The paper shows our preliminary results on the different spectroscopic behavior of three types of gold nanoparticles (obtained respectively by chemical synthesis, laser ablation in pure water and laser ablation in a citrate solution) modified with trans-zeatin, a plant growth regulator, in presence of glutathione. The reaction of ligand substitution of the adsorbed zeatin with glutathione is studied through surface enhanced Raman spectroscopy and is revealed to occur only when citrate-laser ablated gold nanoparticles are employed, making these particles potentially good candidates as vehicles of zeatin inside plant cells for future agricultural applications

Giammanco, F.

Giorgetti, E.

Leva, A.

Margheri, G.

Marsili, P.

Molina-Contreras, J.R.

Muniz-Miranda, M.

Trigari, S.

Zoppi, A.

Electronic Sumy State University Institutional Repository

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  NANOMATERIALS: APPLICATIONS AND PROPERTIES Vol. 4 No 2, 02NNSA02(4pp) (2015)   2304-1862/2015/4(2)02NNSA02(4) 02NNSA02-1  2015 Sumy State University Glutathione-Induced Release of Zeatin From Functionalized Gold Nanovectors  A. Zoppi1,2, J.R. Molina-Contreras3,*, P. Marsili1,2, M. Muniz-Miranda4, G. Margheri2, S. Trigari2, A. Leva5, E. Giorgetti2, F. Giammanco1  1 Physics Department, University of Pisa, Largo B. Pontecorvo 3,56127 Pisa, Italy 2 ISC, CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy 3 Departamento de Ingeniería Eléctrica y Electrónica, Instituto Tecnológico de Aguascalientes,  Av. Adolfo López Mateos No 1801 Ote., Fracc. Bona Gens, 20256, Aguascalientes, Ags, México  4 Department of Chemistry, University of Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy 5 IVALSA, CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy  (Received 15 March 2015; published online 22 August 2015)  The paper shows our preliminary results on the different spectroscopic behavior of three types of gold nanoparticles (obtained respectively by chemical synthesis, laser ablation in pure water and laser ablation in a citrate solution) modified with trans-zeatin, a plant growth regulator, in presence of glutathione. The reaction of ligand substitution of the adsorbed zeatin with glutathione is studied through surface enhanced Raman spectroscopy and is revealed to occur only when citrate-laser ablated gold nanoparticles are em-ployed, making these particles potentially good candidates as vehicles of zeatin inside plant cells for future agricultural applications.   Keywords: Zeatin, Glutathione, Gold Nanoparticles, Raman, SERS.   PACS numbers: 82.70.Dd, 87.14.Lk, 87.64.kp                                                             * jrmolinacon@gmail.com 1. INTRODUCTION  The number of applications in biosensors, sensors and biomedical diagnostics based on the interactions between nanoparticles and biologically relevant mole-cules, has attracted an increasing interest [1-4]. The study of model systems such amino acids or nucleic acids on well characterized nanoparticle-surfaces has represented a useful approach to developing an under-standing of the structure and stability of bio-molecules formed at metal interfaces. In particular, Surface En-hanced Raman Scattering (SERS) turned to be a power-ful investigation method for the detection of low analyt-ical concentrations and for the identification of the con-formation and orientation of molecules adsorbed on metal surfaces [5,6]. In this work we report our preliminary results on the studies of the capability of glutathione to induce the release of adsorbed zeatin from gold nanoparticles (AuNP), using SERS technique.  Zeatin, a derivative of purine, is a natural cyto-kinin which regulates multiple developmental and physiological processes in a plant through its ability to promote cell division, keep meristematic cell iden-tity, maintain high cellular redox potentials during abiotic stress and regulate nutrient sink/source ac-tivity of different organs [7]. The chemical structure of zeatin is basically that of adenine whose SERS spectrum has been measured on both Ag and Au substrates [8-10]. Despite this cytokinin exists as cis- and trans-structures, only trans-zeatin (Fig. 1a) is biologically active and it is used in in vitro culture to stimulate the growth and the bud formation. Howev-er, due to its high cost, it is not recommended for large-scale subministrations.  