Particuology 52 (2020) 97–104
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Particuology
journal homepage: www.elsevier.com/locate/partic
Differently synthesized gold nanoparticles respond differently to
functionalization with L-amino acids
Darshana V. Havaldar, Reshma V. Patil, Disha N. Moholkar, Priyanka S. Magdum,
Akash P. Vadrale, Kiran D. Pawar ∗
School of Nanoscience and Biotechnology, Shivaji University, Kolhapur, Maharashtra, India
a r t i c l e
i n f o
Article history:
Received 13 September 2019
Received in revised form
29 November 2019
Accepted 19 December 2019
Available online 22 February 2020
Keywords:
Gold nanoparticles
Biogenic synthesis
Amino acids
Functionalization
Electrophoretic mobility
a b s t r a c t
The potential utility of gold nanoparticles (AuNPs) synthesized via different methods for biomedical applications vary greatly due to inherent differences in their surface properties. In the present
study, we investigated the functionalization of AuNPs synthesized by chemical reduction, plant extract,
and bacteria-mediated methods with 22 l-amino acids. Nanoparticles produced by bacteria-mediated
(B AuNPs) and plant-mediated (P AuNPs) methods showed good potential, as they were able to be functionalized with six (histidine, lysine, methionine, phenylalanine, tryptophan, and tyrosine) and four
(cystine, tryptophan, tyrosine, and valine) amino acids, respectively. In contrast, AuNPs produced by
chemical reduction (C AuNPs) were not found to be suitable for functionalization. Optimal functionalization conditions were found to be amino acid concentration of 20–25 mM and neutral pH (7) for
P AuNPs, whereas B AuNPs tolerated more variable conditions. The electrophoretic mobility of P AuNPs
after functionalization indicated that these nanoparticles were less sensitive than B AuNPs to the deviations from optimal conditions. A significant change in mobility was observed when B AuNPs were
functionalized with either methionine or tryptophan. Overall, the results of this study suggest that
the suitability of the three differently synthesized AuNPs with amino acids is in the following order:
B AuNPs > P AuNPs > C AuNPs.
© 2020 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of
Sciences. Published by Elsevier B.V. All rights reserved.
Introduction
A phenomenal revolution has taken place in the field of nanotechnology over the past two decades (Zhu & Xu, 2016). At present,
nanoparticles (NPs) are being used in a range of scientific and industrial applications and are also found in consumer products. The
unique properties of NPs are attributable to their size and surfacearea-to-volume ratio, and make them highly valuable for to science
and technology (Soto-Alvaredo et al., 2017). Metal NPs (MNPs),
typically particles of noble metals, are the subject of considerable
research due to their unique optical, electrical, and magnetic properties (Csapó et al., 2014) and the large number of accessible active
sites per unit area (Zhu & Xu, 2016).
Nanoparticles of gold, which are a widely studied group of NPs,
have attracted much attention because of their potential utility
in catalysis (Nita et al., 2016), electronics and sensor technologies
(Han, Park, Chun, & Yoon, 2015), and solar cells (Chen, Wang, Han,
∗ Corresponding author.
E-mail address: kdp.snst@unishivaji.ac.in (K.D. Pawar).
Cheng, & Qian, 2015). Gold NPs (AuNPs) are of particular interest as
they have wide range of properties including a high surface-areato-volume ratio, biocompatibility (Yeh, Creran, & Rotello, 2012),
and low toxicity (Khlebtsov & Dykman, 2011; Murphy et al., 2008).
These unique properties make them an effective tool for various
biomedical applications such as drug and gene delivery (Hussain
& Hussain, 2015), tissue engineering (Vial, Reis, & Oliveira, 2017),
and microbe detection and identification (Syed & Bukhari, 2011).
