Calixarenes functionalised water-soluble iron oxide magnetite nanoparticles for enzyme immobilisation

Article abstract of DOI:10.1080/10610278.2020.1740704

In this study, we first used water-soluble iron oxide nanoparticles for Candida rugosa lipase immobilisation. Moreover, two new complexation phenomena of the prepared water-soluble Fe₃O₄ nanoparticles with an enzyme might address int

Full text of DOI:10.1080/10610278.2020.1740704

Supramolecular Chemistry
ISSN: 1061-0278 (Print) 1029-0478 (Online) Journal homepage: https://www.tandfonline.com/loi/gsch20
Calixarenes functionalised water-soluble iron
oxide magnetite nanoparticles for enzyme
immobilisation
Serkan Sayin, Elif Ozyilmaz, Mehmet Oguz, Rüstem Yusufoglu & Mustafa
Yilmaz
To cite this article: Serkan Sayin, Elif Ozyilmaz, Mehmet Oguz, Rüstem Yusufoglu & Mustafa
Yilmaz (2020): Calixarenes functionalised water-soluble iron oxide magnetite nanoparticles for
enzyme immobilisation, Supramolecular Chemistry, DOI: 10.1080/10610278.2020.1740704
To link to this article: https://doi.org/10.1080/10610278.2020.1740704
Published online: 19 Mar 2020.
Submit your article to this journal
Article views: 37
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=gsch20

SUPRAMOLECULAR CHEMISTRY
https://doi.org/10.1080/10610278.2020.1740704
ARTICLE
Calixarenes functionalised water-soluble iron oxide magnetite nanoparticles for
enzyme immobilisation
a
b
c
c
c
Serkan Sayin , Elif Ozyilmaz , Mehmet Oguz , Rüstem Yusufoglu and Mustafa Yilmaz
a
b
Department of Environmental Engineering, Faculty of Engineering, Giresun University, Giresun, Turkey; Department of Biochemistry, Selcuk
c
University, Konya, Turkey; Department of Chemistry, Selcuk University, Konya, Turkey
ABSTRACT
ARTICLE HISTORY
In this study, we first used water-soluble iron oxide nanoparticles for Candida rugosa lipase
Received 30 December 2019
Accepted 4 March 2020
immobilisation. Moreover, two new complexation phenomena of the prepared water-soluble Fe
nanoparticles with an enzyme might address interesting results in terms of enzyme activity and
3
O
4
KEYWORDS
Calix[n]arene; water-soluble
Fe O ; lipase;
3 4
immobilisation;
nanoparticles
stability in typical enzymatic reactions. The catalytic activity on pH and temperature dependence,
and reusability of the immobilised lipases (p-SCX4-NP-E and p-SCX8-NP-E) were also investigated
with the hydrolysis reaction of p-nitrophenyl palmitate (p-NPP). The results show that the highest
catalytic affinities for p-SCX4-NP-E and p-SCX8-NP-E were obtained at pH 5.0 and pH 7.0,
respectively.
successfully employed as a receptor in biological fields
7–11]. Besides water solubility, indeed these calixarenes
Introduction
[
Researchers dealing with supramolecular assembly have
been recently pointed their attraction on recognition of
small protein, controlled protein assembly, the com-
plexation of the biomaterials with supramolecular com-
pounds, etc. because of the suitability of supramolecular
compounds for inclusion complexation in biological
fields [1–3]. Calixarenes counted third generation of
supramolecular compounds after cyclodextrins and
crown ethers have been most preferred one, because
of their simple and easily synthesis, tailored functionali-
zation, and bearing hydrophilic and hydrophobic units
should be able to form a host-guest inclusion complex
with biomolecules such as haemoglobin, cholesterol,
anthelmintics, testosterone, steroid hormones, lectin,
etc [12–14].
Water-soluble iron oxide nanoparticles have gained
much interest in bio-applications including materials
and life sciences, magnetic fluid hyperthermia, biomedi-
cine, data storage, magnetic separation, and immobilisa-
tion of biomolecules, etc. due to their unique magnetic
properties, low toxicity, biocompatibility, and applicabil-
ity into aqueous media [15–22]. Most fabrication meth-
ods produce hydrophobic iron oxide nanoparticles;
therefore the development of water-soluble iron oxide
nanoparticles is still challenged. However, the procedure
involved ligand exchange reactions are mostly applied
to convert hydrophobic iron oxide nanoparticles to
[4–6]. Of course not all calixarene derivatives suitable for
biological studies, so enzymatic reactions occur in an
aqueous medium; hence, their un-suitable derivatives
require to be turned into water-soluble derivatives. For
the last few years, water-soluble calixarenes have been
CONTACT Mustafa Yilmaz
myilmaz42@yahoo.com
Department of Chemistry, Selcuk University, Konya 42075, Turkey
©
2020 Informa UK Limited, trading as Taylor & Francis Group

