Simultaneous and visual detection of cysteamine based on Michael addition reaction with polydiacetylene liposomes

Article abstract of DOI:10.1039/d0tc02721a

In this study, we report the fabrication of a highly sensitive colorimetric and fluorometric sensor comprising self-assembled polydiacetylene (PDA) liposomes for the measurement of cysteamine concentration. The N-maleimidomethanol (HM) moiety was used as the Michael addition acceptor, which reacts with the thiol groups in cysteamine to generate maleimide-thiol conjugation resulting in the conformational transition of the conjugated backbone. Furthermore, this chemosensor system displays a clear blue-to-red colorimetric transition in the presence of cysteamine among various biothiols with high selectivity and sensitivity owing to the structural specificity of cysteamine. The resulting PDA solutions were analyzed via FE-SEM, UV-vis, fluorescence and Raman spectroscopy, and chromoisomerism for naked-eye visualization. This chemosensor provides a convenient method for the detection of cysteamine in aqueous solution and in real samples. This journal is

Full text of DOI:10.1039/d0tc02721a

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Simultaneous and visual detection of cysteamine
based on Michael addition reaction with
polydiacetylene liposomes†
Cite this:DOI: 10.1039/d0tc02721a
a
ab
Thanh Chung Pham,
Hyun Sung Kim *b and Songyi Lee*ab
Seongman Lee,a Ye Rim Son,b Minseok Kwak,
In this study, we report the fabrication of a highly sensitive colorimetric and fluorometric sensor comprising
self-assembled polydiacetylene (PDA) liposomes for the measurement of cysteamine concentration. The
N-maleimidomethanol (HM) moiety was used as the Michael addition acceptor, which reacts with the thiol
groups in cysteamine to generate maleimide–thiol conjugation resulting in the conformational transition of
the conjugated backbone. Furthermore, this chemosensor system displays a clear blue-to-red colorimetric
transition in the presence of cysteamine among various biothiols with high selectivity and sensitivity owing
to the structural specificity of cysteamine. The resulting PDA solutions were analyzed via FE-SEM, UV-vis,
fluorescence and Raman spectroscopy, and chromoisomerism for naked-eye visualization. This
chemosensor provides a convenient method for the detection of cysteamine in aqueous solution and in real
samples.
Received 8th June 2020,
Accepted 3rd August 2020
DOI: 10.1039/d0tc02721a
rsc.li/materials-c
using several analytical techniques, including high-performance
liquid chromatography (HPLC) with electrochemical,6
Introduction
Intracellular thiols, such as cysteine (Cys), homocysteine (Hcy), fluorescence7 and UV/Vis-absorbance detection,8 capillary
glutathione (GSH) and cysteamine (Cyst), play a crucial role in electrophoresis,9 and gas chromatography with flame photo-
cellular growth, metabolism, and the maintenance of biological metric detection.10 Generally, these techniques have been used
systems.1 However, abnormal concentrations of thiols are in combination in order to increase the sensitivity and selec-
implicated in a variety of health conditions, such as liver tivity of the detection; however, this makes the procedure more
damage, skin lesions, delayed growth and edema.2 Therefore, complex and elaborate besides being time-inefficient and
it is highly important to report changes in thiol concentrations laborious. Recently, Singh et al. reported the detection of Cyst
via real-time monitoring. Cysteamine is an aminothiol com- via UV/Vis spectroscopy using polydentate aromatic nano-
pound (HS–CH2–CH2–NH2) synthesized endogenously via the particles complexed with Cu2+ 11
In addition, Pathak et al.