Scientists have been working to further improve the surface
properties of NPs to create multifunctional NPs functionalized with
specific recognition moieties like enzymes, antigens, antibodies,
proteins (Subbiah, Veerapandian, & Yun, 2010). Surface functionalization of AuNPs alters the physico-chemical properties (Baptista
et al., 2008; Radwan & Azzazy, 2009; Uehara, 2010), which may play
a crucial role in a number of applications, particularly in biomedical
treatments such as cancer therapy (Muddineti, Ghosh, & Biswas,
2015; Yamada, Foote, & Prow, 2015), radiotherapy (Ngwa et al.,
2014), or enable their use as drug carriers (Ghosh, Han, De, Kim, &
Rotello, 2008). The size of nanoparticles limits their use for targeted
drug delivery to particular cells, which they can enter simply by
translocating across the membrane. To resolve this issue, nanopar-
https://doi.org/10.1016/j.partic.2019.12.010
1674-2001/© 2020 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.
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D.V. Havaldar et al. / Particuology 52 (2020) 97–104
ticles have been functionalized with a number of biomolecules
including ligands, amino acids, and proteins (Tiwari, Vig, Dennis,
& Singh, 2011). The conjugation of proteins or amino acids with
MNPs stabilizes the system and increases biocompatibility (Bohara,
Thorat, & Pawar, 2016).
It is noteworthy that the degree to which MNPs, such as AuNPs,
can be surface functionalized vary between differently synthesized
MNPs. For example, AuNPs synthesized via chemical and physical methods may be difficult to surface functionalize owing to
the absence of surface-bound functional groups. In comparison,
biologically synthesized (biogenic) AuNPs may be more easily functionalized, enabling their decoration with molecules for imaging
or therapeutic applications (Mukherjee & Patra, 2017). Biogenic
AuNPs are highly stable and monodisperse compared with NPs synthesized via chemical or physical methods, which increases their
potential utility in various biomedical applications (Mukherjee
& Patra, 2017). The ease of surface functionalization of biogenic
AuNPs may stem from the inclusion of biomolecules such as
proteins, lipids, and carbohydrates, which act as reducing and
capping agents during biogenic synthesis. The presence of these
biomolecules or their functional groups creates an external biomatrix around the surface of the MNPs, which enables easy and
effective binding of drug molecules, thus avoiding the need for
chemical capping agents (Mukherjee & Patra, 2017).
To date, there have been no systematic studies on the surface
functionalization of MNPs such as AuNPs synthesized by different
methods. To address this paucity of information, the present study
investigated the interactions and responses of differently synthesized AuNPs that were functionalized with 22 natural l-amino
acids (AA). To this end, AuNPs were synthesized by three different
routes; namely, chemical (C AuNPs), plant-mediated (P AuNPs),
and bacteria-mediated (B AuNPs) synthesis, and then the success
of functionalization with AAs was tested. Functionalization was
characterized and monitored with ultraviolet visible (UV–vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), agarose
gel electrophoresis (AGE), and transmission electron microscopy
(TEM) imaging.
Material and methods
Materials
Gold (III) chloride trihydrate (HAuCl4 ·3H2 O) was purchased from Hi Media (Mumbai, India). Trisodium citrate
(Na3 C6 H5 O7 ·2H2 O), Luria-Bertani (LB) medium, sodium hydroxide
(NaOH), hydrochloric acid (HCl), and the 22 AAs were procured
from Merck Bioscience (Mumbai, India). All chemicals were of pure
analytical grade, solutions and reagents were prepared in sterile
double distilled water (SDDW).
Methods
Chemical synthesis of gold nanoparticles
Chemical synthesis of colloidal AuNPs was carried out using
Turkevich’s method (Turkevich, Stevenson, & Hillier, 1951), which
is based on HAuCl4 and trisodium citrate. Briefly, 10 mL of an aqueous solution of trisodium citrate (38.8 mM) was added drop-wise to
100 mL of HAuCl4 (0.5 mM) solution under continuous stirring and
boiling conditions. After 5 min, the solution turned from its original
light yellow to a ruby-red color, indicating the synthesis of colloidal
C AuNPs.
Plant-mediated synthesis of gold nanoparticles
Plant-mediated biogenic AuNPs were synthesized using an
aqueous extract of almond (Prunusdulcis) seed coats as a source
of capping and reducing agents. To this end, almond seeds were
soaked in SDDW for 24 h, after which seed coats were manually separated using surgical forceps, air dried, and then crushed into fine
powder. Subsequently, 4–6 g of seed-coat powder was suspended
in 100 mL SDDW, boiled for 2 h, filtered through Whatman No. 1
filter paper and then centrifuged at 10,000 rpm for 10 min. In a typical biogenic synthesis reaction, almond-seed-coat (ASC) extract
was added to HAuCl4 solution (4 mM) in a 2:1 ratio, then the pH
adjusted to 6 with 1 N NaOH. The mixture was incubated at 90 ◦ C
for 5–10 min. The progress of the reaction was monitored visually
by observing the change of color from light brown to ruby red and
recording the surface plasmon resonance (SPR) spectrum.
