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Self-assembled conjugated polymer/chitosan-graft-oleic acid micelles for fast visible detection of aliphatic biogenic amines by a “turn-on” FRET Haoquan Zhong, Chunchen Liu, Wenjiao Ge, Run-Cang Sun, Fei Huang, and Xiaohui Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06168 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 19, 2017

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ACS Applied Materials & Interfaces

Self-assembled conjugated polymer/chitosan-graftoleic acid micelles for fast visible detection of aliphatic biogenic amines by a “turn-on” FRET Haoquan Zhong,† Chunchen Liu,§ Wenjiao Ge, † Runcang Sun, † Fei Huang, § and Xiaohui Wang †,* †

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

Guangzhou, 510640, P. R. China. §

State Key Laboratory of Luminescent Materials and Devices, South China University of

Technology, Guangzhou, 510640, China

ACS Paragon Plus Environment

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ABSTRACT: A fluorescent “turn-on” sensor for fast measuring aliphatic biogenic amines (BAs) was developed based on fluorescent carboxylated polyfluorene (PFTBTCOOH)/ chitosan-graftoleic acid (CS-graft-OA) micelles. In this system, biobased CS-graft-OA micelles prepared by graft modification of chitosan with oleic acid serve as the vector of hydrophobic fluorescent probe. Bright “turn-on” fluorescence was achieved by the intermolecular fluorescence resonance energy transfer (FRET) of the encapsulated PFTBTCOOH driven by the formation of electrostatic complex with aliphatic BAs, which facilitate a visual identification. The peaks intensity ratio of the PFTBTCOOH/CS-graft-OA micelles towards increasing amount of aliphatic BAs was calibrated, giving a linear relationship. The fluorescence response of PFTBTCOOH/CS-graft-OA micelles to aliphatic BAs in milk and yogurt matrix reaches a detection concentration as low as 10 µM, showing that PFTBTCOOH/CS-graft-OA micelles solution is a potential chemosensor for fast and selective detecting trace aliphatic BAs, which is of great significance to public health and food safety.

KEYWORDS: amphiphilic chitosan derivative, micelles, carboxylated functionalized polyfluorene, aliphatic biogenic amines, chemosensor


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INTRODUCTION Biogenic amines (BAs) are low-molecular-weight organic bases containing aliphatic, heterocyclic or aromatic groups generated by decarboxylation of amino acids. BAs are widely found in organisms and foods and play vital roles in varieties of biological processes. Among them, aliphatic BAs including putrescine, cadaverine, spermidine and spermine regulate cell growth and differentiation1, and act as biomarkers for health risks, including bacterial infections and cancer.

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In addition, a number of aliphatic BAs, especially cadaverine and putrescine, are

associated with food spoilage, the primary cause of food poisoning.

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Therefore, the selective

detection and identification of aliphatic BAs with rapid, trustworthy methods are important for public health and food safety. It is difficult to detect and identify aliphatic BAs due to their low concentration in the tissue fluid and the lack of chromophores and chromophore in their molecules.7-8 The current widely adopted analytical route is based on “separation and detection” using high-performance liquid chromatography (HPLC), gas chromatography (GC) and capillary electrophoresis (CE), which often requires a long operation time.7-11 Optical methods based on UV-vis absorption or fluorescence are ideal alternatives because they are fast and feasible.12-16 Colorimetric sensors are attractive for enabling visual identification although they have comparably lower sensitivity.17-18 Fluorescent sensors are mostly based on a quenching mode using the electronrich nature of BAs, which are sensitive but susceptible to interference from probe surroundings, especially for the complex food matrix.19 In comparison, ratiometric fluorescent sensing based on the relative value of signal changes at different wavelengths is more sensitive, selective and accurate for BAs detection.15, 20


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Recently, fluorescent conjugated polymers (CPs) with superior sensitivity relative to small molecular receptors and low toxicity have emerged as promise fluorescent probes and been used in the trace detection of a variety of chemical or biological analytes, such as metal ions, small biomolecules, DNA, and proteins.