Glutathione is a non enzymatic antioxidant which has a role in the defence mechanism and antioxidant capacity in plant under abiotic stress [11]. It is an im-portant constituent of all living cells and the most abundant thiol species at a concentration range of 1−10 mM in the cytoplasm. It is a tripeptide made of glutam-ic acid, cysteine and glycine (Fig. 1b) where the –SH functionality is known to exhibit a strong affinity with metallic gold. For instance, this affinity has been al-ready exploited for drug release from gold nanoparti-cles surfaces [12].    Fig. 1 – Chemical structures of: a) trans-zeatin and b) gluta-thione.  The basic idea is that glutathione, physiologically present in the cells, drives the release of zeatin  mole-cules from hormone-labeled Au nanoparticles, thanks to its higher affinity for gold. Indeed, AuNP have been recently demonstrated to enter and accumulate in plant cells with very low or none toxicity [13, 14]. In this respect, AuNP could offer an efficient tool for the loading and transportation of zeatin inside plant cells, where it can be freed by glutathione exchange. Zeatin-enriched  gold nanoparticles could be thus employed as novel vectors that can potentially deliver a higher amount of phytohormones, reducing costs in plants growth procedures, like for instance in commercial mi-cropropagation protocols. To check the ability of glutathione to induce a lig- A. ZOPPI, J.R. MOLINA-CONTRERAS ET AL. PROC. NAP 4, 02NNSA02 (2015)   02NNSA02-2 and-exchange reaction on zeatin-labeled Au nanoparti-cles,  three different gold aqueous colloidal suspensions were tested: AuNP_chem was obtained by chemical reduction of gold ions with sodium citrate, AuNP_abl was prepared by laser ablation in pure water and AuNP_citr_abl was obtained by laser ablation in a cit-rate solution. The colloidal suspensions were modified with zeatin and glutathione addition was inspected through Raman measurements. We found that ligand exchange occurred only with AuNP_citr_abl and we believe that these results could be useful for the understanding of complex biological systems opening the way to applications in plant sci-ence, agriculture and biotechnology.  2. EXPERIMENTAL  2.1 Sample preparation  AuNP_chem was prepared by Na-citrate reduction of HAuCl4 according to the modified Turkevitch method [15]. Laser ablation was performed by irradiating a gold target with the fundamental wavelength (1064 nm) of a Q-switched Nd:YAG laser (rep. rate 10 Hz, pulse width 17 ns) with 220 mJ pulse energy. The laser was focused on the target in order to obtain a circular spot of 1.5 mm, corresponding to a 124 mJ/mm2 fluence. AuNP_abl was prepared in pure water while AuNP_citr_abl was obtained in a 10-4 M Na-citrate solution.  Trans-zeatin was used as an alkaline solution (pH 12), glutathione was dissolved in pure water. Ultrapure water (18.2 MΩ·cm @ 25°C) was used for all prepara-tions.  2.2 Methods  Extinction spectra were obtained at room tempera-ture using a Cary 5 spectrometer in quartz cuvettes.  Raman spectra were recorded at room temperature using a single grating RM2000 Renishaw micro-Raman apparatus with an air cooled, charge coupled device and coupled with a near IR laser diode emitting at 785 nm. Raman spectra were taken on a dried drop of the colloidal suspension put on an aluminium foil. A 20x magnification objective was used to probe the drop in several points. FT-SERS spectra were recorded using a Multi-Raman (Bruker) apparatus operating at 1064 nm and coupled to a liquid-N2 cooled Ge detector. Irradiation power and spectral resolution were respectively set at 500 mW and 4 cm-1. A 10 mm glass cuvette was used to record the spectra of 0.5 ml of the suspensions, averag-ing over 200 scans. In order to check the glutathione-induced release of zeatin from gold nanoparticles, the colloidal con-centrations were previously adjusted to a 0.8 extinc-tion maximum (optical path 2 mm). Zeatin was add-ed to the colloidal suspension to a final concentration of 10-5 M (pH 4-5) and spectral characterization was performed after ten minutes. The effect of 10-3 M glutathione was then checked. This value of concen-tration was selected according to the glutathione level in living cells.  