2
S. SAYIN ET AL.
hydrophilic so as to be soluble in aqueous environment.
For the construction of water-soluble magnetic nanopar-
(lower and upper rim) for the binding of Fe O nanoparti-
3 4
cles as well as Candida rugosa lipase in aqueous nature.
ticles
with
supramolecular
assembly,
the
p-sulphonatocalix[n]arene-carboxylic acid core [4] is an
attractive starting point. This is a system that calixarene
plays as a molecular glue by providing convenient bind-
ing lobes for the grafting Fe O nanoparticles. Indeed,
Results and discussion
Synthesis and characterisation of water-soluble
calix[n]arene grafted magnetic nanoparticles
3
4
water-soluble calixarenes used through the nanoparticle
immobilisation provide water solubility to Fe O nano-
3
4
The aims of this study were the design, and preparation
particles in this system. To the best of our knowledge,
there are no reports of lipase immobilisation onto water-
soluble iron oxide nanoparticles. Moreover, for the first
time, a complexation of the prepared water soluble-calix
of p-sulphonatocalix[n]arene carboxylic acid-grafted Fe3
O (p-SCX4-NP and p-SCX8-NP), and the complexation
4
of their with C. rugosa lipase via supramolecular assem-
bly in order to investigate enzyme activity and stability
in typical enzymatic reactions. For this purpose, the
syntheses of p-sulphonatocalix[4]arene-tetracarboxylic
acid and p-sulphonatocalix[8]arene-octacarboxylic acid
depicted in Scheme 1 and Scheme 2 were carried out in
four steps. In the first step, p-tert-butylphenol was
[
n]arene grafted Fe O nanoparticles with an enzyme
3 4
might address interesting results in terms of enzyme
activity and stability in typical enzymatic reactions. In
order to see, we wish to prepare the water-soluble calixar-
ene derivatives which have convenient sites at its both rims
Scheme 1. Immobilisation of lipase with p-sulphonatocalix[4,8]arene-coated Fe O nanoparticles. Reaction conditions: (i) methyl-
3
4
bromoacetate, NaH; (ii) KOH, aqueous EtOH; (iii) H SO ; (iv) FeCl , FeCl , NH ; (v) Candida rugosa lipase, phosphate buffer solution
2
4
2
3
3
(50 mM, pH 7.0).