.
degradation of coenzyme A and converted to the neurotrans- and Kim et al. designed a N-doped carbon12 and a three main
mitter hypotaurine by cysteamine dioxygenase.3 Cyst has been component-containing long chain13 through one-photon and
used as a therapeutic agent for the treatment of cystinosis, two-photon fluorescent probes for Cyst detection, respectively.
cystic fibrosis, neurodegenerative disorders such as Hunting- However, most of these methods and materials were developed
ton’s disease and Parkinson’s disease, and nonalcoholic fatty for the analysis of biological fluids with complicated sample
liver disease.4 Cysteamine hydrochloride (HS–CH2–CH2–NH2ꢀ pretreatments, such as derivatization. To our knowledge, no
HCl) is also used in cosmetics as an antioxidant, a hair studies have performed the quantification of Cyst using a
straightening agent, and a hair waving agent. However, recent colorimetric sensor system.
studies in Europe and Japan reported that Cyst acts as an
Conjugated polymer systems are an enabling class of mate-
allergen affecting hairdressers.5 Biothiols have been analyzed rials for various functions such as sensing matrices,14 organic
semiconductors,15 microelectronics,16 and drug delivery sys-
a Department of Industry 4.0 Convergence Bionics Engineering, Pukyong National
University, Yongso-ro, Nam-gu, Busan 48513, Republic of Korea.
E-mail: slee@pknu.ac.kr
tems (DDS).17 Polydiacetylenes (PDAs) are well-known conju-
gated polymers, which have been extensively investigated and
utilized as an attractive platform in sensing applications due
to their unique optical properties.18 The monomers of PDA
easily self-assemble into liposomal structures in aqueous
solution. PDAs can be generated via UV or g irradiation, or by
b Department of Chemistry, Pukyong National University, Busan 48513,
Republic of Korea. E-mail: kimhs75@pknu.ac.kr
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0tc02721a
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plasma treatment of self-assembled diacetylene monomers.19 UV-Visible spectrophotometer (Jasco) at room temperature.
No catalysts or initiators are required for the polymerization, Fluorescence emission spectra were obtained using an F-7000
which ensures that the PDAs are produced with a high degree of fluorescence spectrophotometer (Hitachi High-Tech). The
purity. PDA undergoes a color shift from blue to red upon structures and morphologies were analyzed with a field emis-
environmental stimulation, accompanied by fluorescent transi- sion scanning electron microscope (FE-SEM, CX-200, COXEM,
tion. The stimulus-induced blue-to-red transition and the Republic of Korea; JEM-2100F, JEOL).
fluorescence enhancement of PDA facilitate the development
Synthesis of N-(2-hydroxymethyl)maleimide (HM). Male-
of various chemosensors using the polymer. The dual signal imide (1.0 g, 10.30 mmol) and 36% aq. formaldehyde
generation is mainly attributed to the interfacial perturbation (3.16 mL, 41.20 mmol) were dissolved in 10 mL deionized water.
of PDA caused by external stimuli, which subsequently induce a The mixture was stirred and heated to reflux for 4 h. The solution
conformational change in the PDA-conjugated backbone. was cooled to room temperature, then extracted with EtOAc,
To date, a variety of PDA-based sensors have been developed dried over Na2SO4, and concentrated in a vacuum to obtain a
for the detection of changes in temperature20 and mechanical white residue (yield: 59.2%) with the following spectral details:
force,21 as well as various analytes.22 Moreover, the PDA- 1H NMR (400 MHz, DMSO-d6) d 7.07 (s, 2H), 6.29 (s, 1H), 4.77
containing sensing systems have been adapted to various (s, 2H); 13C NMR (101 MHz, DMSO-d6) d 171.16, 135.46, 60.13. ESI
architectural constructs such as liposomes, fibers, films, and HRMS m/z = 150.0163 [M + Na]+, calc. for C5H5NO3 = 127.03.