21-29

However, the widely used CPs suffer from intrinsic

drawbacks like poor water solubility and aggregation-caused quenching, which limited their practical application.

22, 30-31

Recently, water-soluble CPs and CPs nanoparticles are attracting

increasing attention because they offer an efficient path to deal with the problems of poor water solubility and aggregation-caused quenching.32-35 In our previous works, we established a novel way for dissolving and protecting fluorescent CPs from self-quenching in aqueous medium by encapsulation them in self-assembled

amphiphilic polysaccharides micelles.36-42 The

polysaccharide-based micelles demonstrated excellent safety, biocompatibility and stability, wherein the analysts can be enriched and the receptor-analyte interaction is maximized. 43-44

37-39, 41,

Thus, we envision the amphiphilic polysaccharides micells may be a suitable platform for

the fast detection of BAs with CPs probes. Herein, novel water-phase ratiometric fluorescent detection of BAs is demonstrated using selfamplifying CPs encapsulated in chitosan-graft-oleic acid (CS-graft-OA) nanocarriers. Chitosan (CS) is an abundant natural polysaccharide produced industrially from marine chitin with attractive biocompatibility, non-toxicity and biodegradability.45-46 CS-graft-OA was prepared via graft copolymerization of CS with oleic acid (OA) . In order to increase the detection selectivity to BAs, novel carboxylated CPs with blue-emitting polyfluorene (PF) unit and 5% or 15% doped red-emitting carboxylated benzo [2,1,3] thiadiazole (BTCOOH) units are prepared by Suzuki coupling reaction. The carboxylic groups in CPs interact with the added aliphatic amines via electrostatic interaction leading to an enhanced intermolecular fluorescence resonance energy


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transfer (FRET) from PF to BTCOOH units allowing a quantitative ratiometric detection with high sensitivity, as well as a visually noticeable "turn-on" fluorescence response from blue to red. Interestingly, the enhanced FRET doesn’t happen to the aromatic or heterocyclic BAs enabling a discriminative detection to aliphatic BAs. In this context, the carboxylated CPs are incorporated in the CS-graft-OA micelles to facilitate the host-analyte interaction in aqueous media. This carboxylated CPs micelles sensor can not only combine the advantage of visual identification and high sensitivity, but also discriminate aliphatic BAs from other types BAs, which may provide an alternative method to qualitatively and quantitatively detect aliphatic BAs.

EXPERIMENTAL SECTION Materials. Chitosan (Mw: about 50 kDa) was purchased from the Golden-Shell Biochemical Co.,

Ltd.

Oleic

acid

(OA),

N-Hydroxysuccinimide

(NHS)

and

1-ethyl-3-(3-

dimethylaminopropyyl) carbodiimide (EDC) were obtained from Sigma (USA). The amines were purchased from Aladdin and used without further purification. 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-9,9-dioctylfluorene, dioctylfluorene

and

2,7-dibromo-9,9-

4,7-Bis(2′-(5′-bromothienyl))-2-dodecyl-2,1,3-benzotriazole

were

synthesized according to the literature method,47-48 and were purified by recrystallization. All other chemicals used were of analytical grade and used straightly. Pure water for measurements was obtained from a Millipore Milli-Q purification system. Characterization. Fourier transform infrared (FT-IR) spectra were taken on a Bruker TENSOR 27 spectrophotometer at frequencies ranging from 400 cm-1 to 4000 cm-1. 1H-NMR and

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C-NMR spectra were collected on a Bruker Avance 400 MHz spectrometer using

tetramethylsilane as internal standard. Molecular weights of the samples were measured on a


