Accepted Manuscript A selective fluorescent and colorimetric dual-responses chemosensor for streptomycin based on polythiophene derivative Minhuan Lan, Weimin Liu, Jiechao Ge, Jiasheng Wu, Jiayu Sun, Wenjun Zhang, Pengfei Wang PII: DOI: Reference:

S1386-1425(14)01464-4 http://dx.doi.org/10.1016/j.saa.2014.09.106 SAA 12782

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

22 May 2014 23 July 2014 24 September 2014

Please cite this article as: M. Lan, W. Liu, J. Ge, J. Wu, J. Sun, W. Zhang, P. Wang, A selective fluorescent and colorimetric dual-responses chemosensor for streptomycin based on polythiophene derivative, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.09.106

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A selective fluorescent and colorimetric dual-responses chemosensor for streptomycin based on polythiophene derivative

Minhuan Lan,a,b Weimin Liu,a* Jiechao Ge,a Jiasheng Wu, a Jiayu Sun,a Wenjun Zhang,b Pengfei Wanga a

Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China. b

Center of Super-Diamond and Advanced Films (COSDAF) and Department of Physics and Materials Science, City University of Hong Kong, Hong Kong SAR (P.R. China). *Prof. Weimin Liu, Tel: +86-10-82543475, Fax: +86-10-82543475, Email: [email protected].

Keywords: Streptomycin

Colorimetric

sensor,

Fluorescent,

Polythiophene,

Conformational

change,

ABSTRACT A colorimetric and fluorescent dual-responses chemosensor (PT3, a water-soluble polythiophene) for streptomycin was designed and synthesized. The structure of PT3 was characterized by using infrared spectroscopy, 1H NMR and gel-permeation chromatography analyses. The conformational change of PT3 induced by streptomycin resulted in the red shift of absorption spectra and fluorescent quenching. Moreover, PT3 showed excellent selectivity for streptomycin over other antibiotics and biomolecules. PT3 could quantificationally detect streptomycin in the range of 2 to 70 µM with a detection limit of 0.2 µM (116 ppb), which is lower than the maximum residue limit defined by World Health Organization (200 ppb).

1. Introduction Antibiotics, such as penicillin, neomycin, and streptomycin have been widely used in veterinary practice for treating the bacterial infections of domestic animals and dairy cattle. They are also used as a pesticide, to combat the growth of bacteria, fungi, and algae. Human also often take antibiotics based drug to treat infectious diseases [1]. Thus, antibiotic residues are often encountered in animal-derived food products, drinking water, and even in human body; these residues are now becoming one of the most serious threats to human health [2, 3]. For example, the emergence of antibacterial-resistant super bacteria which has killed thousands of people around the word arises from abuse of antibiotics. Thus, there is a high demand for a selective and efficient approach to track the residual antibiotics. To date, various analytical methods, such as microbiological methods [4, 5], immunochemical methods [6, 7], high-performance liquid chromatography [8-10], and spectral analysis have been developed to detect antibiotic residues [11, 12]. Among these, spectral analysis such as UV-vis and fluorescence spectroscopy is widely used due to its distinctive advantage, such as high selectivity, high sensitivity, short response time, conveniently and on line detection [13-15]. However, few successful examples were satisfied to practical application. Therefore, development of new approaches for quantitative detection of antibiotics with high sensitivity and selectivity is an important topic from both scientific and technical views. As an emerging sensory material, water-soluble conjugated polythiophenes have been receiving much research interest and have been designed for probing many biological relevant targets, such as LPS, DNA, proteins, ATP and so on [16-21]. The conjugated polythiophenes are able to bind analytes through noncovalent bonding, such as the electrostatic interaction, hydrophobic interaction, hydrogen bonds, which is proved to cause conformational changes [22, 23]. When the conjugated polythiophene chain was in nonplanar state, the absorption wavelength is about 400 nm. However, the absorption wavelength of polythiophene can be red shifted due to more planar conformation of the conjugated chain. Therefore, water soluble polythiophene based chemosensors have the advantages over other conjugated polymers in visual detection [24-27]. Additionally, the conformational changes of the polythiophene chain can also result in fluorescent quenching and a redshift in the emission wavelength after binding the target. Along with our continuing efforts in the exploration of chemosensors for the selective