organic–inorganic hybrids, which are used as matrices in
Synthesis of PCDA–HM22b
. Oxalyl chloride (0.7 mL,
biosensing applications.23 A popular strategy to promote ideal 5.51 mmol) was added dropwise to a CH2Cl2 (20 mL) solution
sensing properties has been to introduce special receptors into containing 10,12-pentacosadiynoic acid (PCDA) (0.5 g, 1.34 mmol)
the PDA system. In this approach, the receptor moiety is under N2 at room temperature. After 1 h of stirring, DMF (0.1 mL)
covalently linked to a monomeric diacetylene acid, followed was added to the solution. The resulting mixture was stirred for
by direct self-assembly. Recently, Kim et al. have designed a 4 to 6 h, followed by concentration under reduced pressure to
colorimetric and fluorometric polydiacetylene sensor based on obtain PCDA–Cl, which was used directly without any purifica-
the decomposition of a pyridine-mercury complex.24 However, tion by dissolving in THF (30 mL). To this mixture was added a
the PDA impregnated paper undergoes a blue-to-red color solution containing 125 mg (0.98 mmol) of HM and 0.4 mL
change in the presence of alkyl-, aryl- and bio-thiols, which (4 mmol) of triethylamine in 10 mL of THF. The resulting
has not been expected for selective sensors.
solution was stirred overnight at room temperature under N2.
In the current study, we developed a simple and effective After evaporating the liquid, the crude product was dissolved
analytical method for the selective and sensitive quantification with H2O, extracted with EtOAc and purified via silica gel
of Cyst using a conjugated PDA–HM sensor system carrying the column chromatography (hexane–EtOAc : 1–1) to obtain a white
1
receptor for cysteamine. The reaction of Cyst and the HM- solid (yield: 29.2%) with the following spectral data: H NMR
moiety in the polymer network based on Michael addition (400 MHz, chloroform-d) d 6.80 (s, 2H), 5.52 (s, 2H), 2.27
reaction and the structural specificity of Cyst play an important (t, J = 7.5 Hz, 2H), 2.24–2.17 (m, 4H), 1.57 (p, J = 7.5 Hz, 2H),
role in the color change from blue to red as well as in the 1.52–1.43 (m, 4H), 1.40–1.31 (m, 4H), 1.23 (d, J = 4.9 Hz, 24H),
spectral results of PDA–HM. To the best of our knowledge, this 0.88–0.83 (m, 3H); 13C NMR (101 MHz, chloroform-d) d 169.04,
study reports an unprecedented Cyst sensor with simultaneous 134.89, 60.26, 33.89, 32.01, 29.74, 29.72, 29.71, 29.58, 29.45,
and visual detection.
29.20, 29.11, 29.01, 28.95, 28.82, 28.44, 28.37, 24.65, 22.79,
19.29, 19.27, 14.23. ESI HRMS m/z = 484.3420 [M + H]+, calc.
for C30H45NO4 = 483.33.
Preparation for the diacetylene assembly and
photopolymerization20. The PCDA–HM monomer (9.67 mg,
0.02 mmol) was dissolved in DMF (1 mL) and injected into
Experimental
Materials and methods
We purchased 10,12-pentacosadiynoic acid (PCDA), oxalyl 19 mL of deionized water while shaking the mixed solution to
chloride, maleimide, and formaldehyde from TCI, South Korea. yield a total monomer concentration of 1 mM. The sample was
All organic solvents used in the synthesis were obtained from then sonicated at 80 1C for 40 min. The resulting solution was
Sigma-Aldrich and used without further purification. Real filtered through cotton, yielding a filtrate that was cooled down
samples were purchased from a drugstore, including ‘Biomed to 4 1C for 12 h. Polymerization was carried out at room
– Aloe moisture protein BB PER’, BVI – Ceramide Clinic Perm’, temperature by irradiating the solution with 254 nm UV light
‘Nico Nico – Blue smart wave’, and ‘Nico Nico – Red smart (1 mW cmꢁ2) for 10 min.
wave’, all of which contain cysteamine, and ‘Biomed – Amino
Characterization of PDA–HM liposomes. Before and after
acid L.P.P. Perm’ that does not contain cysteamine. Flash treatment with Cyst, the morphologies of PDA liposomes were
chromatography was carried out on silica gel (230–400 mesh), characterized via FE-SEM. Samples were freshly prepared and
followed by the determination of 1H and 13C NMR spectra using dropped on small fragments of silicon wafers, which were dried
a Bruker Advance 400 MHz and 600 NMR spectrometer. Mass at room temperature overnight. The samples were then coated
spectra were obtained using a maXis HD (Bruker). UV absorp- with 3–4 nm-thick Pt for 40 s before FE-SEM examination at an
tion spectroscopy measurements were conducted using a V-730 accelerating voltage of 15 kV. The Raman spectra were recorded
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using an NRS-5100 (Jasco) Raman microscope with 785 nm
radiation.
