Chinese Journal of Chemical Physics  2016, Vol. 29 Issue (6): 749-753

#### The article information

Jun Luo, Kai-yu Fu, Hai-yan Dong, Dao-yong Chen

Self-suspended Pure Polydiacetylene Nanoparticles with Selective Response to Lysine and Arginine

Chinese Journal of Chemical Physics, 2016, 29(6): 749-753

http://dx.doi.org/10.1063/1674-0068/29/cjcp1605094

### Article history

Accepted on: May 12, 2016
Self-suspended Pure Polydiacetylene Nanoparticles with Selective Response to Lysine and Arginine
Jun Luo, Kai-yu Fu, Hai-yan Dong, Dao-yong Chen
Dated: Received on May 3, 2016; Accepted on May 12, 2016
The State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, Shanghai 200433, China
*Author to whom correspondence should be addressed. Hai-yan Dong, E-mail:donghy215@fudan.edu.cn; Dao-yong Chen, E-mail:chendy@fudan.edu.cn
Abstract: We demonstrate a very convenient access to self-suspended pure poly (10, 12-pentacosadiynoic acid) (PDA) nanoparticles (NPs) simply by adding the ethanol solution of diacetylene monomer to water, followed by UV irradiation. The as-obtained PDA NPs are of high purity because no any initiator, catalyst or stabilizer was used during the whole process. The stabilizer-free PDA NPs are stable in the aqueous suspension. Due to the high purity and stability, the PDA NPs can respond sensitively and selectively to lysine and arginine among 18 kinds of water soluble natural amino acids; without the competitive interaction from the stabilizer, the sensitivity was enhanced.
Key words: Polydiacetylene    Selective response    Lysine    Arginine
Ⅰ. INTRODUCTION

Polydiacetylenes have attracted great interests because of their unique optical and electrical properties [1, 6]. One of the most interesting properties of polydiacetylenes is their chromatic change in response to environmental variations [7]. It has been reported that polydiacetylenescan undergo a color transition from blue to red in response to different types of external stimuli, such as heat [8, 9], pH [10, 11], electric field [12], magnetic field [13], mechanical stress [14], chemical solvent [15], and chemical/biological analytes [16]. This visible color change leads to their potential applications for visional detection of various analytes [17, 18].

Natural amino acids play vital roles in many biological processes [19, 21]. As one of the essential amino acids, lysine has significant effect on the metabolic functions of animals and humans, such as adjusting Krebs-Henseleit cycle and the somatotropic hormone in the human body [22, 23]. Arginine makes a great contribution to cell division, wounds healing, ammonia removal, immune function, hormones release, gene regulation, and glycoprotein targeting [24]. Therefore, selective detection of lysine and arginine is significant.

In our previous work, we prepared the glycine stabilized poly (10, 12-pentacosadiynoic acid) (PDA) nanoparticles (NPs), which responded selectively to Pb2+ and Ag+ [25]. The interaction between the PDA NPs and metallic ions are relatively strong. Therefore, in this system, glycine was required not only for stabilizing the PDA NPs, but also for weakening the interaction between the PDA NPs and metallic ions, leading to the selective response to the ions that can interact with the NPs most strongly. Herein, we report preparation of self-suspended pure PDA NPs for selective detection of natural amino acids; no stabilizer was used based on the consideration that existence of a stabilizer will lower the sensitivity since the interactions between PDA NPs and amino acids are very weak. In the present study, the PDA NPs were prepared by simply adding the ethanol solution of 10, 12-pentacosadiynoic acid (DA) monomer to water, followed by UV irradiation for its topochemical polymerization. The as-prepared PDA NPs have a high purity because there are no any initiator, catalyst or stabilizer used during the preparation. The NPs selectively respond to lysine and arginine among 18 kinds of water soluble natural amino acids, and the stabilizer-free feature of the NPs enhanced the sensitivity considerably.

Ⅱ. EXPERIMENTS

The diacetylenemonomer 10, 12-pentacosadiynoic acid (DA, CH3(CH2)$_{11}$-C$\equiv$C-C$\equiv$C (CH2)8-COOH) was purchased from GFS Chemicals, Inc., and absolute ethanol from Sinopham Chemical Reagent Co., Ltd., China. All the amino acids used in this work (L-lysine (Lys), L-arginine (Arg), L-histidine (His), L-glutamic acid (Glu), L-threonine (Thr), L-valine (Val), L-aspartic acid (Asp), L-leucine (Leu), L-asparagine (Asn), L-alanine (Ala), L-cysteine (Cys), L-glutamine (GLn), glycine (Gly), L-serine (Ser), L-proline (Pro), L -phenylalanine (Phe), L-methionine (Met), L-isoleucine (Ile)) were bought from J & K Scientific Ltd., China.

