A colorimetric and fluorometric polydiacetylene biothiol sensor based on decomposition of a pyridine-mercury complex

Journal Pre-proof A Colorimetric and Fluorometric Polydiacetylene Biothiol Sensor Based on Decomposition of a Pyridine-Mercury Complex Jong Pil Lee, Fadilatul Jannah, Kwangmin Bae, Jong-Man Kim

PII:

S0925-4005(20)30118-0

DOI:

https://doi.org/10.1016/j.snb.2020.127771

Reference:

SNB 127771

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

11 November 2019

Revised Date:

22 January 2020

Accepted Date:

22 January 2020

Please cite this article as: Lee JP, Jannah F, Bae K, Kim J-Man, A Colorimetric and Fluorometric Polydiacetylene Biothiol Sensor Based on Decomposition of a Pyridine-Mercury Complex, Sensors and Actuators: B. Chemical (2020), doi: https://doi.org/10.1016/j.snb.2020.127771

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A Colorimetric and Fluorometric Polydiacetylene Biothiol Sensor Based on Decomposition of a Pyridine-Mercury Complex Jong Pil Lee,a Fadilatul Jannah,a Kwangmin Baea and Jong-Man Kima,b* a

Department of Chemical Engineering, Hanyang University, Seoul 04763, Korea

b

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Institute of Nanoscience and Technology, Hanyang University, Seoul 04763, Korea

Email: [email protected]

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AUTHOR INFORMATION *Corresponding Author

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Highlights

A new strategy for colorimetric and fluorometric biothiol detection was developed based

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

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E-mail address: [email protected]

on decomposition of a pyridine-mercury complex. 

A mercury complexed pyridine-containing polydiacetylene (PDA) displayed a brilliant

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blue-to-red color change and a turn-on fluorescence in the presence of biothiols. 

Incorporation of the sensor in electrospun nanofibers led to an enhanced sensitivity.



The sensor system was found to be more selective for cysteine than for homocysteine and glutathione due to the size effect.



The strategy developed in this study should be applicable to the design of other thiol-

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specific sensors that utilize pyridine-containing conjugated molecules.

Abstract

Biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) participate in numerous cellular functions and physiological processes. Because changes in the levels of these biological important substances are associated with various clinical disorders and diseases,

methods for their selective sensitive and quantitative detection are in high demand. In the current study, we developed a new, selective and sensitive colorimetric and fluorometric biothiol chemosensor that is comprised of a mercury-complexed, pyridine-containing polydiacetylene (PDA). Owing to its high affinity toward sulfur, biothiols promote decomposition of the pyridinemercury complex in the PDA in conjunction with colorimetric and fluorescence changes.

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Specifically, upon addition of biothiols the new chemosensor undergoes a blue-to-red color transition and generates red fluorescence. Moreover, incorporation of the chemosensor in

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electrospun PEO nanofibers leads to an enhanced sensitivity for biothiol detection. The strategy

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developed in this study should be applicable to the design of other thiol-specific sensors that utilize pyridine-containing conjugated molecules.

1. Introduction

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Keywords: polydiacetylene, colorimetric, fluorometric, biothiol, electrospinning

Biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) participate in

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numerous cellular functions and physiological processes. Alternations in the levels of these biothiols can lead to various clinical disorders and diseases. [1-4] For example, a deficiency in Cys, whose plasma levels typically range from 135 to 300 µM, [5] causes a growth rate decrease,

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liver damage, edema, lethargy, loss of muscle and skin lesions. Moreover, elevated levels of Cys correlate with neurotoxicity. [6-8] On the other hand, elevated concentrations of Hcy, a condition termed hyperhomocysteinemia, is an independent risk factor for major pathologies, and the development of dementia and Alzheimer’s disease.[9] GSH has normal plasma levels that are typically in the 1-6 µM range.[10] It is also known that GSH plays an important role in maintaining redox homeostasis for cell growth by controlling the oxidative stress levels. [11] As a consequence,

alternations in the level of GSH are directly linked to various diseases such as cancer, Alzheimer’s and cardiovascular disease. [12-15] Owing to the important functions that biothiols perform in living systems, increasing attention has been given to methods for their detection. Thus far, protocols developed for sensing biothiols have been based mainly on analytical methods such as chromatography, capillary

