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M.O. Oztürk-Oncel et al.
Colloids and Surfaces B: Biointerfaces 196 (2020) 111343
shows that 24 h modification results in the highest nitrogen peak
(Fig. 3C). Thus, 10 mM concentration and 24 h dipping time are
considered to be the optimum modification parameters for both
His-APTES and Leu-APTES and all experiments were performed
accordingly.
zeta potentials between ꢀ 10 and +10 mV are regarded as approximately
neutral, while nanoparticles are regarded to be strongly cationic and
strongly anionic with greater zeta potentials than +30 mV or less zeta
potentials than ꢀ 30 mV, respectively [50]. According to the zeta po-
tential analysis, SNPs exhibited a pH dependent trend like other nano-
particles; the more negative zeta potentials are obtained with increasing
pH [51]. The zeta potential of SNPs at pH = 5.0 showed slightly positive
charge, where His-APTES functionalization resulted strongly cationic
SNP surfaces with an average zeta potential value of +37.9 ± 1.1 mV.
Similarly, surface charge of anionic SNP with an average zeta potential
of ꢀ 27.6 ± 0.9 mV at pH = 7.4 [52] was confirmed to be altered after
His-APTES incorporation, which was found to be approximately neu-
tral/slightly positive with an average zeta potential of +8.6 ± 0.6 mV.
SNPs at pH = 9.0 have the most strongly anionic surface charge among
all others and yet SNPs modified with His-APTES resulted approximately
neutral/slightly negatively charged particles. To sum up, at pH 7.4,
His-APTES modified SNP have shown approximately neutral/slightly
positive surface charge. This finding does not fully support the positively
charged His-APTES molecules at pH = 7.4. The phenomena may be
explained as zeta potential analysis were conducted by using SNPs
instead of PDMS surfaces and aggregation of these nanoparticles may
affect the given results. Also, these findings at pH 7.4 may not represent
the actual cell culture environment. In general, cell culture studies were
performed at pH = 7.4, however pH of the medium could decrease to
more acidic pH values due to metabolic activity of cells and possible cell
debris. Under these conditions, imidazole side chain carrying unpaired
electrons can bind a proton reversibly to make the molecule partially
positively charged. For clarification of this point, cationic MB and
anionic EY adsorption study was also conducted to clarify zeta potential
measurements by using directly His-APTES modified PDMS surfaces.
Fig. 3E indicates the binding energies corresponding to O1s, C1s,
Si2s, and Si2p PDMS-specific peaks at 532.8, 285.1, 154.0, and 102.7 eV
were detected in all samples [44]. Besides, peaks at 401.1 eV appeared
after Leu-APTES and His-APTES modifications, which were assigned to
N1s core line [38]. Observation of these peaks in both modified sub-
strate surfaces was a certain indication for the presence of the molecules
on PDMS substrates. When compared between modified surfaces, the
increase of N1s peak in His-APTES modified PDMS showed relatively
higher surface nitrogen content of the surface, due to the extra nitrogen
atoms in the imidazole ring of the molecule.
The quantitative elemental composition of native and modified
substrates was summarized in Table 1. Compared to native PDMS,
modification with Leu-APTES and His-APTES resulted in increased O%
and N% with a concomitant decrease in Si%. Treatment with oxygen
plasma prior to modification was responsible for the increase in oxygen
content in both functionalized groups. The presence of N atom is an
indication of SAMs modification, and the relation between theoretical
and measured atomic percentages of N provides information about the
surface coverage. In Leu-APTES group, the atomic percentage of N was
expected to be 10.53 if the surface was fully covered, whereas XPS
analysis shows that the surface N content was only 1.35 %. Thus,
approximately 13 % of the surface is covered with Leu-APTES. Similarly,
stoichiometric and measured values of N% in His-APTES modified PDMS
are 19 and 2.96, respectively, giving a surface density of 16 %. Full
surface coverage with SAMs may not be possible due to many issues: i)
low surface densities (the number of activated surface functional
groups), ii) the orientation of SAMs in a way to hinder accessible hy-
droxyl groups and iii) the repulsive forces between SAMs.