SUPRAMOLECULAR CHEMISTRY
3
reacted with formaldehyde in the presence of
a convenient alkaline base so that p-tert-butylcalix[4]
arene [1] and p-tert-butylcalix[8]arene [2] were success-
fully synthesised according to the literature procedure
one multiplet at 3.97–4.18 ppm (36 H), corresponding to
OCH and ArCH Ar signals in the spectrum (see Figure 1).
2
2
The infrared spectrum of both p-sulphonatocalix[4]
arene-tetracarboxylic acid-coated Fe O4 nanoparticles
3
[
23,24]. In the next step, p-tert-butylcalix[4]arene [1] and
(p-SCX4-NP) and p-sulphonatocalix[8]arene-octacar-
boxylic acid-coated Fe O nanoparticles (p-SCX8-NP) is
p-tert-butylcalix[8]arene [2] were alkylated with methyl
bromoacetate in refluxing THF in the presence of NaH as
a base [25,26]. Ester derivatives of p-tert-butylcalix[4]
arene [3] and p-tert-butylcalix[8]arene [4] were then
hydrolysed using KOH in aqueous EtOH to yields tetra-
carboxylic acid derivative 5 or octacarboxylic acid deri-
vative 6 [25,26]. In the last step, treatment of derivative 5
3
4
shown in Figure 2. In FTIR spectra, the characteristic SO
asymmetric (1160 cm for p-SCX4-NP, and 1140 and
2
−
1
−
1
−1
1106 cm for p-SCX8-NP), SO symmetric (1036 cm
2
−
1
for p-SCX4-NP, and 1031 cm for p-SCX8-NP) and
−
1
−1
S-O (875 cm for p-SCX4-NP, and 885 cm for p-SCX8-
NP) stretches for p-SCX4-NP and p-SCX8-NP.
Furthermore, the characteristic carbonyl bands present
in the compound 7 and 8 spectra are shifted from 1726
and 1714 cm to 1625 cm respectively in the FTIR
spectra of p-SCX4-NP and p-SCX8-NP which confirms
the carboxylate form attached to the nanoparticle
surface.
with conc. H SO4 gave p-sulphonatocalix[4]arene-
2
tetracarboxylic acid [7] in high yield [27,28]. Moreover,
p-sulphonatocalix[8]arene-octacarboxylic acid [8] was
synthesised for the first time by the reaction of octacar-
boxylic acid derivative 6 with conc. H SO . The structure
−
1
−1
2
4
of calixarene derivatives was determined using FTIR,
1
H-NMR, and elemental analysis techniques. In addition,
The thermogravimetric analysis was used to elaborate
the structure of the Fe O nanoparticles coated with
the Fe O nanoparticles coated with p-sulphonatocalix
3
4
3 4
[
4]arene-tetracarboxylic acid (p-SCX4-NP) and/or p-sul-
p-sulphonatocalix[4]arene-tetracarboxylate (p-SCX4-NP).
The results represented in Figure 3(a,b) depict that the
first weight loss was attained at 50–250°C because of the
evaporation of water and other solvents. Setting up the
temperature in the range of 300–800°C, the total weight
loss was found about 55% for p-SCX4-NP and 58% for
p-SCX8-NP mass due to the decomposition of p-sulpho-
natocalix[4]arene and p-sulphonatocalix[8]arene-tetracar-
boxylate units, respectively.
phonatocalix[8]arene-octacarboxylic acid (p-SCX8-NP)
were prepared for the first time. Their surface morphol-
ogies were determined by FTIR, TGA, TEM, and XRD
techniques.
1
The HNMR spectroscopy has provided information
to identify the structure and purity of p-sulphonatocalix
[8]arene-octacarboxylic acid [8]. The one singlet at 7.44
ppm (16 H) was assigned to aromatic proton signal and
1
Figure 1. H-NMR spectrum of p-sulphonatocalix[8]arene-octacarboxylic acid.

4
S. SAYIN ET AL.
Figure 2. FTIR spectrum of (a) p-sulphonatocalix[4]arene-tetracarboxylic acid and p-SCX4-NP; (b) p-sulphonatocalix[8]arene-
octacarboxylic acid and p-SCX8-NP.
Self-assembly inclusion complexation of Candida
rugosa on water-soluble p-sulphonatocalix[n]
arene-carboxylate-adorned Fe O nanoparticles
lipase (p-SCX4-NP-E) and/or p-SCX8-NP-immobilised
lipase (p-SCX8-NP-E). The structure of the immobilised
lipases was assessed using TEM and XRD techniques. Zeta
potential analysis was also applied to determine the charge
onto the surface of the immobilised lipases.
3
4
It is well known that the selectively functionalised calixar-
enes play versatile tools for the binding of biomolecules.
Basically, they comprise prominent units which maintain
sensitive and selective inclusion complexation for biomole-
cules at both of their rims. Taking an account, fabricated
Transmission electron microscopy (TEM) of p-SCX4-NP
(Figure 4(a)), p-SCX4-NP-E (Figure 4(b)), p-SCX8-NP (Figure
4(c)), and p-SCX8-NP-E (Figure 4(d)) was used to determine
their particle size and morphology (Figure 4). All TEM micro-
graph show that a uniform spherical with 2–5 nm of aver-
age particle diameter without clustering are observed.
X-ray diffraction (XRD) analysis provided further
insights into the structure and phase purity of p-SCX4-
water-soluble
p-sulphonatocalix[n]arene-carboxylate-
adorned Fe O nanoparticles (p-SCX4-NP and p-SCX8-
3
4
NP) were treated with Candida rugosa lipase for the first
time in aqueous nature to obtain p-SCX4-NP-immobilised

