. synthesized novel pyrylium-containing mesoporous

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materials which could serve as chromo-fluorogenic sensors for biogenic amines

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in aqueous environment.33 With respect to the application of pyrylium salts in

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probing Lys, as far as we know, scarce examples were found. Herein, we have

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developed a simple pyrylium-based compound 1 that can be employed as a

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highly selective colorimetric and fluorimetric chemosensor for Lys in aqueous

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environment. The rationale in the design of compound 1 is explicated as follow.

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Compound 1 mainly consists of two parts: a pyrylium moiety as the receptor

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and a pyrene moiety as the fluorophore, which are linked by a carbon-carbon

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double bond. This is a classical fluorescent photo-induced electron transfer

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(PET) structure. It is well-known that PET process usually causes the

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fluorescence quenching of the fluorophore. Therefore, it is expected that sensor

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1 would show weak fluorescence, namely, the “off” state. Compared with other

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AA, the distinctive structural difference of Lys is the existence of a long-chain

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aliphatic amino group that is far away from the electron-withdrawing group

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carboxyl which could abate the nucleophilicity of a nucleophile. Consequently,

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the introduction of Lys will destroy the pyrylium ring due to the good reactivity

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between pyrylium and Lys, which had already been described before.34-35 As a

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result, the luminescence of the pyrene unit was regained and it led to the 4

the cyanide

anion.32 Bricks and

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fluorescence enhancement of the whole system, that is, the “on” state.

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Meanwhile, the destruction of the pyrylium ring will also break the

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intramolecular charge transfer (ICT) progress from the electron-rich pyrene

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moiety to the electron-poor pyrylium ring. Thus, a blue shift in the absorption

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spectra of sensor 1 accompanying a distinct color change is also expected.

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These could serve as the foundation for the Lys sensing of sensor 1.

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2. Experimental

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2.1 Apparatus

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Absorption spectra were all taken on a Hitachi UV-4100 spectrophotometer.

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Fluorescence spectra were taken on a Jasco FP-6300 spectrofluorometer. The 1H

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NMR and

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and recorded at 400 MHz and 100 MHz respectively. Mass spectra were measured on

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a Agilent 6310 MS spectrometer and a Q-TOF MS spectrometer.

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2.2 Synthesis

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C NMR spectra were measured on a Bruker AVANCE-400 spectrometer,

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All solvents and reagents were commercially available and used without further

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purification unless for special needs. All reactions were magnetically stirred and

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monitored by thin-layer chromatography (TLC). The synthesis of 1 ( Scheme 1) was

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readily achieved by treatment of 2-methyl-4, 6-diphenyl pyrylium tetrafluoroborate

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with pyrene-1-carboxaldehyde in acetic acid by the classic condensation reaction, and

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it gave the corresponding compound 1 in good yield. The structure of 1 was further

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confirmed by 1H NMR, 13C NMR and TOF-ES mass spectra.

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2-methyl-4, 6-diphenylpyrylium tetrafluoroborate and pyrene-1-carboxaldehyde 5

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were prepared by the literature reported methods.36-37

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Pyrene-1-carboxaldehyde (0.276 g, 1.2 mmol) was added to a solution of

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2-methyl-4, 6-diphenylpyrylium tetrafluoroborate (0.334 g, 1 mmol) in acetic acid (20

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mL) under stirring. Then, the reaction mixture was slowly heated to 120 ℃ and kept

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stirring for 4 h under refluxing. The solution turned from light yellow to dark purple.