3. RESULTS AND DISCUSSION   3.1 AuNP_chem colloidal suspension  The citrate-reduced AuNP colloid was very sensitive to the addition of 10-5 M zeatin. This was macroscopi-cally evidenced by the colour changes of the suspension. Fig. 2 compares the extinction spectrum of the pure colloid with those acquired on zeatin@AuNP_chem for different cytokinin concentrations. As evidenced, zeatin addition caused a flattening of the main plasmonic band at 526 nm, that is characteristic of spherical par-ticles, while a new band appears and becomes promi-nent in the 750-800 nm spectral range, attributable to nanoparticle aggregation. Besides, the absorption peak of zeatin occurs at 270 nm.    Fig. 2 – UV-vis spectra of the Au_NP_chem colloidal suspen-sion before and after the addition of zeatin 10-6 M, 10-5 M, 10-4 M. The absorption peak at 270 nm is attributed to the cytokin-in molecule.   The SERS spectra of zeatin at 10-4, 10-5 and 10-6 M concentrations were then taken on dried drops. Fig. 3 compares three typical SERS spectra with the Raman spectrum taken on an aqueous solution of zeatin.   Fig. 3 - SERS spectra (@785 nm) of zeatin@AuNP_chem col-loid at different concentrations of the cytokinin, compared with the observed Raman spectrum of a 210-2 M solution of zeatin (pH 12).  With respect to free zeatin, SERS spectra show an intensification of the 732 cm-1 band which can be asso-ciated to the purine ring breathing mode, as already predicted for SERS spectra of adenine measured on gold [8,9]. Other signals are enhanced such as those  GLUTATHIONE-INDUCED RELEASE OF ZEATIN PROC. NAP 4, 02NNSA02 (2015)   02NNSA02-3 near 1210 cm-1 and 1310 cm-1. On the whole, chemically reduced gold NPs behave as a good SERS substrate for the detection of zeatin giving a fair reproducibility of the measured spectra.  Due to the quite large red shift of the extinction maximum observed as a consequence of the addition of zeatin, FT-SERS spectra were also collected for 10-5 and 10-6 M zeatin (Fig 4). This method had moreover the advantage of making the measurements directly on the colloidal solution which allowed us to check imme-diately the effect of glutathione as well. 10-3 M gluta-thione was tested for the 10-6 M zeatin@AuNP_chem sample. Despite the 1000:1 ratio, glutathione resulted to be ineffective, suggesting a very strong linkage of zeatin molecules to the gold surface. The measured spectrum in fact remained unchanged even one day later.     Fig. 4 – FT-SERS (@1064 nm) spectra of zeatin in 10-5 M and 10-6 M concentrations, adsorbed on AuNP_chem. Effect of 10-3 M glutathione addition to 10-6 M zeatin@AuNP_chem.   3.2 AuNP_abl colloidal suspension  According to the previous results, we considered a different type of colloid to be used as zeatin carrier. In particular we used AuNP prepared through laser abla-tion in pure water. With respect to chemical reduction procedures, laser ablation offers the highest purity and a surfactant-free surface, which allows for example effec-tive and non mediated interaction of the metal with the environment. Moreover, the “naked” AuNPs exhibit an excellent stability and in general a monomodal and mon-odisperse size distribution [16] which may be a challenge with traditional chemical procedures.  The plasmonic band of AuNP_abl suspension was peaked at 522 nm and was less sensitive to zeatin addi-tion with respect to the chemically citrate reduced sample discussed before. 10-5 M zeatin did not produce any significant spectral shift of the plasmonic band, while a small widening was observed after the addition of glutathione (Fig. 5). Despite the assembly of gold nanoparticles mediated by glutathione has been al-ready reported [17], we did not observe this effect on the naked nanoparticles. The scarce reactivity of AuNP_abl towards zeatin anchorage was further confirmed spectroscopically. The Raman spectra appeared very poorly enhanced by the metallic substrate. The effect of glutathione addition was thus not revealed (Fig. 6). 