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electrophoresis, mass spectrometry, and electrochemistry. [16-18] However, the use of these techniques is time-consuming, and it requires that manipulations be performed in instrumentally

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well-equipped laboratories by trained technicians. For this reason, sensor systems that are based

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on colorimetric and fluorometric methods have received special attention because they do not require the use of special instrumentation and, in many cases, they enable naked eye detection with

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modestly high levels of sensitivity and selectivity. [19,20] The vast majority of

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colorimetric/fluorometric biothiol chemosensors developed thus far take advantage of the strongly nucleophilic nature of the thiol group. These sensor systems frequently utilize nucleophilic addition or substitution reactions of thiols at electron-deficient carbons in conjugated molecules to

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bring about changes in absorption and/or emission spectroscopic properties. [21-23] We have carried out several studies aimed at developing applications of polydiacetylene (PDA)-based smart colorimetric and fluorometric functional materials. [24-31] PDAs are

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conjugated polymers that are prepared by topochemical polymerization of self-assembled diacetylene monomers (DAs). [32] Because changes in their absorption and emission properties are promoted by molecular stimulation, ligand-modified PDAs have been extensively used as fundamental components of colorimetric and fluorometric sensors for chemically, environmentally and biologically important target molecules. [33-50] Although numerous PDA based sensors have been described, to the best of our knowledge those that target biothiols have not been developed.

During the course of recent efforts in this area, we formulated a new and intriguing proposal for the design of a sensor for biothiols that is based on a polydiacetylene (PDA) as the sensor matrix. Owing to the high affinity between mercury and sulfur, we believed that it should be possible to fabricate a mercury-complexed, pyridine-containing polydiacetylene (PDA) that would undergo decomposition in the presence of biothiols in a manner that would induce changes in

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absorption and emission spectroscopic properties. Specifically, based on the known formation of Py-Hg(II)-Py complexes, [51,52] we envisaged that the pyridine (Py)-containing diacetylene, DA-

Furthermore,

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expected that

self-assembly

of DA-Hg-DA

followed

by

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HgBr2.

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Py (Scheme 1a), would react to form the complex DA-Hg-DA (Scheme 1b) when treated with

photopolymerization would yield the biothiol sensor Poly(DA-Hg-DA) (Scheme 1c). Hg(II) has

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very high affinities towards biothiols. [53] Thus, we anticipated that following their penetration

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into Poly(DA-Hg-DA) biothiols would bind to mercury ions and cause rupture of the polymer matrix in a way that would bring about changes in the absorption and emission properties of the

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PDA backbone (Scheme 1d).

(a) DA DA-Py

HgBr2

(b) DA-Hg-DA

self-assembly and polymerization

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(c) Poly(DA-Hg-DA)

blue-colored PDA

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RSH

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(d) Thiol Reaction

red-colored PDA

Scheme 1. Schematic for formation of Poly(DA-Hg-DA) and reaction involved in the blue-tored color transition promoted by thiols. Structures of the (a) pyridine-containing diacetylene DAPy, (b) mercury-bridged bisdiacetylene DA-Hg-DA, (c) self-assembled and polymerized DA-

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Hg-DA, and (d) red-colored PDA generated by reaction with thiols. As described below, the results of a study designed to assess these proposals showed that

the pyridine mercury complexed PDA, Poly(DA-Hg-DA), on paper undergoes a brilliant blue-tored color change and fluorescence turn-on upon exposure to thiols. In addition, we observed that

the colorimetric transition is specific to biothiols and that immobilization of Poly(DA-Hg-DA) on electrospun nanofiber results in an increase in biothiol detection sensitivity.

2. Experimental section 2.1 Materials 10,12-Pentacosadiynoic acid was purchased from GFS chemicals (USA). 4-Aminopyridine,

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mercury(II) bromide, L-cysteine, DL-homocysteine, L-glutathione, polyethyleneoxide (PEO) (200,000

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g mol-1) and phosphate buffered saline were obtained from Sigma-Aldrich (Korea). NMR spectra were recorded on a Varian Unitylnova (USA) (300 MHz for 1H and 13C) spectrometer at 298 K in CDCl3.