SUPRAMOLECULAR CHEMISTRY
5
Figure 3. TGA curves of p-SCX4-NP (a) and p-SCX8-NP (b).
NP (Figure 5(a)), p-SCX4-NP-E (Figure 5(a)), p-SCX8-NP
−30.5 mV (pH 7.0), respectively. This can be ascribed to
ionisation of acidic units in both of the immobilised
lipases in response to pH changes.
The change observed in the complex pH depen-
dency may be due to the non-covalent interaction
between lipase/water-soluble p-sulphonatocalix[n]
(Figure 5(b)), p-SCX8-NP-E (Figure 5(c)) (Figure 5).
Figure 5 shows that all of the XRD patterns have six
diffraction peaks which can be indexed to the spinel
crystal structure. Moreover, undetected additional
peaks from other phases indicate that both of the struc-
tures maintain in high purity.
arene-carboxylate-adorned
Fe O
nanoparticles.
3
4
The zeta potential was performed in aqueous media
at various pH values to determine the surface charge
and the complexation type of the immobilised lipases
Accordingly, sulphonic acid groups of p-SCX4-NP
and p-SCX8-NP turn into sulphonate anion forms
at pH 1.0–5.0, and so the anion moieties of p-sul-
(p-SCX4-NP-E and/or p-SCX8-NP-E) (Figure 6). Figure 6
phonatocalix[n]arene-carboxylate-adorned
Fe O
3 4
shows that p-SCX4-NP and p-SCX8-NP exhibited
a negative zeta potential that shifted after complexation
with lipase due to negative charge–positive charge
attraction between sulphonato units of calixarene and
most probably protonated lysine moieties of lipase. As
the pH was increased, the surface charges of the nano-
particles p-SCX4-NP-E and p-SCX8-NP-E became more
negative −23.4 and −22.9 mV (pH 5.0) to −32.2 and
nanoparticles may involve in the complexation pro-
cess of lipase and the interaction takes place via
electrostatic, and hydrogen bonding attraction. In
accord with that, the zero charge surfaces for the
immobilised lipase can be expected if we consider
the protonated lysine units of lipase were equal to
the sulphonato moieties of p-sulphonatocalix[n]
arene-carboxylate-adorned Fe O4 nanoparticles at
3

6
S. SAYIN ET AL.
Figure 4. TEM micrographs of (a) p-SCX4-NP, (b) p-SCX4-NP-E, (c) p-SCX8-NP and (d) p-SCX8-NP-E (e) Histogram of p-SCX4-NP (f)
p-SCX8-NP.
pH 5.0. Yet, zeta potential result shows that the
surface charges of p-SCX4-NP-E and p-SCX8-NP-E
are −23.4 and −22.9 mV at pH 5.0, respectively (see
Figure 6). This finding may address to other possibi-
lities such as the charge motion of the other func-
tional units like –COOH and –SH over lipase that
might also play a crucial role in lipase-water soluble
p-sulphonatocalix[n]arene-carboxylate-adorned Fe3
and 25.5 mg, respectively, each of protein per g of nano-
particles. Figure 7 illustrates the catalytic activity of the
free and immobilised lipases (p-SCX4-NP-E and p-SCX8-
NP-E) at various pH values. It was found that p-SCX4-NP
-E exhibited the highest catalytic affinity at pH 4–6. The
superior catalytic affinity for p-SCX4-NP-E was observed
as about 98% at pH 5.0, whereas the activity efficiencies of
the free lipase and p-SCX8-NP-E were obtained as 46%
and 61%, respectively, at same pH. However, decreasing
pH to 7.0, activity efficiency of p-SCX8-NP-E became
O nanoparticles attraction.
4
1
00% while the efficiency for p-SCX4-NP-E was 85%.
The effect of pH and temperature on the activity of
immobilised lipases
Basically, immobilisation of lipases with p-SCX4-NP and/
or p-SCX8-NP by means of inclusion complexation
improved the pH stability of lipase and extends the applic-
ability of lipase at lower acidic reaction medium.
Temperaturedependent catalytic activities of the free
and immobilised lipases (p-SCX4-NP-E and p-SCX8-NP-E)
at different temperatures are shown in Figure 8. Inclusion
In order to see any changes in the catalytic efficiencies of
lipases, typical hydrolysis reaction of p-nitrophenyl palmi-
tate (p-NPP) by lipase was carried out in aqueous media
[29–33]. In the case of immobilised lipases (p-SCX4-NP-E
and p-SCX8-NP-E) were obtained the loading of 26.2 mg