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Waters GPC 2410 using tetrahydrofuran as the eluent. Thermogravimetric (TGA) measurements were carried out with a NETZSCH (TG209F1) analyzer at a heating rate of 20 °C/min under N2 atmosphere. The size and size distribution of the micelles were determined using a Malvern 90 Plus particle size analyzer. The UV-vis absorption spectra were recorded with a HP 8453 spectrophotometer. Photoluminescence (PL) spectra were collected on a Jobin-Yvon spectrometer. All measurements were made at 25 °C. Synthesis of chitosan graft oleic acid (CS-graft-OA). CS-graft-OA was synthesized via the reaction of the amino group of CS with the carboxyl group of OA as seen in Scheme 1a. Briefly, 1.0 g of CS was dissolved in a mixture solution of 100 ml 1 % (w/v) acetic acid (HAc) water solution and 80 ml methanol. 0.3mL OA was added to the CS solution followed by a drop-wise addition of 0.078 g EDC and 0.039 g NHS methanol solution with stirring at room temperature. After reacting for 24 h, the mixture was dialyzed against distilled water using dialysis membranes (Mwco=3500) for 24 h, and then centrifuged to move the precipitation and the supernatant was lyophilized to obtained CS-graft-OA (yield: 53.4%). The FT-IR spectra and 1H-NMR spectrum of CS and the CS-graft-OA copolymers are shown in Figure S1. Comparing with the FT-IR spectrum of unmodified CS, a new absorption peaks at 1708 cm-1 attributed to the stretching vibration of carbonyl group can be found in the spectrum of CS-graft-OA, illustrating the introduction of acyl group. Moreover, the band at 1456, 2929 and 2856 cm-1 corresponds to the C-H bending of aliphatic chain, suggesting the successful synthesis of CS-graft-OA. The presence of the grafted OA side chain is further confirmed by the 1H-NMR spectrum and 13C-NMR spectrum of CS-graft-OA. 1

H-NMR (600 MHz, D2O): δ (TMS, ppm) 8.36(m, H, internal CONH), 8.15(m, 2H, internal

CHCH), 2.78-4.52(m, 9nH, NAG), 2.08- 1.83(m, 28H, internal CH2), 1.82-1.71(m, H, internal


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OH), 1.45- 0.77(m, 4H, terminal CH3).

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C-NMR (151 MHz, D2O): δ (TMS, ppm) 181.42,

100.88, 77.45, 74.86, 74.51, 72.71, 60.11, 56.33, 23.26, 22.12; calculated degree of OA substitution based on 1H-NMR: DSOA, HNMR= (IHc/3)/IH2 = 7.91%. Synthesis of the carboxylated CPs. The carbohxylated CPs were synthesized according to the literature method. 49The sample with 5% BTCOOH doping was denoted as PFTBTCOOH5, and the sample with 15% BTCOOH doping was denoted as PFTBTCOOH15. GPC: PFTBTCOOH5 Mw 26.7 kDa; Mw/Mn 1.63, PFTBTCOOH15 Mw 10.3 kDa; Mw/Mn 1.55. 1H NMR (600 MHz, CDCl3): δ(TMS, ppm) 7.78-7.63-7.61(6H,C-H, PF units), 7.19(4H,C-H, BT units), 2.05, 1.181.07(14H,CH2, PF units), 0.75-0.73(3H,CH3, PF units). The synthetic routes to PFTBTCOOH are illustrated in Figure S2. PFTBTCOOH5 and PFTBTCOOH15 are readily soluble in polar organic solvents, such as DMSO or THF, but not soluble in water. Molecular weight and Electrochemical property and optical property of the conjugated polymers are depicted in Table S1. The thermal stability of copolymers was characterized by TGA (Figure S3) in N2 atmosphere and the maximum decomposition temperatures (Td) of copolymers were about the same temperature at 396oC, showing a good thermal stability. Preparation of the Fluorescent amphiphilic CS-graft-OA micelles. The micelles selfassembled from CS-graft-OA copolymer in water were prepared by mild ultrasonic treatment and passed through a 0.45 µm membrane filter (Millipore) similar to our previous reports.50-51 The critical micelle concentration (CMC) values of CS-graft-OA copolymer was determined by fluorescent spectroscopy by using pyrene probe. The micelle size and size distribution (PDI) were measured by dynamic light scattering (DLS). The morphology of micelles was observed by TEM.


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CPs encapsulating CS-graft-OA micelles were prepared by a method similar to our previous method.