Bacteria-mediated synthesis of gold nanoparticles
For bacteria-mediated biogenic AuNP synthesis, we used
a previously isolated, screened, and identified bacterial
strain—Pseudomonas stutzeri, which was capable of synthesizing iron oxide magnetic nanoparticles and AuNPs (Desai & Pawar,
2020). This bacterial strain was maintained in our laboratory and
used in the present study. Briefly, P. stutzeri was cultured in LB
broth for 72 h at 37 ◦ C on an orbital shaker, then the biomass was
separated by centrifugation at 10,000 rpm for 10 min. Next, 2 g of
bacterial biomass was re-suspended in 20 mL of SDDW, dispersed
by vortexing gently, and then used as a source of reducing and
capping agents for B AuNP synthesis. Briefly, 10 mL of biomass
solution was mixed with 0.3 mL HAuCl4 (3 mM), and the pH was
adjusted to 9 with 1 N NaOH. The mixture was incubated at 80 ◦ C
for 1 h and the appearance of the ruby-red color monitored by
visual inspection to determine successful synthesis of B AuNPs,
which was confirmed by recording the UV–vis spectrum.
Characterization of gold nanoparticles
To confirm the synthesis AuNPs, UV–vis spectra were recorded
in the range of 250–800 nm on a biospectrophotometer (Eppendorf, USA). To characterize the functional groups on the surface of
C AuNPs, P AuNPs, and B AuNPs, FTIR spectra were recorded from
400 to 4000 cm−1 using FTIR spectrometer (Shimadzu, Japan). The
size and morphology of AuNPs were analyzed by TEM (JEM-2100,
JOEL, USA).
Functionalization and characterization of gold nanoparticles
decorated with amino acids
For functionalization of all NPs, aqueous stock solutions of
each AA (100 mM) were prepared. The functionalization reactions
(200 ␮L) were set up by mixing aqueous stock solutions of each of
AA (25 mM) under study with either C AuNPs, P AuNPs, or B AuNPs
in a 1:4 ratio and left to react for 24 h with stirring at room temperature. These reactions were then monitored and confirmed by
observing the change in color and recording the UV–vis spectra in
the range of 250–800 nm to study spectral shifts and aggregation.
Furthermore, for AuNPs which were successfully functionalized
with AAs (AA@AuNPs), the effects of AA concentration and pH
were studied by varying these two parameters in the ranges of
0.2–25 mM and 1–9 respectively. The AA@AuNPs were then characterized and confirmed by recording and comparing the FTIR spectra.
Effect of variation of amino acid concentration and pH on
electrophoretic mobility
The effects of variation from the optimum AA concentration and
pH on aggregation (size) and surface charge of AuNPs were studied
by assessing the electrophoretic mobility of the AA@AuNPs on AGE.
To this end, P AuNPs and B AuNPs were functionalized with optimum or increased (25 mM) concentrations of AAs and at optimum
and ±1 pH. After functionalization, 20 ␮L each of AA@P AuNPs or
D.V. Havaldar et al. / Particuology 52 (2020) 97–104
AA@B AuNPs were mixed with 5 ␮L of loading buffer (with 30%
sucrose and 0.5% bromophenol blue), loaded into wells of 0.8%
agarose gel slabs and then run at 50 V for 20 min in TAE buffer
(40 mM Tris, 20 mM acetate, and 1 mM ethylene diamine tetra
acetic acid) at pH 7.
Results and discussion
Synthesis and characterization of gold nanoparticles
In the present study, we successfully synthesized C AuNPs,
P AuNPs, and B AuNPs using the Turkevich, ASC-extract, and P.
stutzeri-mediated methods, respectively. The appearance of a rubyred color demonstrating the synthesis of colloidal AuNPs was
observed during all syntheses (Fig. 1, inset images). Synthesis
by the three methods was also confirmed by the characteristic
absorption spectra in the range of 520–540 nm that were recorded.