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The mixture was filtrated off immediately and the precipitate was washed with hot

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acetic acid for several times, which gave the compound 1 as a dark purple solid, and it

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was dried and used without further purification (0.48 g, 85.1% yield); 1H NMR(400

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MHz, DMSO-d6): δ (ppm) 7.82 (m, J=16 Hz, 6H), 8.19(m, 2H), 8.30(d, 1H), 8.40(d,

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J=8.8 Hz, 1H), 8.45(m, J=8.8 Hz, 3H), 8.53(d, J=8.8 Hz, 3H), 8.65(d, 2H), 8.84(d,

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1H), 9.03(d, J=8.8 Hz, 2H), 9.18(d, J=8.8 Hz, 1H), 9.57(d, J=16 Hz, 1H) ;

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NMR(100 MHz, DMSO-d6) δ 114.30, 116.46, 120.99, 123.35, 123.97, 124.58, 125.48,

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126.19, 127.24, 127.45, 128.38, 128.95, 129.57, 129.98, 130.07, 130.16, 130.30,

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130.40, 130.61, 131.02, 131.24, 133.14, 134.19, 135.14, 142.54, 163.30, 168.73,

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171.02; TOF-ES-MS+ m/z Calcd for C35H23O+ 459.1749 [M]+, found 459.1753.

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C

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Scheme 1 The synthetic route of 1

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3. Results and discussion

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In consideration of the solubility of 1 and Lys, a combination of acetonitrile

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and water (1:1, v/v) was chosen to be our test system. Fig.1 shows the changes 6

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in the UV-vis spectrum when Lys was added to CH3CN/H2O (1:1, v/v) solution

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containing sensor 1 (50 µM). As we can see, sensor 1 exhibited two apparent

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absorption bands in the visible region from 350 nm to 700 nm. With increasing

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Lys concentration (0-7 equiv), the absorption peak at 564 nm and 376 nm both

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gradually decreased. The change of absorption peak at 564 nm, which is

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assignable to the π-π* electron transition, demonstrated that the ICT progress

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was turned off upon the addition of Lys. Meanwhile, the decrease of absorption

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peak at 376 nm, which can be ascribed to the characteristic absorption peak of

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pyrylium ring, revealed that the pyrylium ring moiety was destroyed after Lys

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was added to the system. The result perfectly coincided with our design

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concept: due to the good reactivity of the pyrylium ring with Lys, the PET and

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ICT process was blocked after Lys was added, which resulted in the regain of

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pyrene moiety fluorescence as well as a dramatic color change of the system,

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and it was further authenticated by following experiments.

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Fig.1 The UV-vis spectral changes of sensor 1 (5×10-5 mol•L-1 in CH 3CN:H2O=1:1, v/v) upon continuous addition of Lys

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Selectivity is one of the most pivotal requirements for all kinds of detection

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methods. To evaluate the selectivity for Lys, changes in the UV-vis and

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fluorescence intensity of sensor 1 promoted by addition of excess amounts of a

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wide variety of AA were measured. The unique UV-vis change corresponding 7

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to the appearance of a contrasting light yellow was observed exclusively upon

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the addition of Lys, and could be detected by naked eye. Other common AA

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that we tested, including cysteine (Cys), alanine (Ala), arginine (Arg), glutamic

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acid (Glu), leucine (Leu), tyrosine (Tyr), isoleucine (Ile), methionine (Met),

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aspartic acid (Asp), glycine (Gly), threonine (Thr), phenylalanine (Phe),

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histidine (His), serine (Ser), asparagine (Asn), glutamine (Gln), proline (Pro),

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valine (Val) and tryptophane (Trp), did not cause any obvious UV-vis

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spectroscopic changes (See Fig. 2). In the meantime, when the absorption ratios

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at 460 nm and 564 nm were monitored, good selectivity was observed for Lys

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with a more than 10-fold intensity increase in absorbance (See Fig. S1). As we

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can see from Fig. 3, the fluorescent emission of sensor 1 upon excitation at 365

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nm was fairly weak, which was ascribed to the quenching effect of PET

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process. After Lys (9 equiv) was added into the CH3CN/H 2O (v: v, 1:1) system,

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a more than 10-fold fluorescence intensity enhancement of sensor 1 at 457 nm

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system came up as the fluorescent color converted from dark blue to wathet,

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which was a classic turn-on type fluorescent sensor and showed high selectivity

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for Lys, and this point was also supported by fluorescence titration experiment.