36-40, 43-44

Typically, PFTBTCOOH was firstly dissolved in tetrahydrofuran (THF) with

stirring at room temperature. The CS-graft-OA copolymer was dissolved in pure water to obtain a concentration of 1 mg/mL. Specific amounts of PFTBTCOOH/THF solutions were slowly dropped in the CS-graft-OA micelle aqueous solution under vigorous stirring, and then followed by an ultrasonic treatment for 30 min. In the next, THF was removed from the resulting solution by rotary evaporation at 37 oC. Finally the solution was adjusted to CS-graft-OA concentration of 1 mg/mL and the resultant solution was filtered through a 0.45 µm microfilter to remove nonincorporated PFTBTCOOH. The obtained solution was stored at 4 oC for subsequent analysis. The amount of CPs encapsulated in the micelles was determined by UV-vis with reference to a calibration curve of CPs in THF. The encapsulation efficiency (EE, %) and loading content (LC, %) of CPs in the chitosan micelles were calculated by the following equation, respectively. EE (%) =

LC (%) =

×100% (1) ×100% (2)

Fluorescence titration procedure. The analyte induced aggregation experiments were carried out by the successive addition of amine solution to the CS-graft-OA micelles solution of PFTBTCOOH at room temperature in PBS buffer with pH=7.4. For each assay, the analyte solution was spiked into the probe solution in a Quartz cuvette, shaken for 30 s and stored for 3 min before measuring the fluorescence spectra. The fluorescence lifetime spectra were recorded on a Hamamatsu Quantaurus-Tau Compact fluorescence lifetime (Janpan) (λex=403 nm), and the measurements were conducted under air.


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Determination of spermine in milk and yogurt. Milk and yogurt were cheated according to the literature.52-53 10 g aliquots of fresh milk and yogurt were precisely weighed and mixed with 6 ml of 5% perchloric acid with magnetic stirring. Then, the mixture was centrifuged at 5000 rpm for 10 min and the precipitate was rinsed by 4 mL of 5% perchloric acid before a second round of centrifugation. The supernatants were collected and concentrated to a final volume of 10 mL. For each analysis, a 50µL sample was taken and mixed with different concentrations (050 µM)of spermine. RESULT AND DISCUSSION The self-assembly of amphiphilic CS-graft-OA micelles and the loading of carboxylated conjugate polymer. A simple and versatile method was developed to conveniently disperse hydrophobic CPs molecules in water through the self-assembly of amphiphilic chitosan derivative. As depicted in Scheme 1b, CS-graft-OA copolymers self-assemble into core-shell structured micelles in water with OA side chains as hydrophobic inner cores and chitosan as hydrophilic outer shells. The hydrophobic CPs molecules located at the inner cores of micelles can be protected from aggregation by the outer hydrophilic polysaccharide corona, and thus their water dispersibility is greatly improved. The CMC of CS-graft-OA copolymers was determined to be 0.069 mg/mL by Pyrene fluorescent probe method. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were employed to characterize the self-assembled CS-graft-OA micelles, as shown in Figure 1. The DLS data shows the average hydrodynamic diameter of the CS-graft-OA micelles is 158.9 nm with a PDI of 0.246. TEM shows that the selfassembled CS-graft-OA micelles are nearly spherical with diameters ranging from 100 nm to 120 nm, which are a little bit smaller than the hydrodynamic size due to the shrinkage of micelles during the drying process of TEM observation.


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Scheme 1. (a) Preparation of CS-graft-OA. (b) Schematic illustration of the preparation of fluorescent CS-graft-OA micelles.