DA monomer were firstly dissolved in ethanol at 1 mmol/L and then filtered through 0.45 µm filter to remove residual polymer. After that, 10 mL DA ethanol solution was added into 90 mL water under stirring at 25 °C. Then the suspension was kept still at room temperature for 20 h followed by UV exposure at 254 nm for 30 s, resulting in blue PDA.

Sizes of DA and PDA nanoparticles were measured with dynamic light scattering (DLS) using ALV-5000 laser scattering spectrometer ($\lambda$=632.8 nm). Morphologies of DA and PDA were characterized by transmission electron microscopy (TEM), which was performed on a JEOL 2000FX TEM. Color transition was recorded with Ultraviolet-Visible Spectroscopy (UV-Vis) by Shimadzu UV-2550 spectrophotometer.

Ⅲ. RESULTS AND DISCUSSION A. Preparation of PDA nanoparticles with long stability

We first added 10 mL DA ethanol solution into 90 mL water under stirring at 25 °C, and the DA suspension exhibited opalescent. DLS measurements revealed that the hydrodynamic radius of freshly prepared DA NPs was 106 nm, and increased with the aging time (as shown in Fig. 1) and then precipitated after 90 h. TEM observation indicated their morphology was spherical, with diameter particle size growing from~20 nm to~60 nm (Fig. 1).

 FIG. 1 Hydrodynamic radius distributions and TEM images of DA nanoparticles with different aging time.

The difference between the sizes measured by DLS and those by TEM is remarkable, which can be explained by the following two reasons: (ⅰ) for a suspension, the sizes measured by DLS were Zeta-averaged, so that the large particles made a bigger contribution to the size than the smaller ones [26]; (ⅱ) the particles shrunk during drying process [27] resulted in smaller particle size in the TEM images. Similar phenomenon was frequently reported in literature [31, 32].

It is known that for the topochemical polymerization, DA molecules must be regularly packed [28]; sufficiently long aging time is required for the regular packing of DA molecules in the suspension. However, as exhibited in Fig. 1(a), a long aging time may result in too large particles or precipitates. DA suspension with a proper aging time should be selected. The DA suspensions at different aging times were topochemically polymerized. As shown in Fig. 2(a), after the topochemical polymerization, the suspension at the aging time of 20 h has a dark blue color. It is also exhibited in the UV-Vis spectra (Fig. 2(b)) that absorbance intensity of the suspension at aging time of 20 h is much stronger than that of the suspension at aging time less than 20 h.

 FIG. 2 (a) The photographs and (b) UV-Vis spectra of PDA suspensionprepared from the corresponding DA suspensions of different aging time.

Considering that the particles in the suspension at the aging time of 20 h are of a proper size (Fig. 1 and Fig. 3), and that the relatively strong UV-Vis absorbance can give sufficient color contrast when used as a sensor, we selected the polymerized suspension with the aging time of 20 h for further study (denoted as PDA-20). The dark blue color of PDA-20 reveals sufficient regular-packing of DA molecules in the suspension at the aging time of 20 h. We also tracked the polymerized suspensions by DLS measurements. The results demonstrate that, after the polymerization, PDA NPs become stable in the suspension. As exhibited in Fig. 3, no remarkable change in the particle size was detected in the suspension PDA-20 during the storing. The relatively long time stability of PDA NPs is beneficial for their potential application as a sensor [29]. Additionally, Zeta potential of the PDA NPs in PDA-20 was measured to be -24.8 mV. Obviously, the carboxyl groups of PDA partially ionized in the system. The hydrophilicity of the carboxyl groups and the electric repulsion between the NPs are responsible for the stabilization of the PDA NPs in the suspension.

 FIG. 3 (a) Hydrodynamic radius distributions of PDA NPs after storing for different time, (b) TEM images of PDA NPs after storage of 60 h.
B. Selective response of PDA-20 to lysine and arginine

Eighteen kinds of water soluble natural amino acids were used to test the chromatic change of PDA-20. The UV-Vis spectrum shows that the pure PDA-20 has a maximum absorption peak at 650 nm and a shoulder peak at 600 nm, presenting blue appearance. When we added different concentrations of lysine and arginine, the color of the suspension changed to purple/red. In UV-Vis spectra, the maximum absorption peaks shifted to 550 nm (Fig. 4 (A)-(D)). The detection limit could be as low as 10 µmol/L (Fig. 4 (A)-(D)). But for other 16 kinds of the amino acids, the color changed slightly or did not change even the concentration of amino acids reached 5 mmol/L (Fig. 4(E)). In the UV-Vis spectra of the mixtures of PDA-20 with the respective other 16 kinds of the amino acids, no remarkable changes were detected even concentration is 5 mmol/L (Fig. 4(E), (F) ).