SUPRAMOLECULAR CHEMISTRY
7
Figure 5. XRD patterns of (a) p-SCX4-NP (red line) and p-SCX4-NP-E (black line), (b) p-SCX8-NP, and (c) p-SCX8-NP-E.
complex of lipase with p-SCX4-NP brought the optimum
temperature regarding lipase activity from 35°C to 40°C,
while the complex of lipase with p-SCX8-NP exhibited in
high activity at 35°C, which was the result obtained for
free lipase. Nevertheless, increasing temperature to 60°C,
a superior catalytic activity observed for p-SCX8-NP-E.
The results addressed that immobilisation of lipase with
the p-SCX4-NP and p-SCX8-NPenhanced in stability of
C. rugosa lipase at a high temperature.
p-SCX8-NP-E) at 30°C. The experiments for the hydro-
lysis reaction were carried out five times, each time using
the same immobilised lipases and fresh p-NPP solution.
Figure 9 depicts that p-SCX4-NP-E and p-SCX8-NP-E,
when employed as a catalyst for the five times, exhibit
75% and 72% catalytic efficiency, respectively.
Conclusion
To study reusability of immobilised lipases p-SCX4-
NP-E and p-SCX8-NP-E, the hydrolysis reaction of p-NPP
was performed at pH 5.0 (for p-SCX4-NP-E) and 7.0 (for
Two new water-soluble p-sulphonatocalix[n]arene-
carboxylate-grafted magnetic nanoparticles (p-SCX4-
NP and p-SCX8-NP) were successfully fabricated and

8
S. SAYIN ET AL.
Figure 6. Zeta potentials of (a) p-SCX4-NP and p-SCX4-NP-E; (b) p-SCX8-NP and p-SCX8-NP-E.
dependencies, and reusability of immobilised lipases
into model reaction were also performed. The results
show that the immobilised lipase (p-SCX4-NP-E)
became more efficient catalyst in the reactions occurred
at pH 5.0, whereas p-SCX8-NP-E exhibited superior cat-
alytic affinity at pH 7.0. In addition, separation, and
reusability process of the enzymatic reaction can be
performed very easily due to the magnetic properties
of immobilised lipases. In view of that, the current study
might open new doors in the applicability of biocompa-
tible materials, and expand the usability of enzymes.
Figure 7. Effect of substrate pH on residual activity of free lipase,
p-SCX4-NP-E, and p-SCX8-NP-E.
Experimental methods
Chemical and materials
characterised accordingly. Then, p-SCX4-NP and
p-SCX8-NP were immobilised for the first time onto
Candida rugosa lipase by means of anion-cation attrac-
tion force in an aqueous nature. The hydrolytic hydro-
lysis reaction of p-NPP was used to assess the catalytic
efficiency of immobilised lipases (p-SCX4-NP and
p-SCX8-NP) in aqueous media. pH and temperature
A DC Alufolien Kieselgel 60 F254 (Merck) was used for TLC
analyses. All chemicals used were of standard analytical
grade from Aldrich. Anhydrous magnesium sulphate was
used as the drying agent. p-Nitrophenyl palmitate (p-NPP),
and Candida rugosa lipase were purchased from Sigma
Chemical Co.
Figure 8. Effect of temperature on the activity of free lipase, p-SCX4-NP-E, and p-SCX8-NP-E.