Figure 1. The morphology and size distribution of CS-graft-OA micelles determined by (a) DLS and (b) TEM. The hydrophobic CPs PFTBTCOOH can be easily entrapped into the self-assembled CS-graftOA micelles through a mild ultrasonic treatment. The CS-graft-OA micelles show concentrationdependent loading capability towards CPs (Figure 2a,b). The encapsulation efficiencies (EE) of


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CS-graft-OA micelles gradually decrease from 36.97 to 19.62% along with increasing feed concentration of PFTBTCOOH5 from 1 to 5 µg/mL. This indicates that a larger PFTBTCOOH feeding amount would accelerate precipitation resulting in reduced EE value. On the other hand, LC correspond to the exact loading amount of CPs in CS-graft-OA micelles increases from approximately 0.037 to 0.098% upon increasing PFTBTCOOH5 concentration from 1 to 5 µg/mL. PFTBTCOOH15 shows a much higher EE than PFTBTCOOH5 at the same feeding concentration. For example, the EE of PFTBTCOOH15 reaches 88.54% at 1 µg/mL feed concentration, while that of PFTBTCOOH5 is only 36.97%. This phenomenon can be understood by the different molecular size of the two CPs. PFTBTCOOH15 with Mw of 15.9 kDa tends to be entrapped by the CS-graft-OA micelle more easily than the PFTBTCOOH5 with Mw of 43.6 kDa. The influence of CPs loading amount on the particle size and surface proerties of CPs/CSgraft-OA were investigated through DLS. As shown, the size of CPs/CS-graft-OA micelles is obviously smaller than CS-graft-OA micelles, suggesting the loading of CPs in CS-graft-OA micelles is accompanied by a shrinkage of micelle core. In the case of PFTBTCOOH5/CS-graftOA, the micelles particle size significantly decreases from 141 to 108 nm along with increasing CPs concentration from 0.3697 to 0.981 µg/mL. This result demonstrates that the CS-graft-OA micelles containing more CPs have smaller particle size. It is possibly due to aggregate structure is changed with the introduction of CPs.50 With the introduction of CPs, the hydrophobic interaction of the hydrophobic blocks increases, forming a more compact core and thus resulting in a smaller size. The zeta-potential of CPs/micelles with adding CPs loading is also presented in Figure 2. For PFTBTCOOH5/CS-graft-OA, the zeta-potential significantly decrease from approximately +33.5 to +16.7 mV while the loading concentration of CPs is increased from 0 to


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0.981 µg/mL. Similar phenomenon is also observed for PFTBTCOOH15/CS-graft-OA. This is another evidence proving the gradual loading of negatively charged PFTBTCOOH molecules, which balanced the positive charge of chitosan.

Figure 2. (a, b) EE and LC of CS-graft-OA micelles with different feeding amount of CPs. (c, d) Particle size and surface zeta-potential of CPs/CS-graft-OA micelles with different CPs loading amount. Optical properties. The PL and UV-vis spectra of PFTBTCOOH in THF and in CS-graft-OA micelles solution are compared in Figure 3. As shown in Figure 3a, PFTBTCOOH5 in THF exhibit an absorbance peak at 389 nm with a shoulder peak at 473 nm, which may be attributed to polyfluorene (PF) unit. While in CS-graft-OA micelles water solution, the absorption band of PF shows a small red-shift from 389 nm to 402 nm and the shoulder peak at 473 nm is enhanced, which is generally associated with the aggregation or crystalline β-phase formation of


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polyfluorenes.54 The PFTBTCOOH15 in THF has only one absorption band centered at 384 nm probably due to the aggregation of the polymer chains is eliminated by the strong electrostatic repulsion between anionic carboxylate groups. The corresponding PL emission peaks of PFTBTCOOH5 in THF mainly appear at 416 nm, 440 nm and a small peak at 638 nm, while the PL emission profile of PFTBTCOOH5 in the CSgraft-OA micelles water solution exhibiting obviously red-shifted peaks at 437 nm, 467 nm and 655 nm, respectively. The PL profile of PFTBTCOOH15 in THF is similar with that of PFTBTCOOH5 in THF, except for an obviously enhanced emission at 655 nm, which is generally attributed to the BTCOOH unit. zIn contrast, the PL profile of PFTBTCOOH15 in the CS-graft-OA micelle aqueous solution presented red-shifted emission peaks at 437 nm and 700 nm, respectively, and the shoulder peaks originally at 440 nm and 638 nm disappeared. These results should associate with the changed chain conformation driven by the increased inter and/or intra chain interactions of PFTBTCOOH facilitated by the encapsulation of micelles for providing a close proximity.55

Figure3. The UV-vis (a) and PL spectra (λex =380 nm) (b) of PFTBTCOOH in THF (solid lines) and the CS-graft-OA micelle aqueous solution (dash lines).