Precisely, SPR peaks were recorded with maximum intensities at
525, 537, and 527 nm for C AuNPs, P AuNPs, and B AuNPs, respectively (Fig. 1(a)). The morphology and size of NPs were assessed
by TEM imaging, which revealed mostly spherical morphologies,
with sizes ranging between 5 and 30 nm. We observed C AuNPs
and P AuNPs to be mostly spherical with some irregularly shaped
particles, and the preparations were polydisperse in nature, with
an average size of 5–15 nm, whereas B AuNPs were mostly spherical and monodisperse with a narrow size distribution in the range
of 20–30 nm (Fig. 1(b)–(d)).
Functionalization of gold nanoparticles of amino acids
While AuNPs belong to the group of noble-metal NPs, they
surpass other noble-metal NPs in terms of their non-toxicity, exceptional biocompatibility, and inactivity toward the physiological
environment (Boisselier & Astruc, 2009). Although bare AuNPs
possess an excellent range of physical and chemical properties,
they are not always appropriate for biomedical applications due
to a lack of suitable surface groups for loading with molecules
of biomedical importance (Giljohann et al., 2014; Zhang, 2015).
Surface functionalization of AuNPs with biomolecules, particularly
AAs or proteins, may overcome these shortcomings, meaning that
functionalized AuNPs may be more suitable for various biomedical applications. Recently, researchers have begun to functionalize
AuNPs with peptides and proteins (Chen & Rosi, 2010; Dickerson,
Sandhage, Kenneth, & Naik, 2008) to manufacture NPs with desired
properties. Because AAs are the building blocks of peptides and
proteins, a better understanding of how they react with AuNPs
could inform the design of NPs for various biomedical applications. The wide variety of side chains and functional groups of
AAs can enable investigations into the binding abilities of AAs with
AuNPs. Thus, the different chemical and physical properties of the
20 AAs can lead to distinct NP preferences (Shao & Hall, 2016)
with respect to chemical, plant-mediated, and bacterial-mediated
AuNPs.
In the present study, we compared the surface functionalization
of C AuNPs, P AuNPs, and B AuNPs with 22 L-AAs. Surface plasmon
resonance is an important optical property of MNPs, which depends
on the absorption of specific bands of light by colloids of metals
(Zarabi, Arshadi, Farhangi, & Akbarzadeh, 2014). In SPR, the incident
electromagnetic field causes resonance of conduction band electrons on the surface of the particles, resulting in a strong absorption
band in the spectrum (Link & El-Sayed, 2003). The size, shape, and
aggregation of MNPs determine the width, sharpness, and position
of the absorption peak (Dutta & Hofmann, 2004; Link & El-Sayed,
2003). Owing to this mechanism, UV–vis spectroscopy confirms the
conjugation of AuNPs and AAs (Doyen, Goole, Bartik, & Bruylants,
99
2016). Therefore, in the present study, we monitored surface functionalization by recording the SPR of functionalization reactions
and monitoring the color change during the reaction. Recent studies indicate that successful surface functionalization of MNPs leads
to visible color changes of the functionalized colloidal solution and
bathochromic shifts of >10 nm in the SPR peaks without aggregation (Bohara et al., 2016; Ghosh & Pal, 2007). Thus, reactions that
exhibited visible color changes, bathochromic shifts of >10 nm in
the SPR peaks, and no aggregation were considered successful in
the present study.
Initial screening with 22 AA for functionalization of C AuNPs,
P AuNPs, and B AuNPs showed variable responses in terms of
visible color changes, bathochromic shifts in SPR peaks, and aggregation of AuNPs (Fig. 2; Table 1). Distinct color changes and
aggregation were observed when C AuNPs were reacted with nine
of the 22 AAs; namely, arginine (Arg), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), histidine (His), isoleucine (Ile), leucine
(Leu), methionine (Met), and lysine (Lys). The SPR peaks of the
resulting preparations showed bathochromic shifts in the range
of 16–175 nm (Fig. 2(a); Table 1). These larger shifts (>50 nm),
together with visual observation, clearly indicate that C AuNPs
aggregate upon interaction with these nine AAs, which was confirmed by the observed color change. The rest of the tested AAs
neither caused color changes nor bathochromic shifts in SPR
peaks (Fig. 2(a); Table 1). Ideally, functionalization of MNPs with
biomolecules should not induce aggregation or large shifts in the
absorption maxima of MNPs (Bohara et al., 2016). Based on our
observations, C AuNPs do not appear to be suitable for functionalization with AAs.