SUPRAMOLECULAR CHEMISTRY
9
Figure 9. Reusability of p-SCX4-NP-E and p-SCX8-NP-E.
The synthesis of organic compounds
dichloromethane was vigorously stirred at rt for 7 days.
The volatile was evaporated, and the remaining was gently
p-tert-Butylcalix[4]arene [1], p-tert-butylcalix[8]arene [2],
treated with Et O. The obtained precipitate was collected
2
5
,11,17,23-tetra-tert-butyl-25,26,27,28-tetrakis[(methox-
ycarbonyl)methoxy]-calix[4]arene [3], 5,11,17,23,29,35,
1,47-octa-tert-butyl-49,50,51,52,53,54,55,56-octakis
(methoxycarbonyl)methoxy]-calix[8]arene [4], tetra-
by filtration, and dried in an oven. Light violet powder was
−1
obtained in a yield of 63%. m.p. > 350 °C. FTIR (ATR) cm :
714 (VC=O), 1140, 1106 (V asymmetric), 1031 (VSO2 sym-
4
[
1
SO2
1
metric) and 885 (VS-O). H-NMR (400 MHz, D O): δ 3.97–4.18
2
carboxylic acid derivative 5, octacarboxylic acid deri-
vative 6, p-sulphonatocalix[4]arene-tetracarboxylic
acid [7] were synthesised according to the published
experimental procedures [23–28]. p-Sulphonatocalix[8]
arene-octacarboxylic acid [8], Fe O nanoparticles
(
m, 36 H, Ar-CH -Ar, and OCH ), 7.44 (s, 16 H, ArH).
2 2
General preparation of Fe O nanoparticles coated
3
4
with p-sulphonatocalix[n]arene-carboxylic acids
3
4
coated with p-sulphonatocalix[4]arene-tetracarboxylic
acid (p-SCX4-NP), p-sulphonatocalix[8]arene-octacar-
A typical chemical co-precipitation of Fe(III) and Fe(II)
ions in the presence of calixarene derivatives was
applied to produce p-SCX4-NP and/or p-SCX8-NP. For
boxylic acid-coated Fe O4 nanoparticles (p-SCX8-NP),
3
p-SCX4-NP-immobilised lipase (p-SCX4-NP-E), and
p-SCX8-NP-immobilised lipase (p-SCX8-NP-E) were pre-
pared for the first time.
that, a mixture of each 25 mL of 1.0 M FeCl and 1.75 M
2
FeCl solutions was injected into a solution of 0.1 g
3
p-sulphonatocalix[n]arene-carboxylic acid in 20 mL
Milli-Q water at 60°C. Fifteen minutes later, 15 mL conc.
NH was added to the reaction mixture in order to form
iron oxide in the system. The reaction mixture was
heated for 1 h at 60°C. Magnetic decantation was carried
3
Synthesis of p-sulphonatocalix[4]arene-
tetracarboxylic acid [7]
White colour powder. Yied: 92%, m.p. > 350°C. FTIR
out to collect the Fe O4 nanoparticles coated with
3
−
1
(
ATR) cm : 3133 (VOH), 1726 (VC=O), 1625 (VH2O), 1497,
p-sulphonatocalix[n]arene-carboxylic acid, then washed
with water and dried at rt under vacuum for overnight.
1
1
403, and 1375 (VC=C), 1153, 1111 (V
034 (VSO2 symmetric) and 781 (VS-O). H-NMR (400 MHz,
asymmetric),
SO2
1
D O): δ 3.32 (brs, 8 H, Ar-CH -Ar), 4.16 (brs, 8 H, OCH ),
2
2
2
General preparation of the immobilised lipases
7
.17 (s, 8 H, ArH).
Fifty-milligram of Fe O4 nanoparticles coated with
3
p-sulphonatocalix[n]arene-carboxylic acid (p-SCX4-NP
and/or p-SCX8-NP) were added to a suspension of
Synthesis of p-sulphonatocalix[8]arene-
octacarboxylic acid [8]
6
0 mg Candida rugosa lipase in 390 µL phosphate buf-
A solution of octacarboxylic acid derivative 6 (0.2 g,
.113 mmol) and conc. H SO4 (1.7 mL) in 10 mL
fers (50 mM, pH 7.0). The mixture was incubated at 30°C
and stirred at 150 rpm for 30 min after magnetic
0
2