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Olympus BX 51W/DP74 (Japan) was used to obtain optical and fluorescence microscopic images. SEM

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images were obtained using a HORIBA EX-250 (Japan) operated at a beam energy of 15kv. Raman spectra were recorded on a LabRAM HR Evolution Raman spectrometer (Horiba Scientific, 785 nm

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laser source). UV-vis absorption spectra were recorded using an USB2000 miniature fiber-optic spectrometer (Ocean optics, USA). IR spectra were recorded on a Thermo Nicolet NEXUS 470 FTIR

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using an ATR accessory (Thermo Fisher Scientific, Inc, USA). High resolution electrospray ionization mass spectrometry (HR-ESI MS) data were acquired using a Q-TOF mass spectrometer (SYNAPT G2, Methanol). XRD spectra were recorded with a minFlex600 (Rigaku, Germany). ESR200R2V

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(NanoNC) was used for fabrication of electrospun fiber.

2.2 Synthesis.

Synthesis of N-(pyridin-4-yl) pentacosa-10,12-diynamide (DA-Py). A propylphosphonic anhydride (3.20 mmol, 50% wt) in 2 mL of ethyl acetate was added

to a solution containing 10,12-pentacosadiynoic acid (1.00 g, 2.67 mmol), triethylamine (300 µL, 2.16 mmol) and 4-aminopyiridine (0.30 g, 3.20 mmol) in 15 mL of anhydrous dichloromethane.

The resulting mixture was stirred overnight at room temperature. The mixture was washed with brine (3x 10 mL), dried (Na2SO4), filtered and the filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography on silica gel (50% ethyl acetate/hexane) to yield DA-Py (1.1 g, 90 %). IR (KBr): ν = 715, 815, 997, 1160 1207, 1290, 1326, 1379, 1415, 1469, 1511, 1600, 1704,

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2848, 2915, 3151, 3234 (cm-1). 1H NMR: (300MHz, CDCl3): δ = 0.88 (t, 3H), 1.26-1.56 (m, 32H), 2.25 (t, 2H), 2.44 (m, 4H), 7.44 (d, 2H), 8.53 (d, 2H). 13C NMR (75 MHz, CDCl3): δ 171.9, 150.7,

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144.8, 113.4, 77.2, 65.2 37.8, 31.9, 29.6, 29.5, 29.4, 29.3 29.1, 29.0, 28.8, 28.7 28.3, 28.2, 25.2,

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22.7, 19.2, 19.1, 14.1; MS (HR-ESI-MS, m/z): calcd. for C30H46N2O [M+H]+ 450.36 found,

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451.3681.

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Synthesis of [(C12H26C≡C−C≡CC9H20C(O)NH-4-C5H4N)2 (HgBr2)] (DA-Hg-DA). HgBr2 (0.04 g, 0.11 mmol) was added to a solution of DA-Py (0.10 g, 0.22 mmol) in 3 mL of methanol. After stirring for several minutes, the formed precipitate was collected by filtration, and

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dried under vacuum to yield 120 mg (80%) DA-Hg-DA . IR (KBr): ν = 723, 829, 1022, 1095, 1160, 1211, 1330, 1382, 1434, 1463, 1515, 1589, 1614, 1702, 2846, 2923 (cm-1). 1H NMR: (300MHz, CDCl3): δ = 0.88 (t, 6H), 1.26-1.56 (m, 64H),

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2.26 (t, 4H), 2.26 (t, 4H), 2.45 (m, 8H), 7.61 (d, 4H), 7.95 (s, 2H), 8.52 (d, 4H).

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C NMR (75

MHz, CDCl3): δ 172.4, 148.7, 147.1, 114.1, 77.7, 65.2, 65.1 37.8, 31.9, 29.6, 29.4, 29.3, 29.1, 29.0, 28.8, 28.6, 28.3, 28.2, 25.0, 22.6, 19.1, 14.1 MS (HR-ESI-MS, m/z): calcd. for C60H92BrHgN4O2 [M+H]+ 1181.61 found 1181.6072

2.3 Fabrication of Paper Type Sensor.

A 0.5 cm diameter round-shaped filter paper was prepared using a puncher. A 5 µL portion of DA-Hg-DA solution in tetrahydrofuran (10.0 mg/mL) was dropped on to the filter paper and photopolymerization was carried out by UV irradiation (254 nm, 1 mW/cm2) for 70 s.