detection of antibiotics based on both small molecule and polythiophenes [28-30], herein, we reported poly(3-thiophene acetic acid) (PT3) as a colorimetric and fluorescent dual-responses chemosensor for streptomycin. The details of streptomycin binding characteristics of the PT3 have been investigated by UV-vis and fluorescence spectroscopy. The selective and sensitive of PT3 toward streptomycin arise from the strong electrostatic interaction bonds between carboxyl group and guanidyl group. The limit of detection was calculated to be 0.2 µM (116 ppb), which is lower than maximum residue limits defined by World Health Organization (200 ppb) [31]. 2. Experimental 2.1. Materials and general methods Dry FeCl3, 3-thiophene acetic acid, phosphate, pyrophosphate, adipic acid, aspartic acid, and glutamic acid were purchased form Alfa Aesar. Carbenicillin, chloromycetin, penicillin, neomycin, erythromycin, ampicillin, and streptomycin were purchased from INALCO. Other reagents were purchased from Beijing Chemical Regent Co. All reagents and chemicals were AR grade and used directly without further purification unless otherwise noted. CHCl3 was distilled from CaH2 under nitrogen. The water was purified by Millipore filtration system. All UV-vis and fluorescence spectra in this work were recorded in Hitachi U3010 and Hitachi F4500 fluorescence spectrometers at 25 oC. All spectral characteristics were carried out in DMF/ HEPES buffer (5/95, v/v) solution. The stock solution of PT3 was prepared in DMF (1.0 × 10-3 M), stored at room temperature in the dark before use. The antibiotics solutions (1.0 × 10-2 M) were prepared in HEPES buffer solution. To a quartz cell (1 cm of optical path length) filled with 2 mL of PT3 (DMF/HEPES buffer (5/95, v/v) solution) was added with the stock solution of antibiotics dropwise using a micro-syring. The volume of these added antibiotics stock solutions was less than 100 µL to remain the concentration of PT3 unchanged. All pH measurements were made with a Sartorius basic pH-meter PB-10. 1H NMR (400 MHz) spectrum was determined on a Bruker Advance-400 spectrometer with chemical shifts reported as ppm (tetramethylsilane as internal standard). The gel-permeation chromatography (GPC) was performed using Polystyrene as the standard, and THF was employed as eluent. FT-IR spectrum in KBr was collected on a Varian Excalibur 3100 FTIR spectrometer. 2.2. Synthesis 6.6 g dry FeCl3 was dissolved in 30 mL of dry CHCl3 under nitrogen, and then 1.42 g

3-thiophene acetic acid dissolved in 20 mL dry CHCl3 was added dropwise. The reaction mixture was stirred at room temperature for 2 days. The resulting precipitate was collected, wash with methanol, and finally dried under vacuum to give the desired polythiophene as a brown solid (0.85 g, Yield: 59.8%). 1H NMR (400 MHz, NaOD-D2O, TMS, ppm): δ 3.23~3.82 (br), 6.83~7.32 (br). IR (KBr pellet, cm-1) 3430, 2924, 1706, 1628, 1398, 1200. Gel-permeation chromatography analysis (GPC): Mn=32,952 g mol-1, PDI=1.67.

3. Results and discussion 3.1. The design and synthesis of chemosensor PT3 Water-soluble polythiophene-based chemosensors transduced by simply conformational change upon binding target molecule provide an ideal platform for the design of colorimetric sensors [32, 33]. A streptomycin molecule bears two guanidyl groups with pKa values up to 13.5, which means that the groups can bear positive charge in the aqueous solution [34]. Moreover, the extreme affinity of carboxyl group to guanidyl group over other cation could improve the selectivity and specificity of polythiophene-based chemosensors for streptomycin [31, 35]. Based on the above considerations, we designed a simple but selective and sensitive chemosensor for streptomycin sensing on the basis of strong electrostatic interaction between carboxyl-modified polythiophene and streptomycin. As shown in scheme 1, the conformation of PT3 in aqueous solution (5% DMF) is freed disorderly. Upon complexation with streptomycin, the electrostatic interaction between carboxyl and guanidyl groups may induce the conformational change in the polythiophene chain. Meanwhile, the binding can also restrict the rotation of two thiophene units, resulting in more rigid, rod-like amphiphilic macromolecules and thus enhancing the propensity of aggregate formation in aqueous solution. These conformational changes will significant affect the absorption spectrum and fluorescence spectrum. PT3 was prepared via an oxidative polymerization under nitrogen in the presence of FeCl3 in 59.8% yield. The structure of PT3 was characterized by IR, 1H NMR and GPC analyses.