1
0
S. SAYIN ET AL.
decantation and the final material was washed with
water. The fabricated lipases (p-SCX4-NP-E and/or
p-SCX8-NP-E) were then lyophilised and stored at 4°C
until use. The protein concentration of the immobilised
enzyme was calculated using Bradford’s method [34].
Mehmet Oguz
Mustafa Yilmaz
http://orcid.org/0000-0002-3999-620X
http://orcid.org/0000-0003-2904-160X
References
[1] Ahire VK, Malkhede DD. Interaction studies of haemo-
globin with p-sulfonatocalix[8]arene by spectrophoto-
metric methods. Chem Phys Lett. 2019;731:136597.
2] Perret F, Coleman AW. Biochemistry of anionic calix[n]
arenes. Chem Comm. 2011;47(26):7303–7319.
3] Dong Z, Luo Q, Liu J. Artificial enzymes based on supramo-
lecular scaffolds. Chem Soc Rev. 2012;41(23):7890–7908.
4] Bhatti AA, Oguz M, Yilmaz M. One-pot synthesis of
Fe3O4@Chitosan-pSDCalix hybrid nanomaterial for the
detection and removal of Hg2+ ion from aqueous
media. Appl Surf Sci. 2018;434:1217–1223.
5] Bhatti AA, Oguz M, Yilmaz M. Magnetizing calixarene: azo
dye removal from aqueous media by Fe3O4Nanoparticles
fabricated with carboxylic-substituted Calix[4]arene. J Chem
Eng Data. 2017;62(9):2819–2825.
6] Moridi N, Danylyuk O, Suwinska K, et al. Monolayers of
an amphiphilic para-carboxy-calix[4]arene act as tem-
plates for the crystallization of acetaminophen.
J Colloid Interface Sci. 2012;377(1):450–455.
7] Shetty D, Skorjanc T, Olson MA, et al. Self-assembly of
stimuli-responsive imine-linked calix[4]arene nanocap-
sules for targeted camptothecin delivery. Chem Comm.
2019;55(60):8876–8879.
8] Yilmaz M, Karanastasis AA, Chatziathanasiadou MV, et al.
Inclusion of quercetin in gold nanoparticles decorated
with supramolecular hosts amplifies its tumor targeting
properties. ACS Appl Bio Mater. 2019;2(7):2715–2725.
9] Wang J, Ding X, Guo X. Assembly behaviors of
calixarene-based amphiphile and supra-amphiphile and
the applications in drug delivery and protein recognition.
Adv Colloid Interface Sci. 2019;269:187–202.
Catalytic activity
[
[
[
Hydrolytic activity was carried out according to the pre-
vious method [35]. Briefly; 25 mg of immobilised lipase
was added to 1 mL of phosphate buffer solution (pH 7.0)
and 1 mL of 12.0 mM p-nitrophenyl palmitate solution
prepared in 2-propanol and stirred for 5 min at room
temperature. The termination of the reaction was done
by adding 2 ml of 0.5 M Na CO . The immobilised enzyme
from the reaction medium was separated by a magnet
and the concentration of p-NP formed was measured
using a UV–visible spectrophotometer at 405 nm.
2
3
[
[
[
[
[
Reuse of the immobilised enzyme
To examine the reusability of the immobilised enzymes, it was
treated by using p-NPP as an assay. After used the immobilised
lipase it was magnetically separated and washed with phosphate
buffer (pH 7) and this procedure is repeated for up to five cycles.
Acknowledgments
We would like to thank the Scientific and Technological
Research Council of Turkey (TUBITAK-Grant Number 119Z131),
and the Research Foundation of Selcuk University (SUBAP-Grant
Number: 18201080) for financial support of this work.
[
[
[
10] Kumar R, Sharma A, Singh H, et al. Revisiting fluorescent
calixarenes: from molecular sensors to smart materials.
Chem Rev. 2019;119(16):9657–9721.
11] Yilmaz M, Sayin S. Calixarenes in organo and biomimetic
catalysis. In: Neri, P., Sessler, J., & Wang, MX. (eds),
Calixarenes Beyond. Springer, Cham. 2016;719–742.
12] Coleman AW, Perret F, Moussa A, et al. Calix [n] arenes as
protein sensors. In: Schrader T. (eds), Creative Chem Sens
Syst. Topics in Current Chemistry, Springer, Berlin,
Heidelberg. 2007;31–88.
Disclosure Statement
There are no conflicts to declare.
Funding
The authors thank the Scientific and Technological Research
Council of Turkey [119Z131] and the Scientific Research
Projects Foundation of Selcuk University [18201080] for finan-
cial support of this work.
[
13] Korchowiec B, Korchowiec J, Orlof-Naturalna M, et al. Two
antibacterial nalidixate calixarene derivatives in cholesterol
monolayers: molecular dynamics and physicochemical
effects. Colloids Surf B Biointerfaces. 2016;145:777–784.
14] Silva ED, Valmalle C, Becchi M, et al. J Inclusion Phenom
Macrocyclic Chem. 2003;46(1/2):65–69.
[
ORCID
Serkan Sayin
Elif Ozyilmaz
http://orcid.org/0000-0003-0518-3208
http://orcid.org/0000-0003-4360-4165
[15] Majeed MI, Lu Q, Yan W, et al. Highly water-soluble
magnetic iron oxide (Fe3O4) nanoparticles for drug