3.3 AuNP_citr_abl colloidal suspension  The same procedure of laser ablation was repeated in a 10-4 M Na-citrate solution to prepare AuNP_citr_abl nanoparticles.     Fig. 5 – UV-vis spectra of AuNP_abl, before and after the addition of 10-5 M zeatin and 10-3 M glutathione.    Fig. 6 – Representative SERS spectrum of zeatin@AuNP_abl before and after addition of glutathione (zeatin 10-5 M, gluta-thione 10-3 M).  The plasmonic band, centred at 517 nm, moved to 520 nm after the addition of 10-5 M zeatin and shifted to 620 nm after glutathione addition (Fig. 7). Glutathi-one promoted the assembly of not-labelled nanoparti-cles as well. Moreover, zeatin gave strong SERS signals, which disappeared after the addition of glutathione, attesting a strong cleavage effect (Fig. 8). The occurrence of an interaction between gold sur-face and the ligand molecules was apparently favoured by the presence of citrate ions. This could be tentatively correlated to the mild reducing properties of citrate ions as they kept gold surface free from oxidised species not available for the ligand adsorption. This is even better evidenced by comparing the free-zeatin absorp-tion peaks measured before and after glutathione addi-tion. Fig. 9a shows that a higher ligand adsorption oc-curs for AuNP_citr_abl in accordance with a higher reactivity of the nanoparticles surface. Besides, after glutathione addition, free-zeatin signal increases only for AuNP_citr_abl, due to the ligand-exchange reaction (Fig. 9b).  A. ZOPPI, J.R. MOLINA-CONTRERAS ET AL. PROC. NAP 4, 02NNSA02 (2015)   02NNSA02-4   Fig. 7 – UV-vis spectra of AuNP_citr_abl, before and after the addition of 10-5 M zeatin and 10-3 M glutathione.   Fig. 8 – SERS spectra taken on zeatin@AuNP_citr_abl, before and after addition of glutathione (10-5 M zeatin, 10-3 M gluta-thione).  Fig. 9 – Comparison of the free-zeatin absorption peak (meas-ured in the supernatant solution) before (a) and after (b) addi-tion of 10-3 M glutathione to AuNP_abl and AuNP_citr_abl colloidal suspensions with 10-5 M zeatin.  Coming to the conclusions of this work, we found that glutathione-induced release of zeatin occurred solely on citrate-laser-ablated nanoparticles.  At this stage we are not able to explain other questions, such as why AuNP_chem behaved differently. This aspect is set aside for future developments.   REFERENCES  1. W. Russ Algar, D.E. Prasuhn, M.H. Stewart, T.L. Jennings, J.B. Blanco-Canosa, P.E. Dawson, I.L. Medintz, Bioconju-gate Chem. 22, 825 (2011). 2. R.A. Sperling, W.J. Parak, Phil. Trans. R. Soc. A 368, 1333 (2010). 3. Y. Wang, B. Yan, L. Chen, Chem. Rev. 113, 1391 (2013). 4. C. Kim, E.C. Cho, J. Chen, K.H. Song, L. Au, C. Favazza, Q. Zhang, C.M. Cobley, F. Gao, Y. Xia, L.V. Wang, ACS Nano 4 No8, 4559 (2010). 5. S.E.J. Bell, N.M.S. Sirimuthu, J. Am. Chem. Soc. 128, 15580 (2006). 6. J.S. Suh, M. Moskovits, J. Am. Chem. Soc. 108, 4711 (1986). 7. D. Zalabák, H. Pospíšilová, M. Šmehilová, K. Mrízová, I. Frébort, P. Galuszka, Biotechnol. Adv. 31, 97 (2013). 8. M. Pagliai, S. Caporali, M. Muniz-Miranda, G. Pratesi, V. Schettino, J. Phys. Chem. Lett. 3, 242 (2012). 9. J. Kundu, O. Neumann, B.G. Janesko, D. Zhang, S. Lal, A. Barhoumi, G.E. Scuseria, N.J. Halas, J. Phys. Chem. C 113, 14390 (2009). 10. M. Muniz-Miranda, C. Gellini, M. Pagliai, M. Innocenti, P.R. Salvi, V. Schettino, J. Phys. Chem. C 114, 13730 (2010). 11. G. Noctor, A. Mhamdi, S. Chaouch, Y. Han, J. Neukermans, B. Marquez-Garcia, G. Queval, C.H. Foyer, Plant Cell Envi-ron. 35, 454 (2012). 12. K. Ock, W.I. Jeon, E.O. Ganbold, M. Kim, J. Park, J.H. Seo, K. Cho, S.W. Joo, S.Y. Lee, Anal. Chem. 84, 2172 (2012). 13. I. Kokinaa, V. Gerbredersb, E. Sledevskisb, A. Bulanovsb, J. Biotechnol. 165, 127 (2013). 14. G. Zhai, K.S. Walters, D.W. Peate, P.J.J. Alvarez, J. L. Schnoor, Environ. Sci. Technol. Lett. 1, 146 (2014). 15. J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, A. Plech, J. Phys. Chem. B 110, 15700 (2006). 16. M. Muniz-Miranda, C. Gellini, E. Giorgetti, G. Margheri, P. Marsili, L. Lascialfari, L. Becucci, S. Trigari, F. Giammanco, Thin Solid Films 543, 118 (2013). 17. I.S. Lim, D. Mott, W. Ip, P.N. Njoki, Y. Pan, S. Zhou, C.J. Zhong, Langmuir 24, 8857 (2008).  