Streptomycin Rigid, rod-like amphiphilic state

Free disorderly S

S

S

S

S

n

HOOC

PT3

n

HOOC

HOOC NH

NH

HN C NH2

H2N C NH OH HO

HOOC NH

HOOC NH HN C NH2

H2N C NH OH HO

OH

CHO

CHO

OH O OH O CH2OH NHCH3

OH O OH O CH2OH NHCH3

OH

OH

O O

O O

OH

Scheme 1. Molecular structures of PT3, streptomycin, and the proposed interaction mechanism between PT3 and streptomycin.

3.2. Spectral characteristics To get an insight into the interaction between PT3 and streptomycin, UV-vis spectral measurement of PT3 (20 µM, calculated on monomers basis) were carried out with the addition of incremental concentration of streptomycin in HEPES buffer solution (10 mM, pH 6.8, 5% DMF) at room temperature. As shown in Fig. 1a, the UV-vis spectrum of PT3 in HEPES solution shows an absorption band from 300 to 500 nm, which corresponds to the polythiophene band. Upon incremental addition of streptomycin (0-70 µM), the absorption intensity gradually decreased with red shift and finally displayed a broader peak at 425 nm. This red shift along with hypochromism may be attributable to the streptomycin-induced conformational change of the conjugated chain through the electrostatic interaction. The sensing process of PT3 to streptomycin could be evaluated quantitatively by analyzing the dependence of the absorbance intensity ratio (A490/A403) on the concentration of streptomycin. As shown in Fig. 1a, the (A490/A403) ratio increased gradually upon addition of streptomycin. By plotting the (A490/A403) ratio versus the logarithm of streptomycin concentration, a good linear relationship was obtained for the streptomycin concentration ranging from 2 to 70 µM. (Fig. 1b, inset). Based on that, the limit of detection was calculated to be 0.2 µM (equal to 116 ppb), which is lower than the maximum residue limits (MRL) defined by food administrations (200 ppb).

Fig. 1. (a) Absorption spectra of PT3 (20 µM) in the presence of different concentration of streptomycin (0 to 70 µM) in HEPES buffer solution (10 mM, pH 6.8, 5% DMF). (b) The changes of the absorbance intensity ratio of PT3 (A490/A403) vs. the streptomycin concentration range from 0 to 70 µM in HEPES buffer solution (10 mM, pH 6.8, 5% DMF). The inset in panel shows linear relationship between intensity ratio (A490/A403) and the logarithm of streptomycin concentration in the range from 2 to 70 µM.

The corresponding fluorescence spectra are shown in Fig. 2a. The PT3 solution in the absence of streptomycin exhibits a strong kelly emission peaked at 560 nm when excited at 485 nm. However, the fluorescence of PT3 solution was quenched significantly with increasing streptomycin concentration. Particularly, the fluorescence intensity at 560 nm started quenching steadily and slight red-shift to 565 nm. Meanwhile, the fluorescent color of the mixture solution was changed from kelly to yellow. Such variations in the spectra are associated with the streptomycin-induced conformational change due to the electrostatic interaction. By plotting the

FL intensity at 560 nm versus the concentration of streptomycin, a good linear relationship with streptomycin concentration from 0 to 15 µM was obtained (Fig. 2b). However, we prefer to use absorbance spectra as the quantitative detection due to its ratiometric changes and little influence on environment.

Fig. 2. (a) Fluorescence spectra of PT3 (20 µM) in the presence of different concentration of streptomycin (0 to 70 µM) in HEPES buffer solution (10 mM, pH 6.8, 5% DMF). The inset in panel shows the fluorescent images of PT3 (20 µM) solution with streptomycin solution of 0 and 70 µM at room temperature. (b) The changes of the FL intensity at 560 nm of PT3 aqueous solution vs. the streptomycin concentration range from 0 to 70 µM in HEPES buffer solution.