Fig. S4 shows the amount of MB and EY dyes adsorbed (Q,
μg) on
His-APTES modified PDMS. At pH = 5.0, His-APTES modified PDMS
showed the weakest adsorption for cationic MB, due the repulsive forces
between dye cations and positively charged His-APTES molecules on the
PDMS surface; where a slight decrease in the positive charge was
expressed at pH = 7.4, and at pH = 9.0 the highest adsorption capacity
was obtained (due to the increased number of negative charges on
His-APTES modified PDMS surface). Furthermore, the anionic EY dye
adsoption study also confirmed the positively charged His-APTES mol-
ecules on the PDMS surface via electrostatic interactions. Surface charge
of His-APTES modified PDMS at pH = 5.0 and 7.4 was considered to be
positive, as anionic EY adsorption capacity at these pHs were found to be
higher than pH = 9.0. The lowest amount of EY adsorption was found on
surfaces at pH = 9.0, which demonstrates the decrease in the positive
surface charge. Here, the pH sensitivity of His-APTES modified PDMS
surface’s charge is clearly shown and the change in the surface charge is
found to be not as sensitive to pH as histidine amino acid [48,49]. Zeta
potential measurements, together with MB & EY adsorption study
confirmed the stability of positively charged His-APTES and related
hydrophilic nature of His-APTES modified PDMS at physiological pH
and cell culture conditions. In contrast, hydrophobic leucine amino acid
conjugated SAMs modification leads to an increased WCA value that fell
into the hydrophobic region [53].
Water contact angles (WCA) of hydrophilic and hydrophobic amino
acid conjugated SAMs modified PDMS substrate surfaces were utilized to
confirm the alterations in surface wettability, as shown in Fig. 3F. In
agreement with the literature, WCA of native PDMS was found in the
hydrophobic region, which was measured to be 106.2 ± 3.1◦ [45]. After
oxygen plasma treatment for 1 min, WCA was measured below 14◦ [46],
–
showing the presence of Si OH bonds. These bonds facilitated amino
acid conjugated APTES modification of PDMS surfaces. After incorpo-
ration of Leu-APTES and His-APTES, very low WCA of plasma oxygen-
ated PDMS was increased to 85.0 ± 4.9◦ and 43.5 ± 5.3◦, creating
hydrophobic and hydrophilic surfaces, respectively. Previous studies
documented the hydrophilic nature of APTES modified PDMS, which is a
–
result of functional NH2 groups of the molecule [47]. Here, both
His-APTES and Leu-APTES have these amino functional groups, and
thereby both of these SAMs modification resulted in surfaces with hy-
drophilic properties, partially. Histidine is known as a hydrophilic
amino acid with pH responsive characteristics due to its imidazole ring
side chain [48] and in this study His-APTES modified PDMS demon-
strated hydrophilic surfaces, as expected. The isoelectric point (pI) of
histidine is 7.59, where pKa value 3N-H of the imidazole ring is
pH = 6.0. Below pI, the net charge of the histidine amino acid will be
positive [49]. However, in this study, histidine is conjugated to the
APTES molecule`s amino group through its carboxylic acid group. Due
to the amide bond formation and loss of the acidic functional group
(COOH), it is not possible to accurately calculate the pI of His-APTES
molecules. Imidazole ring will be responsible for the protonation and
deprotonation of the His-APTES molecule during SAM-cell interactions.
In S1, pH responsibility of SAMs from His-APTES was investigated via
zeta potential measurements and cationic methylene blue (MB) &
anionic eosin y disodium salt (EY) adsorption study at different pH
values (pH = 5.0, 7.4 and 9.0). Zeta potential measurements of
His-APTES functionalized silica nanoparticles (SNPs) are given in
Fig. S3. In the literature, it is very well-know that, nanoparticles having
The stability of SAMs modification was confirmed under constant
flow regime for different time duration via XPS analyses and WCA
measurements (SI). Fig. S2.A shows the XPS survey spectra of His-APTES
modified PDMS surfaces under flow for 0.5 and 4 h with a control group.
All His-APTES modified PDMS–related peaks corresponding to O1s, N1s,
C1s, Si2s and Si2p were detected in all groups with no significant dif-
ferences between the signal intensities. Furthermore, similar atomic
percentages of N atom in all experimental groups are a direct evidence
for stable His-APTES modification, regardless of applied flow for
different duration. The standard deviation between measured N atomic
percentages of His-APTES modified PDMS before or after constant flow
was calculated as ± 0.08 %. Supporting the XPS analyses, WCA mea-
surements of His-APTES modified PDMS surfaces (Fig. S2.B) shows no
7