2.4 Electrospinning of DA-Hg-DA and Polyethyleneoxide (PEO).

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A solution of chloroform and tetrahydrofuran (3:1 by volume, 4 mL), containing DA-Hg-DA (50 mg) and PEO (100 mg, Mw = 200,000 g mol−1), was delivered (0.5 mL/h using a KD Scientific

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model 200 series syringe pump) through a 25 G metal syringe needle at to which a high voltage

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(10 kV) was applied. The formed nano/microfibers were collected on the surface of a grounded aluminum plate (the working distance between the tip of the needle and the collector was 15 cm).

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The as-spun fibers were dried in air for an hour and then subjected to photopolymerization by UV

2.5 Sensor Study.

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light (254 nm, 1 mW/cm2) irradiation for 3 min.

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The paper type sensor described above was immersed into 10 mL of 0.05 M solutions of each amino acid in phosphate-buffered saline for 30 min. Alternatively, a 5 uL solution of each amino acid solution was dropped onto an electrospun fiber. The colorimetric responses (CR) of

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the sensor were calculated using the following equations, where Ablue is the absorbance at 650 nm and Ared is the absorbance at 540 nm.

Fluorescence intensities were quantitatively determined using the Adobe Photoshop program. Images taken by Fluorescence microscope have a red intensity between a minimum of 0

and a maximum of 255.

3. Results and Discussion 3.1 Preparation of Polydiacetylene containing Pyridine-Mercury Complex The pyridine-containing diacetylene DA-Py (Scheme 1) was readily prepared by amide

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bond-forming coupling of commercially available 10,12-pentacosadiynoic acid (PCDA) with 4aminopyridine. The mercury complexed diacetylene monomer DA-Hg-DA was generated in high

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yield by treatment of DA-Py with HgBr2. In order to investigate the photopolymerization process,

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DA-Hg-DA was immobilized on a filter paper by using a drop-casting method (see Experimental Section). Irradiation of the monomer on the paper with 254 nm UV light (1 mW/cm2) for 70 s

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resulted in the generation of a blue-colored polymer (Fig. 1a), which strongly indicates that

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formation of Poly(DA-Hg-DA) had occurred. As determined by analysis of powder X-ray diffraction data (Fig. 1b), Poly(DA-Hg-DA) has a highly ordered lamellar structure with d-spacing of 36.5 Å. The peak at 2θ =21.6° (d = 4.1 Å) corresponds to the interchain distance between two

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monomer units. Raman spectroscopic analysis (Fig. 1c, top showed that the spectrum of the monomer DA-Hg-DA contains peaks (C≡C (2112 cm-1) and C=C (1458 cm-1) bond stretching) associated with Poly(DA-Hg-DA) even before it is subjected to UV irradiation, indicating its high

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sensitivity toward the light. In addition, the Raman peak of DA-Hg-DA at 2268 cm-1 almost completely disappears after UV irradiation for 70 s (Fig. 1c, bottom). A saturation curve for UV-

induced polymerization of DA-Hg-DA is displayed in Fig. 1d. It is evident from the inspection of

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a curve that the degree of the polymerization reaches nearly maximum value after 60 s irradiation.

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Fig. 1. (a) Color change of DA-Hg-DA before (top) and after (bottom) UV irradiation (1 mW/cm2, 70 s). (b) Powder XRD spectrum of Poly(DA-Hg-DA). (c) Raman spectra of DA-Hg-DA before (black line) and after (blue line) UV irradiation (1 mW/cm2, 70 s). (d) Absorbance at 640 nm as a

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function of UV irradiation time for DA-Hg-DA.