Glutathione-Induced Release of Zeatin From Functionalized Gold Nanovectors

SocioEconomic Challenges Journal (Sumy State University)

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  
NANOMATERIALS: APPLICATIONS AND PROPERTIES 
Vol. 4 No 2, 02NNSA02(4pp) (2015) 
 
 
2304-1862/2015/4(2)02NNSA02(4) 02NNSA02-1  2015 Sumy State University 
Glutathione-Induced Release of Zeatin From Functionalized Gold Nanovectors 
 
A. Zoppi1,2, J.R. Molina-Contreras3,*, P. Marsili1,2, M. Muniz-Miranda4, G. Margheri2, S. Trigari2, 
A. Leva5, E. Giorgetti2, F. Giammanco1 
 
1 Physics Department, University of Pisa, Largo B. Pontecorvo 3,56127 Pisa, Italy 
2 ISC, CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy 
3 Departamento de Ingeniería Eléctrica y Electrónica, Instituto Tecnológico de Aguascalientes,  
Av. Adolfo López Mateos No 1801 Ote., Fracc. Bona Gens, 20256, Aguascalientes, Ags, México  
4 Department of Chemistry, University of Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy 
5 IVALSA, CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy 
 
(Received 15 March 2015; published online 22 August 2015) 
 
The paper shows our preliminary results on the different spectroscopic behavior of three types of gold 
nanoparticles (obtained respectively by chemical synthesis, laser ablation in pure water and laser ablation 
in a citrate solution) modified with trans-zeatin, a plant growth regulator, in presence of glutathione. The 
reaction of ligand substitution of the adsorbed zeatin with glutathione is studied through surface enhanced 
Raman spectroscopy and is revealed to occur only when citrate-laser ablated gold nanoparticles are em-
ployed, making these particles potentially good candidates as vehicles of zeatin inside plant cells for future 
agricultural applications.  
 
Keywords: Zeatin, Glutathione, Gold Nanoparticles, Raman, SERS. 
 