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Sensing of aliphatic amines. As a proof-of-concept experiment, to better understand the interactions between the carboxylated polymer and diamines, we explored the fluorescence response of PFTBTCOOH/CS-graft-OA micelles to 1, 2-Ethylene glycol (EG) and 1, 2Ethylenediamine (EDA). As shown in Figure 4, the fluorescent PFTBTCOOH/CS-graft-OA micelles are almost unresponsive to EG. However, obvious differences are observed with the addition of EDA. In the case of PFTBTCOOH5/CS-graft-OA (Figure 4c), as the concentration of EDA increases ([EDA] = 0 to 50 µM), the blue emission band of PFTBTCOOH5 at 437 nm exhibits a weak fluorescence quenching along with a progressive growth in the red emission bands at 615 nm and 696 nm. The same phenomenon is observed for the PFTBTCOOH15/CSgraft-OA micelles, except more pronounced growth of red emission at at 696 nm. The changes in PL are so obvious that can be visually observed easily. According to the insets in Figure 4a-d, the solution of PFTBTCOOH5/CS-graft-OA and PFTBTCOOH15/CS-graft-OA show dull yellow and dull red fluorescence under UV light, respectively. They turn a little bit darker in the presence of EG while turn blight orange and blight red in the presence of EDA, respectively, in line with the PL results. The “turn-on” fluorescence response was clearly observable by naked eyes even at a low EDA concentration of 50 µM. The depressed blue emission and enhanced red emisssion in the two cases suggest an increased FRET effect from PF unit to BTCOOH unit happening to the CPs in the micelles. The noticeable FRET effect is probably a result of the electrostatic interactions between the carboxylic groups in CPs and the amino groups in EDA. The formation of electrostatic complex decreases the distance between the PF donor and the BTCOOH acceptor. As a result, efficient FRET happens to the PFTBTCOOH in CS-graft-OA micelles in presence of amines. Since PFTBTCOOH15 contains more carboxyl group than PFTBTCOOH5, PFTBTCOOH15/CS-graft-OA aqueous solution is more sensitive than


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PFTBTCOOH5/CS-graft-OA aqueous solution. The PL changes of PFTBTCOOH5 and PFTBTCOOH15 in THF as a function of increasing EDA concentration were also explored (Figure. 4e, f). It can be seen there are slight PL decrease in the blue region and slight PL increase in the red region. Accordingly, a little bit lighter fluorescence can be observed under UV with the addition of EDA (Figure 4e, f), but the fluorescence change is not obvious in compare with the PFTBTCOOH/CS-graft-OA aqueous system. The significant difference can be explained by the different chain conformations of PFTBTCOOH in micelles solution and THF. Compared with a random-coil conformation in THF solution, the CPs have a more aggregated structure in the micelles, which further facilitate the FRET, as seen in Scheme 2.


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Figure 4. PL spectra of PFTBTCOOH upon titration of EDA and EG in micelles (a,b,c,d) aqueous solution (λex =380 nm) and EDA in PFTBTCOOH THF solution(λex =380 nm) (e,f). The pictures of the PFTBTCOOH micelle solution with 0 and 50µM EG (a,b) and 50µM (c,d) EDA are shown in the inset.

Scheme 2. Sensing mechanism of aggregation-induced FRET of PFTBTCOOH entrapped in CSgraft-OA micelles by aliphatic amines.