Results and discussion
Design and synthesis of the monomer and preparation of the
polymer
The chemosensor was designed according to the following
principles: (1) the chromatic transition of polydiacetylene is
very sensitive to terminal perturbations, and (2) maleimide–
thiol conjugation via Michael addition occurs under mild
conditions. The synthetic route of the monomer PCDA–HM is
depicted in Scheme 1. The reaction of maleimide and formal-
dehyde afforded a detection group HM with a 59.2% yield.25 In
addition, the HM moiety reacted with PCDA to yield PCDA–HM
as a white solid with a 29.2% yield, followed by column
chromatography with hexane : EtOAc = 50 : 50 and further treat-
Scheme 2 Self-assembly and polymerization of PCDA–HM and the
schematic illustration of Cyst analysis using PDA liposomes.
ment using Sephadex-LH 20 with chloroform : MeOH
=
60 : 40.22b The synthesized PCDA–HM monomer was character-
1
ized using H NMR, 13C NMR and ESI HR-MS (ESI†).
spherical particles with a diameter of B200 nm (Fig. 1a and
Fig. S8, ESI†). The addition of Cyst (100 mM) induced structure-
less images, possibly due to the aggregation, as presented in
Fig. 1b. The conjugated backbone of PDA–HM liposomes was
subjected to Raman spectroscopy, since the chromatic transi-
tion of PDA from blue to red is highly related to the conforma-
tional change of the PDA backbone. To obtain an insight into
the polymer backbone of PDA–HM liposomes in response to
Cyst, Raman spectra were then recorded before and after the
treatment with Cyst at room temperature (Fig. 1c). The char-
acteristic CRC and CQC stretching frequencies of blue phase
PDAs appear at 2120 and 1516 cmꢁ1, which shift to 2079 and
1452 cmꢁ1, respectively, after treatment with Cyst. This result
indicates that the conjugated backbone in the red phase was
twisted after the Cyst treatment, and the conjugation in the
blue phase at room temperature was greater than that in the
red phase.
The self-assembled monomers of PCDA–HM were converted
to PDA polymers (PDA–HM) via exposure to 254 nm UV irradiation
for 1 min, resulting in a clear blue-colored solution (Scheme 2).20
The individual PCDA–HM monomer self-assembled and was
polymerized easily because of the extra p–p interaction and
the less steric hindrance associated with the relatively small
head group.
As illustrated in Scheme 2, after the self-assembly of PCDA–
HM into liposomes, the suspension showed a blue color.
Furthermore, Cyst treatment of the blue PDA liposomes directly
restored the fluorescence accompanied by a clear color change
from blue to red. Finally, to demonstrate the practical applica-
tion of our chemosensor, the PDA system was successfully used
to detect Cyst in aqueous solutions with high sensitivity and
selectivity.
Characterization of PDA–HM liposomes
The prepared PDA liposomes were characterized via FE-SEM
and Raman spectroscopy. The SEM images of PDAs revealed
Cysteamine detection using PDA–HM liposomes
The detection of Cyst among various thiols (Fig. 2) using the
PDA–HM sensor system was investigated via colorimetric
changes and altered UV absorption and fluorescence emission
in HEPES buffer (10 mM, pH 7.4). As shown Fig. 3a and b, we
investigated the colorimetric responses of PDA–HM (400 mM)
with Cyst (1 eq.) and other thiols (10 eq.), such as GSH, Cys,
Hcy, sodium pyrosulfate (NasS2O7), potassium thiocyanate
(KSCN), thioglycolic acid, 2-mercaptoethanol and hydrogen
sulfide (H2S) (10 eq.). Among the various analytes, only Cyst
immediately induced the blue-to-violet color transition at 1 eq.