 FIG. 4 Photographs of (A) from 1 to 4: PDA-20 with 1 mmol/L, 100 µmol/L, 10 µmol/L, 1 µmol/L Lys and 5: pure PDA-20; (B) from 1 to 4: PDA-20 with 1 mmol/L, 100 µmol/L, 10 µmol/L, 1 µmol/L Arg and 5: pure PDA-20; (C) from 1 to 16: PDA-20 with 5 mmol/L His, Glu, Thr, Val, Asp, Leu, Asn, Ala, Cys, Gln, Gly, Ser, Pro, Phe, Met, Lle and 17: pure PDA-20. UV-Vis spectra of (D) PDA-20 with different concentrations of Lys; (E) PDA-20 with different concentrations of Arg; (F) PDA-20 with 5 mmol/L of other 16 kinds of amino acids, (a)-(p) His, Glu, Thr, Val, Asp, Leu, Asn, Ala, Cys, Gln, Gly, Ser, Pro, Phe, Met, Lle.

The chromatic change of PDA NPs after adding lysine and arginine is attributed to the change of PDA chain induced by the interaction between the carboxyl group on PDA side chain and the amino acids, resulting in a blue shift in absorption and the color change from blue to purple/red. In our previous work [25], we prepared glycine stabilized PDA NPs which selectively respond to Pb2+ and Ag+, in which the glycine played a vital role in both stabilizing the PDA NPs and weakening the interaction between PDA NPs and metal ions, so that only the Pb2+ and Ag+ which have strongest interaction with PDA NPs could be selectively detected.

However, in the present study, when the glycine stabilized PDA NPs were used for detection of the amino acids, no apparent chromatic change was observed by either naked eyes or UV-Vis spectrometer. As indicated in Fig. 5, mixing the glycine stabilized PDA NPs with lysine (Fig. 5 (a), (b)) or arginine (Fig. 5(c), (d) ) at the concentration of the amino acid up to 1 mmol/L (Fig. 5 (a), (c)) did not result in the chromatic change. Obviously, the interaction between PDA NPs and amino acids is weak. In the presence of glycine, the interaction between glycine and PDA competes and thus further weakens the interaction between PDA and other amino acids. This can explain why the pure PDA NPs are necessary for the sensitive detection of lysine and arginine. The selective detection of lysine and arginine by the pure PDA NPs can be understood by considering the fact that, among 18 kinds of water soluble natural amino acids, lysine and arginine have the highest pI (pH at the isoelectric points, 9.74 and 10.76, respectively [30]), i.e., they were with the most positive charges under the conditions for the interaction with PDA-20. Since the interaction between PDA NPs and the natural amino acids are weak, only lysine and arginine that have much more positive charges can interact with the negatively charged PDA NPs with the sufficient interaction strength to cause the chromatic change of PDA NPs.

 FIG. 5 Photographs of (a) from 1 to 4: PDA-Gly with 1 mmol/L, 100 µmol/L, 10 µmol/L, 1 µmol/L Lys and 5: PDA-Gly; (b) from 1 to 4: PDA-Gly with 1 mmol/L, 100 µmol/L, 10 µmol/L, 1 µmol/L Arg and 5: PDA-Gly. UV-Vis spectra of (c) PDA-Gly with different concentrations of Lys and (d) PDA-Gly with different concentrations of Arg.
Ⅳ. CONCLUSION

Pure poly (10, 12-pentacosadiynoic acid) nanoparticles (PDA NPs) were successfully prepared by non-covalently connected micelle method, simply by adding the ethanol solution of diacetylene monomers to water, followed by the topochemical polymerization via UV irradiation. The product has high purity because there is no any initiator, catalyst, or stabilizer used during the whole process. The stabilizer-free PDA NPs makes the nanoparticles sensitive to lysine and arginine that interact weakly with the nanoparticles, which benefits the promising application of PDA NPs as biosensors.

Ⅴ. ACKNOWLEDGMENTS

This work is supported by the National Natural Science Foundation of China (No.21334001 and No.91127030).

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