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Figure 5. PL spectra of PFTBTCOOH15/CS-graft-OA micellar solutions (λex =380 nm) upon titration of (a) putrescine, (b) cadaverine, (c) spermine, (d) spermidine, (e) tyramine and (f) histamine. The fluorescence reponses of PFTBTCOOH15/CS-graft-OA micellar solutions to aliphatic BAs including putrescine, cadaverine, spermine and spermidine, heterocyclic BAs histamine and acromatic BAs tyramine were investigated and presented in Figure 5. As shown, upon the addition of aliphatic BAs, the blue emission band of the PFTBTCOOH15/CS-graft-OA micellar solution at 437 nm display a slight quenching along with the strong progressive growth of the red


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emission band at 696 nm with an isosbestic point at about 500 nm, proving the enhancement of FRET effect from PF unit to BTCOOH unit.13 Meanwhile, the emission peak at 696 nm in the red region exhibits gradual blue-shift to 677, 673, 658 , 653 nm for putrescine, cadaverine, spermine and spermidine, respectively. This phenomenon can be understood by the difference in molecular chain length of the amines, which may affect the distribution of electron cloud. With the longer chain length, more obvious blue-shift was observed. The chain length of aliphatic BAs also affects the degree of energy transfer. The aliphatic BAs with shorter chain length (e.g. putrescine vs cadaverine) induce stronger red emission band increase, probably due to forming closer complex. It was also noticed that the fluorescence color change induced by spermine and spermidine (Figure 5c,d) was more pronounced, which can be explained by their polyamines structure endowing them stronger binding affinities to -COOH groups. In contrast, the enhanced FRET was not observed with the important aromatic BAs. According to Figure 5e, addition of tyramine resulted in partial fluorescence quenching of both blue emission band and red emission band. The structural hindrance and weaker electrostatic interaction may result in a loser polymer aggregation and thus a non-efficient FRET. The fluorescence quenching is probably caused by the charge-transfer interactions between tyramine and PFTBTCOOH15.19 On the other hand, the blue fluorescence was gradually recovered while the red emission was quenched upon addition of histamine. This phenomemon may be explained by the weakly basic nature of histamine (Table S2 in the SI). In neutral PBS solution, the aliphatic amino group (pKa = 9.80) will be protonated, whereas the second nitrogen of the imidazole ring (pKa = 5.94) will be negatively charged.56 The zeta potential of histamine micelle solution was determined to be -25 mV, suggesting histamine would not induce aggregation, on the contrary it releases the aggregation of CPs in micelles. As a result, the blue emission belong to PF unit was recovered, but the red


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emission was slightly quenched due to the hydrogen bonds between the carboxylic acids and amino groups.57 The proposed mechanism for “turn-on” detection of aliphatic BAs based on aggregation induced enhancement in FRET of PFTBTCOOH is illustrated in Scheme 2. Upon the addition of aliphatic BAs, the PFTBTCOOH15 in CS-graft-OA micelles might be cross-linked via electrostatic interactions and hydrogen bonds between the carboxylic acid in CPs and the amino groups of the aliphatic BAs to form supramolecular aggregate, leading to more efficient FRET from PF segments to BT units and thus resulting in a brighter fluorescence emission. As a result, PFTBTCOOH15/CS-graft-OA micelle solution presents a visible shift in the fluorescence color from blue to orange, enabling a convenient visual identification.

Figure 6. Lifetime decay curves recorded for PFTBTCOOH15/CS-graft-OA in the absence (a) (b) and presence (c) (d) of spermine (50µM). The decay curves were measured under air atmosphere. ( (a) (c): λem =437nm, (b) (d): λem=696nm)


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To further prove the occurrence of FRET from the PF to BTCOOH, we recorded the decay curves of PFTBTCOOH in the aqueous micelles solution before and after adding spermine (Figure 6). With the absence of spermine, the average lifetime of PF (Figure 6a) and BTCOOH (Figure 6b) was determined to be 0.764 ns and 1.325 ns, respectively. With the addition of spermine, the average lifetime of the PF emission peak shows an obvious decrease from 0.764 ns to 0.524 ns (Figure 6c), while an obvious increase from 1.325 ns to 3.186 ns happens to the BTCOOH emission peak (Figure 6d). Generally, the FRET efficiency (E) can be determined by comparing the lifetime of the donor with (τD) and without the presence of the acceptor (τDA) using the relationship E = 1 - [(τDA)/(τD)].58-59 With the addition of spermine, the FRET efficiency was increase from 0.423 to 0.836. This provides another evidence for the enhanced FRET in the sensing system by spermine.