 PACS numbers: 82.70.Dd, 87.14.Lk, 87.64.kp 
 
 
                                                          
* jrmolinacon@gmail.com 
1. INTRODUCTION 
 
The number of applications in biosensors, sensors 
and biomedical diagnostics based on the interactions 
between nanoparticles and biologically relevant mole-
cules, has attracted an increasing interest [1-4]. The 
study of model systems such amino acids or nucleic 
acids on well characterized nanoparticle-surfaces has 
represented a useful approach to developing an under-
standing of the structure and stability of bio-molecules 
formed at metal interfaces. In particular, Surface En-
hanced Raman Scattering (SERS) turned to be a power-
ful investigation method for the detection of low analyt-
ical concentrations and for the identification of the con-
formation and orientation of molecules adsorbed on 
metal surfaces [5,6]. 
In this work we report our preliminary results on 
the studies of the capability of glutathione to induce the 
release of adsorbed zeatin from gold nanoparticles 
(AuNP), using SERS technique.  
Zeatin, a derivative of purine, is a natural cyto-
kinin which regulates multiple developmental and 
physiological processes in a plant through its ability 
to promote cell division, keep meristematic cell iden-
tity, maintain high cellular redox potentials during 
abiotic stress and regulate nutrient sink/source ac-
tivity of different organs [7]. The chemical structure 
of zeatin is basically that of adenine whose SERS 
spectrum has been measured on both Ag and Au 
substrates [8-10]. Despite this cytokinin exists as cis- 
and trans-structures, only trans-zeatin (Fig. 1a) is 
biologically active and it is used in in vitro culture to 
stimulate the growth and the bud formation. Howev-
er, due to its high cost, it is not recommended for 
large-scale subministrations.  
Glutathione is a non enzymatic antioxidant which 
has a role in the defence mechanism and antioxidant 
capacity in plant under abiotic stress [11]. It is an im-
portant constituent of all living cells and the most 
abundant thiol species at a concentration range of 1−10 
mM in the cytoplasm. It is a tripeptide made of glutam-
ic acid, cysteine and glycine (Fig. 1b) where the –SH 
functionality is known to exhibit a strong affinity with 
metallic gold. For instance, this affinity has been al-
ready exploited for drug release from gold nanoparti-
cles surfaces [12]. 
 
 
 
Fig. 1 – Chemical structures of: a) trans-zeatin and b) gluta-
thione. 
 
The basic idea is that glutathione, physiologically 
present in the cells, drives the release of zeatin  mole-
cules from hormone-labeled Au nanoparticles, thanks 
to its higher affinity for gold. Indeed, AuNP have been 
recently demonstrated to enter and accumulate in 
plant cells with very low or none toxicity [13, 14]. In 
this respect, AuNP could offer an efficient tool for the 
loading and transportation of zeatin inside plant cells, 
where it can be freed by glutathione exchange. Zeatin-
enriched  gold nanoparticles could be thus employed as 
novel vectors that can potentially deliver a higher 
amount of phytohormones, reducing costs in plants 
growth procedures, like for instance in commercial mi-
cropropagation protocols. 
To check the ability of glutathione to induce a lig-
 A. ZOPPI, J.R. MOLINA-CONTRERAS ET AL. PROC. NAP 4, 02NNSA02 (2015) 
 
 
02NNSA02-2 
and-exchange reaction on zeatin-labeled Au nanoparti-
cles,  three different gold aqueous colloidal suspensions 
were tested: AuNP_chem was obtained by chemical 
reduction of gold ions with sodium citrate, AuNP_abl 
was prepared by laser ablation in pure water and 
AuNP_citr_abl was obtained by laser ablation in a cit-
rate solution. The colloidal suspensions were modified 
with zeatin and glutathione addition was inspected 
through Raman measurements. 
We found that ligand exchange occurred only with 
AuNP_citr_abl and we believe that these results could 
be useful for the understanding of complex biological 
systems opening the way to applications in plant sci-
ence, agriculture and biotechnology. 
 