When similar functionalization reactions were performed with
P AuNPs, seven AAs-Cys, cysteine free base (CysFB), glutamic acid
(Glu), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine
(Val)—caused a slight color change from dark ruby to purple and
bathochromic shifts in the range of 10–15 nm. However, CysFB,
Glu, and Thr induced aggregation of P AuNPs (Table 1), whereas
the remaining four AA did not. Thus, the observed slight color
change and marginal bathochromic shifts without aggregation
demonstrated the good potential of P AuNP for surface functionalization with Cys, Trp, Tyr, and Val. In comparison, P AuNPs showed
good potential for functionalized with AA than C AuNP (Fig. 3(a);
Table 1).
We found B AuNPs to be most suitable for functionalization, as
we were able to successfully functionalize the surface of these NPs
with six AAs. Initially, 12 AAs; namely, asparagine (Asn), Asp, Cys,
CysFB, His, hydroxy-l-proline (Hypro), Met, phenylalanine (Phe),
Trp, Tyr, Val, and Lys; showed color changes and bathochromic
shifts in the range 10–28 nm. Of these, His, Met, Phe, Trp, Tyr, and
Lys did not induce aggregation, indicating that functionalization
of B AuNPs was successful. Functionalization was confirmed by
UV–vis. absorption spectroscopy, where amino-acid-related peak
intensities were found to be decreased in the spectra of separated
functionalized AuNPs.
The present study therefore demonstrates that biogenic AuNPs
such as P AuNPs and B AuNPs are excellent candidates for functionalization, as we were able to successfully functionalize these
NPs with four and six AA, respectively (Fig. 3).
Effect of amino acid concentration and pH on functionalization
The influence of AA concentration and pH on the functionalization of P AuNPs and B AuNPs with these four and six AAs,
respectively, were investigated. Our experiments of varying AA
concentration and recording the pH of the system revealed
that AA concentration did not affect the pH of the system
(Tables S1 and S2. For Tables S1–S6, see the Supporting Material).
100
D.V. Havaldar et al. / Particuology 52 (2020) 97–104
Fig. 1. Results of synthesis of gold nanoparticles using three different methods. (a) Absorption spectra of gold nanoparticles produced by chemical reduction, plant-mediated
production, and bacteria-mediated production (C AuNPs, P AuNPs, and B AuNPs, respectively). Transmission electron microscopy (TEM) images of (b) C AuNPs, (c) P AuNPs,
and (d) B AuNPs (inset images show the color changes during the three different reactions used for synthesis). Abbreviations: ASC, almond seed coat; BB, bacterial biomass.
Table 1
Bathochromic shifts, color changes, and aggregation during the functionalization of gold nanoparticles produced by chemical reduction (C AuNP), plant-mediated production
(P AuNP), and bacteria-mediated methods (B AuNP) with 22 amino acids (AAs).