Figure 7. I2/I1 ratio of PFTBTCOOH15/ CS-graft-OA micellar solution against the concentration of (a) spermine, (b) spermidine, (c) putrescine, (d) cadaverine.


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Figure 7 illustrated the plot of the ratiometric emission between two peaks of PFTBTCOOH15/ CS-graft-OA micellar solution against the aliphatic amines concentrations, which denote as the I2/I1 ratio. Over the entire titration process (0–50 µM ), the I2/I1 ratio increases from 0.51 to 2.16, 1.86, 1.58 and 1.28 for spermine, spermidine, putrescine and cadaverine, respectively. The increase of the I2/I1 ratio is propotional to the enhanced degree of aggregation. Spermine showns the highest enhancement of the FRET, followed by spermidne, putrescine, and cadaverine, which seem to be associated with the pKa value of each amine. What’s more, a linear relationship was observed in all cases in the range of 0–50 µM, indicating this system could be potentially used for quantification of the amount of amines in aqueous solution. The minimum detection concentration was determined as low as 10 µM. These results suggest that PFTBTCOOH / CS-graft-OA micelles solution can serve as a general and effective chemosensor for fast detect trace aliphatic amines, which is of great significance to food safety detection. Sensing of spermine in milk and yogurt. To illustrate the utility of this method for real samples, the probe was applied to sense spermine in milk and yogurt. As shown in Figure 8, the PL profiles of the probe in water and in the fresh milk and yogurt extraction are almost overlap, showing the food matrix has little effect to the fluorescent signals. The PL profiles of milk and yogurt extraction with successive addition of spermine show obviously increased I2/I1 ratio, just like the aqueous samples. The calculated spermine concentrations in milk and yogurt extraction based on the plot determined in Figure 7 were comparable to the spiked amount of spermine as shown in Table S3, suggesting this sensing system is possible to detection aliphatic BAs in milk and yogurt.


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Figure 8. PL spectra of PFTBTCOOH15/CS-graft-OA micellar solutions (λex =380 nm) upon titration of (a) milk, (b) yogurt with and without spermine.

CONCLUSIONS In summary, a “turn-on” fluorescent sensing system based on CPs/CS-graft-OA micelles was developed for fast and visiable detection of aliphatic amines. The active materials in this system are fluorescent carboxylated polyfluorenes encapsulated in the self-assembled amphiphilic CSgraft-OA micelles, which tend to identify amines through electrostatic interactions. It was interesting to find that a partial FRET fluorescence of the carboxylated polyfluorenes was observed when they were encapsulated in the micelles, and the FRET was significantly enhanced with the presence of trace amines, giving a visible “turn-on” fluorescence response. The CPs containing more carboxyl groups showed higher sensitivity. We investigated the I2/I1 ratio in respond to the increasing concentration of different aliphatic amines. A linear relationship was obtained with all of the aliphatic amines samples in the concentration range of 0–50 µM, showing this sensing system can also be used for quantitative detect trace aliphatic amines. This is very meaningful for fast food safety detection and health monitoring, because the timeconsuming extraction and separtion steps can be saved.


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ASSOCIATED CONTENT Supporting Information. Supporting Information Available: FT-IR spectra, 1H-NMR spectrum of chitosan (CS) and CSgraft-OA, the preparation and electrochemical, optical properties of PFTBTCOOH and the pKa values of tested amines in water are included. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author E-mail address: [email protected].

∗

Tel./Fax: +86 20 87111861.

ACKNOWLEDGMENT This work was financially supported by the National Science Foundation of China (51673072), the Independent Study Projects of the State Key Laboratory of Pulp and Paper Engineering (2016TS01, 2017C04), the Science and Technology Program of Guangzhou, China (201504010033),

the

Fundamental

Research

Funds

for

the

Central

Universities,

SCUT(2017ZD077) and the National Program for Support of Top-notch Yong Professionals of China.


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