2. EXPERIMENTAL 
 
2.1 Sample preparation 
 
AuNP_chem was prepared by Na-citrate reduction 
of HAuCl4 according to the modified Turkevitch method 
[15]. 
Laser ablation was performed by irradiating a gold 
target with the fundamental wavelength (1064 nm) of a 
Q-switched Nd:YAG laser (rep. rate 10 Hz, pulse width 
17 ns) with 220 mJ pulse energy. The laser was focused 
on the target in order to obtain a circular spot of 1.5 
mm, corresponding to a 124 mJ/mm2 fluence. AuNP_abl 
was prepared in pure water while AuNP_citr_abl was 
obtained in a 10-4 M Na-citrate solution.  
Trans-zeatin was used as an alkaline solution (pH 
12), glutathione was dissolved in pure water. Ultrapure 
water (18.2 MΩ·cm @ 25°C) was used for all prepara-
tions. 
 
2.2 Methods 
 
Extinction spectra were obtained at room tempera-
ture using a Cary 5 spectrometer in quartz cuvettes.  
Raman spectra were recorded at room temperature 
using a single grating RM2000 Renishaw micro-Raman 
apparatus with an air cooled, charge coupled device 
and coupled with a near IR laser diode emitting at 785 
nm. Raman spectra were taken on a dried drop of the 
colloidal suspension put on an aluminium foil. A 20x 
magnification objective was used to probe the drop in 
several points. 
FT-SERS spectra were recorded using a Multi-
Raman (Bruker) apparatus operating at 1064 nm and 
coupled to a liquid-N2 cooled Ge detector. Irradiation 
power and spectral resolution were respectively set at 
500 mW and 4 cm-1. A 10 mm glass cuvette was used to 
record the spectra of 0.5 ml of the suspensions, averag-
ing over 200 scans. 
In order to check the glutathione-induced release 
of zeatin from gold nanoparticles, the colloidal con-
centrations were previously adjusted to a 0.8 extinc-
tion maximum (optical path 2 mm). Zeatin was add-
ed to the colloidal suspension to a final concentration 
of 10-5 M (pH 4-5) and spectral characterization was 
performed after ten minutes. The effect of 10-3 M 
glutathione was then checked. This value of concen-
tration was selected according to the glutathione 
level in living cells. 
 
3. RESULTS AND DISCUSSION  
 
3.1 AuNP_chem colloidal suspension 
 
The citrate-reduced AuNP colloid was very sensitive 
to the addition of 10-5 M zeatin. This was macroscopi-
cally evidenced by the colour changes of the suspension. 
Fig. 2 compares the extinction spectrum of the pure 
colloid with those acquired on zeatin@AuNP_chem for 
different cytokinin concentrations. As evidenced, zeatin 
addition caused a flattening of the main plasmonic 
band at 526 nm, that is characteristic of spherical par-
ticles, while a new band appears and becomes promi-
nent in the 750-800 nm spectral range, attributable to 
nanoparticle aggregation. Besides, the absorption peak 
of zeatin occurs at 270 nm. 
 
 
 
Fig. 2 – UV-vis spectra of the Au_NP_chem colloidal suspen-
sion before and after the addition of zeatin 10-6 M, 10-5 M, 10-4 
M. The absorption peak at 270 nm is attributed to the cytokin-
in molecule.  
 
The SERS spectra of zeatin at 10-4, 10-5 and 10-6 M 
concentrations were then taken on dried drops. Fig. 3 
compares three typical SERS spectra with the Raman 
spectrum taken on an aqueous solution of zeatin. 
 
 
Fig. 3 - SERS spectra (@785 nm) of zeatin@AuNP_chem col-
loid at different concentrations of the cytokinin, compared 
with the observed Raman spectrum of a 210-2 M solution of 
zeatin (pH 12). 
 