Studies were conducted to assess the use of Poly(DA-Hg-DA) as a thiol sensor. First, the

PDA-immobilized filter paper was exposed to 100 ppm of various amines and thiols. As can be seen by inspection of Fig. 2a, the sensor paper undergoes a clear color transition from blue-to-red only in the presence of thiols. The process responsible for the chromic transitions was explored using 1H NMR spectroscopy. In Fig. 2c are displayed aromatic regions of 1H NMR spectra of DA-

Py, DA-Hg-DA, butanethiol-treated DA-Hg-DA and the HBr salt of DA-Py. By viewing the spectra, it can be seen that a large downfield shift of a pyridine ring proton occurs in the 1H NMR spectrum of DA-Hg-DA when butanethiol is present. This change suggests that a pyridinium ion is formed by HBr protonation of the decomplexed pyridine moiety in the PDA (Fig. 2b). This conclusion is supported by observations that the chemical shift of the pyridine ring proton in the H NMR spectrum and frequencies for bands in the solid-state FTIR spectrum (Fig. 2d) of the

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product resulting from reaction of DA-Hg-DA with butanethiol are similar to those of the closely

100 ppm

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Butylamine

TEA

Butanethiol

Dodecanethiol Thiophenol

(d) DA-Py Transmittance

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(c)

Pyridine

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(b)

Decylamine

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(a)

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related pyridinium salt prepared by treatment of DA-Py with HBr.

DA-Hg-DA

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DA-Hg-DA + Thiol

DAPy-HBr salt 1800

1600

1400

1200

-1

Wavenumber (cm )

Fig. 2. (a) Photographs of Poly(DA-Hg-DA) coated filter papers upon exposure to 100 ppm of amines and thiols. (b) Schematic representation of the thiol-induced destruction of pyridinemercury complex. (c) 1H NMR (CDCl3) and (d) FT-IR spectra of DA-Py, DA-Hg-DA, butanethiol treated DA-Hg-DA and independently prepared DA-Py HBr salt.

3.2 Colorimetric and Fluorometric Detection of Biothiols Next, we determined if the thiol-specific color transition of the Poly(DA-Hg-DA) impregnated paper can be promoted by the biothiols cysteine (Cys), homocysteine (Hcy) and glutathione (GSH). The initial phase of this study focused on assessing whether Cys-induced

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promotes a blue-to-red color change of the polymer impregnated paper. Inspection of the photographic images displayed in Fig. 3a shows that among the 20 naturally occurring amino acids

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only Cys (1 mM in PBS buffer, pH 7.4) causes a color change. In Fig. 3d has displayed a plot of

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the Cys concentration dependent increase of the CR value of the PDA-immobilized paper, which shows that the sensor has a calculated Cys detection limit of 51 µM. This value should be

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contrasted with the average level of biothiols in human plasma that is ca. 50-100 µM. In addition,

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the Cys-induced color transition is accompanied by the generation of red fluorescence (Fig. 3b). Moreover, Raman spectroscopy was utilized to investigate the effect of thiol exposure on the eneyne conjugated structure of Poly(DA-Hg-DA). As can be seen by viewing the spectra in Fig. 3c,

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the untreated PDA displays peaks corresponding to C≡C and C=C bond stretching at 2102 cm-1 and 1460 cm-1, respectively. After exposure of Poly(DA-Hg-DA) to Cys, the C=C bond stretching band shifts to higher to a higher frequency of 1522 cm-1 and a shoulder associated with C≡C bond

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stretching appears at 2129 cm-1. Importantly, these Raman spectral changes are typical of those for PDAs undergoing blue-to-red transitions. More information about the process responsible for the Cys-induced colorimetric transition of Poly(DA-Hg-DA) was gained from analysis of the free Hg2+ content of a solution produced by dipping the PDA impregnated paper in a PBS buffered Cys solution. The results showed that the concentration of free Hg2+ in the solution increases in a manner that is expected for thiol promoted decomposition of the Py-Hg2+-Py complex (Fig. S1).

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Fig 3. (a) Photographs and (b) fluorescence microscope images of Poly(DA-Hg-DA) after exposure to 1 mM of various amino acids in PBS buffer solution (pH 7.0). (c) Raman spectra of Poly(DA-Hg-DA) before (black line) and after (red line) exposure to 1 mM of cysteine. (d) Color

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response (CR) value of Poly(DA-Hg-DA) as a function of cysteine concentration.