AA
Ala
Arg
Asp
Asn
Cys
Cys FB
Glu
Gln
Gly
His
Hypro
Ile
Leu
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
Lys
C AuNP (525 nm)
P AuNP (537 nm)
B AuNPs (527 nm)
Bathochromic
shift (nm)
Color
change
Aggregation
Bathochromic
shift (nm)
Color change
Aggregation
Bathochromic
shift (nm)
Color
change
Aggregation
0
143
121
0
175
0
0
16
0
143
0
175
175
143
0
0
0
0
0
0
0
122
No
Yes
Yes
No
Yes
No
No
Yes
No
Yes
No
Yes
Yes
Yes
No
No
No
No
No
No
No
Yes
No
Yes
Yes
No
Yes
No
No
Yes
No
Yes
No
Yes
Yes
Yes
No
No
No
No
No
No
No
Yes
2
0
0
0
11
11
15
1
0
2
0
0
0
0
0
2
0
12
10
12
10
0
No
No
No
No
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
No
No
No
No
No
No
Yes
Yes
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
2
2
13
10
28
10
3
2
1
21
16
2
2
16
18
1
2
2
21
21
21
13
No
No
Yes
Yes
Yes
Yes
No
No
No
Yes
Yes
No
No
Yes
Yes
No
No
No
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
Yes
No
When surface functionalization of P AuNPs was carried out with
Cys, Trp, Tyr, and Val, 20–25 mM of these AA and neutral pH (7)
were the optimum conditions. Color change and bathochromic
shifts in the range of 10–12 nm with no aggregation were observed
(Tables S3 and S4). In contrast, optimal functionalization of B AuNPs
required more variable conditions. Functionalization with His was
most successful at 4 mM in acidic conditions of pH 5, whereas Lys
functionalization was optimal at 6 mM and neutral pH (7). The
functionalization of Met and Trp required 0.6 mM of AA and pH
5. Similarly, functionalization with Phe and Tyr was optimal at 6
and 0.4 mM and pH 5 and 3, respectively (Tables S5 and S6). Amino
acids with isoelectric points (pI) in the range of 5–6 were found to
be most suitable for functionalization of P AuNPs, whereas B AuNPs
can be functionalized with AAs with a broader range of pIs (5–10).
D.V. Havaldar et al. / Particuology 52 (2020) 97–104
101
Fourier transform infrared analysis
Fourier transform infrared analysis of functionalized
plant-mediated gold nanoparticles
In the present study, FTIR analyses were performed and the
results compared with the literature to confirm functionalization
of NPs with AA. The FTIR analysis of Cys@P AuNPs (Fig. S1. For Figs.
S1–S10, see the Supporting Material) showed an intense IR band
at 1370 cm−1 , which can be attributed to the vibrational stretching of C O bonds. A number of small IR bands in the 600–800
cm−1 region were observed due to C–S vibrational stretching. Additionally, an IR band was identified at 1035 cm−1 , related to C–NH2
bonds (Huang, Lan, & Chen, 2011). When P AuNPs were functionalized with Trp, vibrational stretching due to CH deformation
modes in the benzene and pyrrole ring were evident from the
IR bands observed at 922 and 868 cm−1 , respectively (Fig. S2). In
addition, a peak was observed at 1139 cm−1 , which could have
occurred due to C–H stretching in the benzene and pyrrole rings
(Pajovića et al., 2015). The FTIR spectrum of Tyr@P AuNPs (Fig.
S3) exhibited a band at 1748 cm−1 , which can be attributed to
carbonyl stretching (Dubey et al., 2015). Likewise, the IR spectrum of Val@P AuNPs (Fig. S4) showed adsorption bands in the
600–800 cm−1 region. In addition, IR bands were observed at 926,
1340, 1395, and 1516 cm−1 corresponding to vibrational stretching of C C deformation, C C O deformation, C O stretching,
COO– asymmetric stretching, and NH3 deformation, respectively
(Sangeetha, Mariappan, Madhurambal, & Mojumdar, 2012). These
IR spectra clearly confirm the successful functionalization of
P AuNPs with Val (Sangeetha et al., 2012).
Fig. 2. Absorption spectra and color changes during functionalization of gold
nanoparticles produced by (a) chemical reduction, (b) plant-mediated production,
and (c) bacteria-mediated production (vials are numbered in line with amino acid
numbering on the image).
Fourier transform infrared analysis analyses of functionalized
bacteria-mediated gold nanoparticles
The FTIR spectrum of B AuNPs functionalized with His (Fig. S5)
exhibited adsorption peaks in the 600–750 cm−1 region, which
were attributable to the bending vibrations of sp2 CH groups, and
bands at 1398 and 1426 cm−1 were attributed to the symmetric
stretching mode of COO− groups (Liu et al., 2010; Mesu, Visser,
Soulimani, & Weckhuysen, 2005; Ogura, Kobayashi, Nakayama,
& Miho, 1999). Functionalization of B AuNPs with Lys was confirmed by the FTIR spectrum (Fig. S6), which showed bands at
1340, 1418, and 1507 cm−1 corresponding to NH2 vibration, free Lys
molecules, and carboxyl double bonds, respectively (Barth, 2001;
Bonor, Reddy, Akkiraju, Dhurjati, & Nohe, 2014).