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(See Fig. S2) In addition, the fluorescence quantum yield of sensor 1 in

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CH 3CN/H2O (v: v, 1:1) was calculated to be 2.64% and the molar absorption

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coefficient ε (M-1 cm-1) at 564 nm and 376 nm were calculated to be 36 000 and

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32 700, respectively. The calculative details can be found in the supplementary

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data. 8

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Fig. 2 UV-vis spectra of sensor 1 (5×10-5 mol•L-1 in CH 3CN:H2O=1:1, v/v)

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upon addition of 9 equiv Lys and other AA. Inset: a color change photograph

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for Lys (the last one) and other AA.

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Fig. 3 Fluorescent spectra (excitation at 365 nm) of sensor 1 (5×10-5 mol•L-1

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in CH3CN:H 2O=1:1, v/v) upon addition of 9 equiv Lys and other AA. Inset: a

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fluorescence color change photograph for Lys and other AA.

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On the other hand, in order to determine the response time of sensor 1 toward

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Lys, a Time-Dependent UV-vis study was carried out in the presence of Lys

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(Fig. S3). The kinetic study shows that upon the addition of Lys, the absorption

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peak at 564 nm decreased very fast. This suggested that the pyrylium reacted

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instantly after Lys was added into the system. As time went on, the reaction

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speed slowed down gradually. In about 7 minutes, the absorption peak at 564

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nm remained stable and the reaction was completed. However, at 460 nm, the

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absorption peak did not change obviously in the beginning up to 2 minutes.

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After 2 minutes, it enhanced rapidly which might indicated the formation of a

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new species, and in 6 minutes, the absorption peak stayed steady. All in all, the

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kinetic study revealed that sensor 1 can react with Lys moderately within a few

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minutes under the experimental condition.

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The fluorescence titration data was also used to calculate the detection limit

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based on a literature reported method.38 As is shown in Fig. S4, the detection

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limit of sensor 1 for Lys is calculated to be 3.61×10-5 mol•L-1. Therefore, the

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result suggests that it can fulfil the detection of Lys at a low concentration

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compared with reported literature.21

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In the end, to test the capability of sensor 1 to detect Lys in real samples, we

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fabricated a Lys test paper as following steps: two well cut filter paper were

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soaked in the CH3CN solution of sensor 1(5×10-5 mol•L-1), and then dried out

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in the air as blue test paper (See Fig.4). After that, the two test paper were

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saturated in a blank urine sample and a control urine sample containing

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Lys(5×10-5 mol•L-1) separately. After 6 minutes, as we can see from Fig 4, the

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test paper in the control sample distinctly turned into light yellow while the

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other one remained almost unchanged, which supported our idea that sensor 1

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can detect Lys in real samples.

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Fig. 4 Lys test paper: (a) before the test (b) added to the urine without Lys (c) added to the urine containing Lys

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4. Conclusion

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In conclusion, a prominent pyrylium-based sensor 1 for Lys was presented,

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which could selectively detect Lys in aqueous environment and real sample.

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The sensor exhibits relatively fast response and high selectivity toward Lys 10

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over other tested AA, which could be detected by naked eye. The simplicity of

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synthesis and analysis suggests that this new chemosensor may find application

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in a variety of different environments where simple and rapid determination of

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Lys might be required.

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Acknowledgments

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The authors are grateful for financial support from the National Natural

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Science Foundation of China (No. 21206016) and the Fundamental Research

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Funds for the Central Universities (DUT11LK13).

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Scheme. 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Highlights 1. The synthetic procedures are quite simple, only 3 steps. 2. The exclusive reactivity between lysine and pyrylium salt made this detection selective. 3. Compared with reported literatures, this detection process has a clear enhancement in fluorescence intensity and a distinct color change.