3.3. Selectivity Achieving high selectivity toward the target over the other competitive species coexisting in the sample was a very important feature to evaluate the performance of a chemosensor. To further study the practical application of this sensor, the selectivity of PT3 was studied by monitoring ratio A490/A403 response in the presence of other antibiotics and biologically important species in HEPES buffer solution. As depicted in Fig. 3 (black column), at the same concentration as

streptomycin (70 µM), other antibiotics including: carbenicillin, chloromycetin, penicillin, neomycin, erythromycin and ampicillin, did not induce significantly change of the absorption spectra in the absence of streptomycin. By contrast, only the addition of an equimolar amount of streptomycin increased A490/A403 to 0.7. Concerning the antibiotics were used in animals or other living body where is rich in anionic biomolecules and protein, thus phosphate, pyrophosphate, adipic acid, aspartic acid, and glutamic acid were used as the potential competitive species. The results showed that these anionic species could not induce obvious change of the value of A490/A403. Furthermore, we also conducted the competition experiments in streptomycin detection which was mixed with the above competitive species. Significant increase of the value of A490/A403 were observed upon addition of streptomycin regardless of the presence of competing species (Fig. 3, red column), which demonstrate that the sensing streptomycin by PT3 is not affected by other species. The outstanding selective and specificity can be attributed to the extreme affinity of carboxyl group to guanidyl group through electrostatic interaction as illustrated in Scheme 1.

Fig. 3. Selectivity of PT3 (20 µM) to streptomycin (70 µM) over competing some antibiotics and other important biological species in 10 mM HEPES buffer solution (10 mM, pH 6.8, 5% DMF).

3.4. pH effect In addition, the influence of pH on the absorbance of PT3 in the absence and presence of streptomycin was investigated. As shown in Fig. 4, the value of A490/A403 of PT3 was increased upon increasing the pH value either in absent or present of streptomycin. However, under basic

conditions, as expected, the probe did not show good response as that under acidic conditions probably due to the less protonation of guanidyl groups. This is why we choose pH value 6.8 as the optimized condition.

Fig. 4. Effect of pH on the absorbance intensity ratio (A490/A403 ) of free PT3 (20 µM) (black stars) and PT3 (20 µM) / streptomycin (6 µM) mixtures (red dots) at room temperature.

4. Conclusion In summary, a water-soluble polythiophene-based chemosensor was synthesized and it exhibited fluorescent and colorimetric dual-responses to streptomycin at submicromole level (0.2 µM) with high selectivity. Addition of streptomycin to PT3 solution shows large fluorescence quenching and red shift in both absorption and emission spectra. The selective and sensitive towards streptomycin of the sensor was demonstrated to originate from the good affinity of carboxyl group to guanidyl group through electrostatic interaction, resulting in significant conformational change of the conjugated polymer chain.

Acknowledgements The work was supported by the NNSF of China (Grant Nos. 21373250 and 61227008) and the Chinese Academy of Sciences.

References [1] S. Selwyn, R. W. Lacey, M. Bakhtiar, The beta-Lactam Antibiotics: Penicillins and Cephalosporins in Perspective. Hodder and Stoughton, London, 1980. [2] UK Ministry of Agriculture, Fisheries and Food. A Review of Antimicrobial Resistance in the Food Chain, UK Department of Environment, Food and Rural Affairs, London, 1998. [3] World Health Organization (WHO). WHO Annual Report on Infectious Disease: Overcoming Antimicrobial Resistance, World Health Organization, Geneva, Switzerland, 2000. [4] S. E. Charm, R. Chi, J. Assoc. Off. Anal. Chem. 71 (1988) 304-316. [5] D. J. Everest, R. Jackman, L. Thorne, S. J. Everest, Analyst 119 (1994) 2727-2729. [6] E. Märtlbauer, E. Usleber, E. Schneider, R. Dietrich, Analyst 119 (1994) 2543-2548. [7] H. Font, J. Adrian, R. Galve, M. C. Estévez, M. Castellari, M. Gratacós-Cubarsi, F. Sánchez-Baeza, M. P. Marco, J. Agric. Food Chem. 56 (2008) 736-743. [8] J. Haginaka, J. Wakai, Analyst 110 (1985) 1185-1188. [9] H. Abbasi, K. E. Hellenäs, Analyst 123 (1998) 2725-2727. [10] K. Basavaiah, K. Tharpa, N. R. Prasad, S. G. Hiriyanna, K. B. Vinay, Indian J. Chem. Techn. 16 (2009) 265-271. [11] Z. H. Alrawi, S. Tabaqchali, Antimicrob. Agents. Ch. 20 (1981) 25-29. [12] P. H. Chan, H. B. Liu, Y. W. Chen, K. C. Chan, C. W. Tsang, Y. C. Leung, K. Y. Wong, J. Am. Chem. Soc. 126 (2004) 4074-4075. [13] Q. Dai, W. M. Liu, X. Q. Zhuang, J. S. Wu, H. Y. Zhang, P. F. Wang, Anal. Chem. 83 (2011) 6559-6564. [14] V. Kumar, E. V. Anslyn, J. Am. Chem. Soc. 135 (2013) 6338-6344. [15] D. Kraskouskaya, M. Bancerz, H. S. Soor, J. E. Gardiner, P. T. Gunning, J. Am. Chem. Soc. 136 (2014) 1234-1237. [16] M. H. Lan, J. S. Wu, W. M. Liu, W. J. Zhang, J. C. Ge, H. Y. Zhang, J. Y. Sun, W. W. Zhao, P. F. Wang, J. Am. Chem. Soc. 134 (2012) 6685-6694. [17] K. P. R. Nilsson, O. Inganäs, Nat. Mater. 2 (2003) 419-424. [18] H. A. Ho, M. Béra-Abérem, M. Leclerc, Chem. Eur. J. 11 (2005) 1718-1724. [19] Y. L. Tang, F. D. Feng, F. He, S. Wang, Y. L. Li, D. B. Zhu, J. Am. Chem. Soc. 128 (2006) 14972-14976.