The FTIR spectrum of Met@B AuNPs (Fig. S7) showed a broad
peak at 865 cm−1 due to C C stretching, in addition to a number
of bands attributable to C CH bending (921 cm−1 ), CH3 rocking
(975 cm−1 ), C C stretching (1340 cm−1 ), CO2 – symmetric stretching (1425 cm−1 ), and C NH bending (1519 cm−1 ) (Grunenberg &
Bougeard, 1987; Grunenberg & Bougeard, 1986; Ramachandran
& Natarajan, 2006). However, the FTIR spectra of Phe@B AuNPs
and Trp@B AuNPs contained fewer bands. The IR spectrum of
Phe@B AuNPs (Fig. S8) displayed two small, characteristic bands at
1424 and 1564 cm−1 which were related to carboxylate symmetric and asymmetric stretching, respectively (Carubelli, Massabni,
& Leite, 1997). This confirmed the success of functionalization of
B AuNPs with Phe. In contrast, the FTIR spectrum of Trp@B AuNPs
(Fig. S9) exhibited IR bands at 922 and 865 cm−1 due to CH
deformation modes in the benzene and pyrrole rings, respectively
(Pajovića et al., 2015). It is well established that the gold surface can form electrostatic interactions with carboxylate (Park &
Shumaker-Parry, 2014), or covalent bonds with amines via under-
102
D.V. Havaldar et al. / Particuology 52 (2020) 97–104
Fig. 3. Absorption spectra and color changes indicating the successful functionalization of gold nanoparticles produced by (a) plant-mediated production and functionalized
with Cys, Trp, Tyr, and Val, and (b) produced by bacteria-mediated production and functionalized with His, Lys, Met, Phe, Trp, and Tyr.
Fig. 4. Transmission electron microscopy images of gold nanoparticles produced by bacteria-mediated production and functionalized with (a) histidine and (b) tyrosine.
Aggregation and changes in the particle size/shape can be seen.
Fig. 5. Electrophoretic mobilities of gold nanoparticles produced by bacteria- and plant-mediated production (P AuNPs and B AuNPs, respectively) and functionalized using
optimized or increased amino acid (AA) concentrations, and at optimum or ±1 pH. (a) The electrophoretic mobilities of P AuNPs functionalized with optimum (left-hand
side well) or increased AA concentration (right-hand side well). (b) The electrophoretic mobilities of P AuNPs functionalized at optimum (middle well), optimum +1 (righthand side well) or optimum −1 pH (left-hand side well). (c) The electrophoretic mobilities of B AuNPs functionalized using optimum (left-hand side well) or increased AA
concentration (right-hand side well). (d) The electrophoretic mobilities of B AuNPs functionalized at optimum (middle well), +1 (right-hand side well), or −1 pH (left-hand
side well). The numbers on the figures indicate the pH of functionalization reaction.
D.V. Havaldar et al. / Particuology 52 (2020) 97–104
103
coordinated gold atoms (Hoft, Ford, McDonagh, & Cortie, 2007).
Similar to Tyr@P AuNPs, the IR spectrum of Tyr@B AuNPs (Fig. S10)
showed a band at 1740 cm−1 corresponding to carbonyl stretching
(Dubey et al., 2015), which confirmed successful functionalization.
can be used as linker molecules for functionalization with drugs
that would otherwise be difficult to attach to AuNPs.
Effect of amino acid concentration and pH on electrophoretic
mobility
We synthesized AuNPs via three different routes to study
their functionalization with 22 AAs. Our results demonstrate that
P AuNPs can be successfully functionalized with four particular
AAs: Cys, Trp, Tyr, and Val, while B AuNPs are most easily functionalized with six AAs: His, Lys, Met, Phe, Trp, and Tyr. Notably,
C AuNPs did not show any potential for conjugation with AA.