An extremely imperceptible change was detected with H2S
(10 eq.), but no changes with other reagents. Cyst penetrates into
the polymer network of PDA–HM easily and rapidly, probably due
to its small size and flexible structure, leading to a reaction with
the CQC double bond of the maleimide moiety via Michael
addition to generate maleimide–thiol conjugation,26 which might
Scheme 1 Synthesis of the monomer PCDA–HM.
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Fig. 1 SEM images of (a) 100 mM PDA–HM after 254 nm UV irradiation and (b) PDA–HM following treatment with 100 mM of Cyst. (c) Raman spectra of
PDA–HM liposomes at room temperature (the blue line) and after treatment with Cyst (the red line).
Fig. 2 Chemical structures of various thiols.
Scheme 3 A suggested mechanism for the production of maleimide–
thiol conjugation.
could not change the color of PDA–HM from blue to red even
though it has a similar structure to Cyst. A reaction mechanism
that generates maleimide–thiol conjugation is suggested in
Scheme 3.28 This phenomenon suggests that PDA–HM can be
used in the detection of Cyst with high selectivity. Fig. 2b shows
the colorimetric titration of PDA–HM in the presence of various
levels of Cyst in HEPES buffer (10 mM, pH 7.4).
The analysis of the UV/Vis absorption spectra of an aqueous
Fig. 3 (a) Colorimetric responses of PDA–HM (400 mM) in the presence
solution of PDA–HM suggests that the original 640 nm absorp-
tion band of PDA–HM decreased with a concomitant increase at
540 nm upon binding with Cyst (Fig. 4a and Fig. S10, ESI†).
Furthermore, the presence of Cyst was also monitored by the
increase in fluorescent emission because the color transition
was accompanied by fluorescence emission (Fig. 4b). We mea-
sured the fluorescence of PDA–HM (200 mM) immersed in Cyst
aqueous solutions at different concentrations. As shown in
Fig. 4b, the emission intensity of PDA–HM increased gradually
with the increase in the Cyst concentration from 0 to 600 mM.
Moreover, the detection limit represents a significant para-
meter for molecular recognition. The limit of detection was
calculated according to a procedure reported in the literature.29
The linear calibration curve assumed that the response y is
linearly related to the concentration x for a limited range of
concentration and expressed as y = a + bx. This model was used
to determine the sensitivity (b) and the LOD value. The limit of
detection (LOD) was estimated at 22.83 mM, which was calcu-
lated using the following equation:
of Cyst (1 eq.) and other thiols (10 eq.), and NasS2O7 (10 eq.), and KSCN
(10 eq.) in HEPES buffer (10 mM, pH 7.4). (b) Colorimetric responses of
PDA–HM (400 mM) in the presence of various amounts of Cyst in HEPES
buffer (10 mM, pH 7.4).
then perturb the backbone of the PDA polymer, allowing the
release of the strain on the alkyl side chains generated during
polymerization. The release of the side chain strain induces the
partial distortion of the arrayed p-orbitals, which leads to the
observed changes in optical properties.27 Thiol groups play a
significant role in the blue-to-red phase transition in response
to Cyst. When PDA–HM solutions are treated with ethylenedia-
mine, which is similar to Cyst in structure but lacks the thiol
groups, the blue color of PDAs remains unchanged (Fig. S9, ESI†).
Despite the presence of the –SH functional group, due to the
larger structures of GSH, Cys, Hcy and thioglycolic acid, it is hard
for them to penetrate into the supramolecular system, and hence
they do not react with the densely packed PDA–HM polymer. H2S
reacts with the alkene bond of the maleimide moiety; however, it
is not large enough to completely induce configurational disorder
of the polymer backbone. Furthermore, the –NH2 groups act as
LOD ¼ 3 ꢂ Sa ꢃ b;
nucleophiles, leading to a reaction of maleimide with Cyst via where Sa is the standard deviation of the response and b is the
Michael addition. It is also demonstrated that 2-mercaptoethanol slope of the calibration curve. The standard deviation of the
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