3.3 Sensitivity Enhancement via Electrospinning Method.

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Immobilizing a PDA sensor system onto a three-dimensional platform typically increases the surface area of the system that consequently leads to an enhancement of sensing performance. [51-54] To test this proposal, we prepared Poly(DA-Hg-DA)-immobilized electrospun nanofibers. A schematic for the electrospinning method used is displayed in Fig. 4a and an SEM image of the generated Poly(DA-Hg-DA)-immobilized PEO nanofibers is shown in Fig. 4b. In order to demonstrate that the immobilized PDA nanofiber platform is a more sensitive sensing system, the

intensity of fluorescence emitted from the nanofibers was determined as a function of Cys concentration (10-90 µM). Inspection of the plot of the data (Fig. 4c) demonstrates that a strong

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linear and positive correlation exists between Cys concentration and emission intensity.

Fig. 4. (a) Schematic representation of the preparation of Poly(DA-Hg-DA)-embedded electrospun fibers. (b) SEM image of the Poly(DA-Hg-DA)-embedded electrospun fibers. (c) Plot

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of relative fluorescence intensities of Poly(DA-Hg-DA) fiber as a function of cysteine concentration (10-90 μM). (d) Graph of relative fluorescence intensities of Poly(DA-Hg-DA) paper type (white bars) and electrospun fiber (red bars) as a function of cysteine concentration (1090 μM).

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To compare sensitivities, changes in the relative fluorescence intensities brought about by

the addition of various Cys concentrations (50, 70 and 90 µM) to Poly(DA-Hg-DA) impregnated paper vs. an electrospun fiber were determined (Fig. 4d). Bar graphs of the data clearly show that the Poly(DA-Hg-DA) complex immobilized on an electrospun nanofiber has a higher sensitivity than the immobilized on paper. Because PDAs immobilized on paper tend to form aggregates that

prevent analyte accessibility, the large increase in the sensitivity caused by immobilized the Poly(DA-Hg-DA) complex on an electrospun nanofiber is presumably a consequence of a significant increase in Cys access to the Py-Hg2+-Py center in the PDA complex.

Finally, the biothiol selectivity (Cys, Hcy vs. GSH) of the Poly(DA-Hg-DA)-immobilized electrospun nanofiber sensor was assessed. Inspection of the plots relative intensities of

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fluorescence from the sensor as a function of biothiol concentration in the 10-90 µM range shows

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that the emission efficiencies increase as the concentrations of Cys, Hcy and GSH increase. Moreover, the detection limits for the respective bithiols are 2.97, 20.5 and 33.0 µM, respectively

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(Fig. S2). The higher sensitivity for Cys compared to those for Hcy and GSH is presumably a result

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of the smaller size of the former substance that enables it to enter the highly ordered polymer

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matrix in a more facile manner.

Fig. 5. Plots of relative fluorescence intensities of electrospun Poly(DA-Hg-DA) fiber vs. concentrations of Cys (black), Hcy (red) and GSH (blue). 4. Conclusion In the effort described above, which was carried out to assess a new strategy for the design of a mercury ion sensor, we synthesized the mercury complexed pyridine-

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containing diacetylene monomer DA-Hg-DA. Furthermore, we showed that this DA-HgDA can be drop casted on a cellulose acetate filter paper and then photopolymerized to

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generate blue colored Poly(DA-Hg-DA) on paper. The PDA impregnated paper undergoes

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a distinct blue-to-red color change in the presence of alkyl-, aryl- and biothiols, which was shown to be associated with decomposition of the pyridine mercury complex Py-Hg2+-

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Py within Poly(DA-Hg-DA). The sensor more sensitively detects Cys than it does the two

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other biothiols, Hcy and GSH, as a consequence of a size effect on access to the Py-Hg2+Py site. A more sensitive biothiol sensor can be created by incorporation of Poly(DA-HgDA) into PEO by using an electrospinning method. The pyridine-mercury complex-based

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strategy developed in this study should be useful in the design of new colorimetric and fluorometric thiol sensors.

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Notes

The authors declare no competing financial interest.

Acknowledgments This study was supported financially by the National Research Foundation (NRF) of Korea (NRF-2017R1A2A1A05000752 and 2012R1A6A1029029).

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:

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