[20] C. Li, M. Numata, M. Takeuchi, S. Shinkai, Angew. Chem., Int. Ed. 44 (2005) 6371-6374. [21] Z.Yao, X. Feng, W. Hong, C. Li, G. Shi, Chem. Commun. (2009) 4696-4698. [22] H. A. Ho, A. Najari, M. Leclerc, Acc. Chem. Res. 41 (2008) 168-178. [23] M. H. Lan, W. M. Liu, Y. Wang, J. C. Ge, J. S. Wu, H. Y. Zhang, J. H. Chen, W. J. Zhang, P. F. Wang, ACS Appl. Mater. Interfaces 5 (2013) 2283-2288. [24] T. M. Swager, Acc. Chem. Res. 31 (1998) 201-207. [25] D. T. McQuade, A. E. Pullen, T. M. Swager, Chem. Rev. 100 (2000) 2537-2574. [26] S. W. Thomas, G. D. Joly, T. M. Swager, Chem, Rev. 107 (2007) 1339-1386. [27] S. Wang, Chin. Polym. Bull. 10 (2011) 120-129. [28] L. T. Zeng, W. M. Liu, X. Q. Zhuang, J. S. Wu, P. F. Wang, W. J. Zhang, Chem. Commun. 46 (2010) 2435-2347. [29] M. H. Lan, W. M. Liu, J. C. Ge, J. S. Wu, H. J. Wang, W. J. Zhang, Y. F. Bi, P. F. Wang, Chem. Commun. 48 (2012) 6818-6820. [30] G. Y. Liu, M. H. Lan, W. M. Liu, J. S. Wu, J. C. Ge, H. Y. Zhang, Chin. J. Polym. Sci. 31 (2013) 1484-1490. [31] J. Y. Sun, J. C. Ge, W. M. Liu, Z. Y. Fan, H. Y. Zhang, P. F. Wang, Chem. Commun. 47 (2011) 9888-9890. [32] X. L. Feng, L. B. Liu, S. Wang, D. B. Zhu, Chem. Soc. Rev. 39 (2010) 2411-2419. [33] C. L. Zhu, L. B. Liu, Q. Yang, F. T. Lv, S. Wang, Chem. Rev. 112 (2012) 4687-4735. [34] A. Gobbi, G. Frenking, J. Am. Chem. Soc. 115 (1993) 2362-2372. [35] A. Echavarren, A. Galan, J. M. Lehn and J. De Mendoza, J. Am.Chem. Soc. 111 (1989) 4994-4995.

GRAPHICAL ABSTRACT

Streptomycin Rigid, rod-like amphiphilic state

Free disorderly S

S

S

S

S

n

HOOC

PT3

n

HOOC

HOOC NH

NH HN C NH2 H2N C NH OH HO OH

HOOC

NH NH HN C NH2 H2N C NH OH HO OH

O O

O

OH O O CH2OH NHCH3

OH

O

CHO

CHO OH

HOOC

OH

OH O O

CH2OH NHCH3 OH

HIGHLIGHTS A new polythiophene-based chemosensor for streptomycin has been developed. PT3 displays colorimetric and fluorescent dual-responses toward streptomycin. The sensing mechanism may be the conformational change of PT3 induced by streptomycin binding.