SUPRAMOLECULAR CHEMISTRY
11
delivery: enhanced in vitro therapeutic efficacy of dox-
orubicin and MION conjugates. J Mat Chem B. 2013;1
molecular receptors. J Am Chem Soc. 1989;111
(23):8681–8691.
(
22):2874–2884.
[26] Ten’kovtsev AV, Razina AB. Nonionogenic star-shaped
calix[8]arenes: synthesis and ionophore properties.
Russ J Appl Chem. 2009;82(9):1615–1619.
[
16] Laurent S, Dutz S, Häfeli UO, et al. Magnetic fluid
hyperthermia: focus on superparamagnetic iron oxide
nanoparticles. Adv Colloid Interface Sci. 2011;166
[27] Shinkai S, Araki K, Tsubaki T, et al. New syntheses of
(1–2):8–23.
calixarene-p-sulphonates
and
p-nitrocalixarenes.
[
[
[
17] Lartigue L, Oumzil K, Guari Y, et al. Water-soluble
rhamnose-coated Fe3O4 nanoparticles. Org Lett.
J Chem Soc Perkin Trans. 1987;1:2297–2299.
[28] Casnati A, Ting Y, Berti D, et al. Synthesis of water soluble
molecular receptors from calix[4]arenes fixed in the cone
conformation. Tetrahedron. 1993;49(43):9815–9822.
[29] Sayin S, Akoz E, Yilmaz M. Enhanced catalysis and enan-
tioselective resolution of racemic naproxen methyl ester
by lipase encapsulated within iron oxide nanoparticles
coated with calix[8]arene valeric acid complexes. Org
Biomol Chem. 2014;12:6634–6642.
2
009;11(14):2992–2995.
18] Xu Y, Qin Y, Palchoudhury S, et al. Water-Soluble Iron
Oxide Nanoparticles with High Stability and Selective
Surface Functionality. Langmuir. 2011;27(14):8990–8997.
19] Amstad E, Gillich T, Bilecka I, et al. Ultrastable iron oxide
nanoparticle colloidal suspensions using dispersants
with catechol-derived anchor groups. Nano Lett.
2
009;9(12):4042–4048.
[30] Chiou S-H, Wu W-T. Immobilization of Candida rugosa
lipase on chitosan with activation of the hydroxyl
groups. Biomaterials. 2004;25(2):197–204.
[31] Cui Y, Li Y, Yang Y, et al. Facile synthesis of amino-silane
modified superparamagnetic Fe3O4 nanoparticles and
application for lipase immobilization. J Biotechnol.
2010;150(1):171–174.
[32] Ozyilmaz E, Sayin S, Arslan M, et al. Improving catalytic
hydrolysis reaction efficiency of sol–gel-encapsulated
Candida rugosa lipase with magnetic β-cyclodextrin nano-
particles. Colloids Surf B Biointerfaces. 2014;113:182–189.
[33] Romero CM, Spuches FC, Morales AH, et al. Design and
characterization of immobilized biocatalyst with lipase
activity onto magnetic magnesium spinel nanoparticles:
[
20] Korpany KV, Mottillo C, Bachelder J, et al. One-step
ligand exchange and switching from hydrophobic to
water-stable hydrophilic superparamagnetic iron oxide
nanoparticles by mechanochemical milling. Chem
Comm. 2016;52(14):3054–3057.
[
21] Lee S, Fursina A, Mayo JT, et al. Electrically driven phase
transition in magnetite nanostructures. Nat Mater.
2
008;7:130.
[
22] Nagesha DK, Plouffe BD, Phan M, et al. Functionalization-
induced improvement in magnetic properties of Fe3O4
nanoparticles for biomedical applications. J Appl Phys.
2
009;105(7):07B317.
[
23] Gutsche CD, Nam KC. Calixarenes. 22. Synthesis, proper-
ties, and metal complexation of aminocalixarenes. J Am
Chem Soc. 1988;110(18):6153–6162.
A
novel platform for biocatalysis. Colloids Surf
B Biointerfaces. 2018;172:699–707.
[
24] Gutsche CD, Dhawan B, No KH, et al. Calixarenes. 4. The
synthesis, characterization, and properties of the calix-
arenes from p-tert-butylphenol. J Am Chem Soc.
[34] Bradford MM. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein dye binding. Anal Biochem.
1976;72(1–2):248–254.
1
981;103(13):3782–3792.
[
25] Arnaud-Neu F, Collins EM, Deasy M, et al. Synthesis, x-ray
crystal structures, and cation-binding properties of alkyl
calixaryl esters and ketones, a new family of macrocyclic
[35] Chiou S-H, W-T W. Immobilization of Candida rugosa
lipase on chitosan with activation of the hydroxyl
groups. Biomaterials. 2004;25(2):197–204.