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O.C. Bodur, E. Hasanog˘lu Özkan and Ö. Çolak et al. / Journal of Molecular Structure 1223 (2021) 129168
such as glutaraldehyde etc. For example, glucose sensitive sen-
sors have been designed using PAMAM dendrimers [8,9]. Den-
drimeric structures have been investigated for the detection of
important biomarkers in the early stage diagnosis of some dis-
eases, using an enzyme-linked immunosorbent assay (ELISA) [10].
Li and Kwak immobilized tyrosinase on pamam-dendrimer for the
penicillamine sensor [11]. It is seen that acetylcholinesterase is
used on pamam-dendrimer for carbofuran, trichlorphone and tra-
the surface with the special pad. Finally, it was washed again with
distilled water and made ready for use.
Modifying the carbon paste electrode; unlike the above prepa-
ration, the paste (graphite powder + nujol) is prepared with
PAMAM-SAL. The modification process can be realized by adding
and mixing the appropriate amount of PAMAM-SAL and filling the
2.4. Enzyme immobilization
As understood from the sources, the applications of dendrimers,
which have a wide range of use in sensor fields, have increased
due to their high stability and low variability [14,15]. Abbreviated
as PAMAM, polyamidoamine dendrimer can be used in new sensor
designs by interacting with substituted groups. In sensor design, it
is very important to achieve superior sensor performance and to
minimize the total cost of a sensor. For this purpose, an acetyl-
choline biosensor containing modified PAMAM-dendrimer, acetyl-
cholinesterase and choline oxidase enzymes has been designed and
the results have been discussed [16].
A mixture of 1.0 mg BSA (bovine serum albumin), 60.0 μL
buffer solution, 10.0 μL AChE (200.0 units / mL), 200 μL ChO
(10.0 units / mL) and 30.0 μL gluteraldehyde (2.5%) was prepared.
This mixture was dropped onto the surface of the modified car-
bon paste electrode prepared by mixing graphite powder, nujol
and PAMAM-Sal and dried at room temperature. The prepared en-
zyme electrode was first washed with distilled water and then
with buffer solution and stored in the refrigerator at +4 °C in the
buffer solution when not in use.
2.5. Amperometric measurements
2. Experimental
Firstly, the carbon paste electrode was modified with PAMAM-
Sal. Then, AChE and ChO enzymes were immobilized to the sur-
face of the modified electrode using cross-linking with glutaralde-
hyde and BSA. Electrochemical measurements; AChE enzyme, hy-
drolyzes acetylcholine to acetic acid and choline. ChO, on the other
hand, oxidizes the released choline to betaine to form H2O2. The
amount of ACh is directly proportional to the anodic currents of
the released H2O2. The determination of ACh with the prepared
biosensor was made on the basis of measuring oxidation currents
of H2O2 released as a result of enzymatic reactions at +0.4 V.
Biochemical reactions occur between AChE and ChO enzymes
immobilized on the modified carbon paste electrode surface and
ACh. Firstly; ACh is hydrolyzed to choline (Ch) and acetic acid with
the help of AChE. Then Ch is oxidized to betaine with ChO. FAD,
the prosthetic group of ChO, is reduced to FADH2 by taking elec-
trons.
2.1. Equipment and reagents
The electro chemical studies were carried out by using CHI
1230-B electrochemical analyzer with
The working electrode was carbon paste electrode (custom
made of teflon, 0.8 cm diameter). Auxiliary and reference elec-
trodes were Pt wire and Ag/AgCl electrode (3.0 KCl), re-
a three electrode cell.
a
M
spectively. The pH values of the buffer solutions were mea-
sured with an ORION Model 720A pH/ionmeter. Temperature
control was achieved with GrantGD120 thermostat. 20 wt% in
methanol PAMAM dendrimer(ethylenediamine core, generation),
salicylaldehyde were purchased from Aldrich. respectively. Acetyl-
cholinesterase (E.C.3.1.1.7, purified from Electrophorus electricus
(electric eel) Type V-S activity of 200. 0 unit/mL) and choline ox-
idase (E.C.1.1.3.17, purified from the Alcaligenes species, activity of
10. 0 unit/mL) were purchased from Sigma. Acetylcholine chloride
was also purchased from Sigma, graphite powder were supplied
from Merck. All other chemicals were obtained from Sigma. All of
the solutions were prepared by using pure water.
FADH2 in the enzyme is oxidized by giving electrons to the oxy-
gen in the solution to make the reaction reversible. Thus, the en-
zyme returns to its initial form. Oxygen is reduced to H2O2 by tak-
ing electrons. As a result of these biochemical reactions, H2O2 is
oxidized at the MCPE surface to oxygen. ACh determination can be
made by measuring the anodic current of H2O2 on the electrode
2.2. Synthesis of denritic macromolecule (PAMAM-Sal)
The dendritic structure was synthesized by template method.
0.4 mmol of salicylaldehyde (41.7 mL, d = 1,17 g/mL) in ethanol
(15 mL) was added dropwise to the solution of PAMAM dendrimer
(0.05 mmol/g) dissolved in DMF (15 mL). Prosess of boiling and
stirring was continued for 24 h under reflux (Fig. 1).
3. Results and discussion
3.1. FT-IR, UV-GB and 1H NMR spectra of dendritic macromolecule
(PAMAM-Sal)
The mixture was cooled to the room temperature and purifica-
tion by acetone. The resulting solid was filtered and dried in the
oven for one day. The yield of (PAMAM-Sal) was 0.2923 g (72%).
Analytical, physical, electronic and characteristic IR spectral
data of the Schiff bases are given in Table 1. Element analy-
sis is compatible with the proposed structure. PAMAM-Sal spec-
trum shows medium broad bands in the region 3274, 3062 and
2937 cm−1 assigned to the υOH, υNH(sym) and υNH(asym), re-
spectively [17]. The appearance of bands in the 1495 cm−1 and
2.3. Preparation of working electrode: carbon paste electrode (CPE),
modified carbon paste electrode (MCPE) preparation
1276 cm−1 regions due to δ-C-C(as) and δ-C-C H(bv), respectively
–
The carbon paste electrode was cleaned with distilled water,
first. The graphite powder was weighed with the help of preci-
sion scales, and the amount of the carbon paste electrode content
was constant (0.0650 g). The mineral oil (nujol), which has a re-
tention capability, was taken with a micro pipette and added to
the graphite powder in a constant volume (30 μL) for each newly
prepared electrode. Then the carbon paste was filled into the cavity
with the aid of an auxiliary tool, so that it was easily and lossless.
After filling, the carbon paste electrode was cleaned and smoothed
=
[18]. The azomethine stretching band υ(CH N) is observed in
1629 cm−1. The IR showed the disappearance of the NH2 bands
=
together with the presence of CH N band which occurs the reac-
tion of salicylaldehyde with amine. It shows that formation of the
PAMAM-Sal.
The electronic spectra o f PAMAM-Sal in DMSO (10−4 M) show
∗
–
band 214 nm are attributed to the C N (n→ σ ) transition (Fig. 4).
The bands at higher energies (260, 286, 317 nm) are associated