Functionalized B AuNPs had increased electrophoretic mobility
compared with functionalized P AuNPs. Functionalization of AuNPs
with high concentrations of AAs such as His and Tyr resulted in
changes in the size and shape of AuNPs, and very limited electrophoretic mobility. The results of the present study suggest
the suitability of AuNPs synthesized by the three methods presented here for functionalization to be in the following order:
B AuNPs > P AuNPs > C AuNPs. Thus, the present study successfully
demonstrates that functionalization of MNPs with AAs can be a
good approach to impart functionality and conjugate further with
biologically active agents and drugs.
Adjusting the pH of the functionalization reaction can be used
to control the aggregation of functionalized AuNPs which, in turn,
may influence the electrophoretic mobility of the particles (Daniel
& Astruc, 2004). In fact, changes in the mobility of functionalized
AuNPs may indicate changes in either the size or shape, which will
affect their ability to react and aggregate in solution (Tiwari et al.,
2011).
Functionalization using optimum or increased (25 mM) AA concentration revealed that the concentration of AAs did not affect
the electrophoretic mobilities of Cys@P AuNPs, Trp@P AuNPs,
Tyr@P AuNPs, and Val@P AuNPs. Similarly, functionalization carried out at either optimum or ±1 pH demonstrated that this
parameter did not influence the electrophoretic mobility. The
P AuNPs appear to be less sensitive to the AA concentration
and pH then the other B AuNPs, as the latter were found to
respond to changes in these conditions. Lower electrophoretic
mobilities were observed for His@B AuNPs and Tyr@B AuNPs that
were functionalized with higher AA concentrations, indicating
aggregation and reduced negative charge. We therefore investigated whether the reduced mobility was due to aggregation
of B AuNPs by TEM imaging, which revealed that high concentrations of His or Tyr induced aggregation of functionalized
B AuNPs (Fig. 4). In comparison with Lys, high His concentrations also resulted in altered sizes and shapes of some of the
B AuNPs (Fig.5(c)). The electrophoretic mobility was marginally
increased for Met@B AuNPs and Lys@B AuNPs, indicating a slight
increase in negative charge. However, electrophoretic mobility
was unchanged for Phe@B AuNPs and Trp@B AuNPs (Fig. 5(c)).
Notably, varying the reaction pH had a larger effect on the mobilities of His@B AuNPs, Met@B AuNPs, Lys@B AuNPs, Trp@B AuNPs,
although this did not affect the mobilities of Phe@B AuNPs or
Tyr@B AuNPs (Fig.5(d)). For Met@B AuNPs and Trp@B AuNPs,
changing the pH by 1 reduced the mobility, indicating reduced
negative charge and aggregation, whereas increasing the pH by 1
increased the mobility. Decreasing the pH by 1 did not affect the
mobility of Lys@B AuNPs. Decreasing the pH reduced the mobility
of His@B AuNPs, and the electrophoretic mobility of Trp@B AuNPs
was reduced when functionalization was carried out at the optimum pH ± 1. In line with our observations, a previous report
by Zare, Akbarzadeh, and Bararpour (2010) suggests that the
interaction of Glu- and glutamate-capped AuNPs affects the electrophoretic mobility, as Glu-capped AuNPs exhibited less mobility
toward the positive pole than glutamate-capped AuNPs (Davood
Zare, Khoshnevisan, Barkhi, & Tahami, 2014). Furthermore, Zare
et al. (2010) demonstrated the effect of pH on the interaction of
Arg and Asp with AuNPs using AGE, confirming that low pH neutralizes the negative charge of AuNPs upon conjugation with AAs such
as Arg and reduces electrophoretic mobility (D Zare et al., 2010).
Taken together, the results of the present and previous studies
indicate that the pH of the functionalization reaction is an important parameter which determines the success of functionalization,
surface charge, aggregation, and electrophoretic mobility of functionalized MNPs.
Our results clearly demonstrate that bacteria-mediated biogenic AuNPs have good potential in terms of their suitability for
functionalization with biomolecules and drugs with various properties and functional groups. In addition, this functionalization of
bacteria-mediated biogenic AuNPs can be further extended as AAs
Conclusions
Conflicts of interest
None.
Acknowledgements
This research did not receive any specific grants from funding
agencies in the public, commercial, or-not-for profit sectors.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in
the online version, at doi:https://doi.org/10.1016/j.partic.2019.12.
010.
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