With respect to free zeatin, SERS spectra show an 
intensification of the 732 cm-1 band which can be asso-
ciated to the purine ring breathing mode, as already 
predicted for SERS spectra of adenine measured on 
gold [8,9]. Other signals are enhanced such as those 
 GLUTATHIONE-INDUCED RELEASE OF ZEATIN PROC. NAP 4, 02NNSA02 (2015) 
 
 
02NNSA02-3 
near 1210 cm-1 and 1310 cm-1. On the whole, chemically 
reduced gold NPs behave as a good SERS substrate for 
the detection of zeatin giving a fair reproducibility of 
the measured spectra.  
Due to the quite large red shift of the extinction 
maximum observed as a consequence of the addition of 
zeatin, FT-SERS spectra were also collected for 10-5 
and 10-6 M zeatin (Fig 4). This method had moreover 
the advantage of making the measurements directly on 
the colloidal solution which allowed us to check imme-
diately the effect of glutathione as well. 10-3 M gluta-
thione was tested for the 10-6 M zeatin@AuNP_chem 
sample. Despite the 1000:1 ratio, glutathione resulted 
to be ineffective, suggesting a very strong linkage of 
zeatin molecules to the gold surface. The measured 
spectrum in fact remained unchanged even one day 
later.  
 
 
 
Fig. 4 – FT-SERS (@1064 nm) spectra of zeatin in 10-5 M and 
10-6 M concentrations, adsorbed on AuNP_chem. Effect of 10-3 
M glutathione addition to 10-6 M zeatin@AuNP_chem.  
 
3.2 AuNP_abl colloidal suspension 
 
According to the previous results, we considered a 
different type of colloid to be used as zeatin carrier. In 
particular we used AuNP prepared through laser abla-
tion in pure water. With respect to chemical reduction 
procedures, laser ablation offers the highest purity and a 
surfactant-free surface, which allows for example effec-
tive and non mediated interaction of the metal with the 
environment. Moreover, the “naked” AuNPs exhibit an 
excellent stability and in general a monomodal and mon-
odisperse size distribution [16] which may be a challenge 
with traditional chemical procedures.  
The plasmonic band of AuNP_abl suspension was 
peaked at 522 nm and was less sensitive to zeatin addi-
tion with respect to the chemically citrate reduced 
sample discussed before. 10-5 M zeatin did not produce 
any significant spectral shift of the plasmonic band, 
while a small widening was observed after the addition 
of glutathione (Fig. 5). Despite the assembly of gold 
nanoparticles mediated by glutathione has been al-
ready reported [17], we did not observe this effect on 
the naked nanoparticles. 
The scarce reactivity of AuNP_abl towards zeatin 
anchorage was further confirmed spectroscopically. The 
Raman spectra appeared very poorly enhanced by the 
metallic substrate. The effect of glutathione addition 
was thus not revealed (Fig. 6). 
3.3 AuNP_citr_abl colloidal suspension 
 
The same procedure of laser ablation was repeated 
in a 10-4 M Na-citrate solution to prepare 
AuNP_citr_abl nanoparticles.  
 
 
 
Fig. 5 – UV-vis spectra of AuNP_abl, before and after the 
addition of 10-5 M zeatin and 10-3 M glutathione. 
 
 
 
Fig. 6 – Representative SERS spectrum of zeatin@AuNP_abl 
before and after addition of glutathione (zeatin 10-5 M, gluta-
thione 10-3 M). 
 
The plasmonic band, centred at 517 nm, moved to 
520 nm after the addition of 10-5 M zeatin and shifted 
to 620 nm after glutathione addition (Fig. 7). Glutathi-
one promoted the assembly of not-labelled nanoparti-
cles as well. 
Moreover, zeatin gave strong SERS signals, which 
disappeared after the addition of glutathione, attesting 
a strong cleavage effect (Fig. 8). 
The occurrence of an interaction between gold sur-
face and the ligand molecules was apparently favoured 
by the presence of citrate ions. This could be tentatively 
correlated to the mild reducing properties of citrate 
ions as they kept gold surface free from oxidised species 
not available for the ligand adsorption. This is even 
better evidenced by comparing the free-zeatin absorp-
tion peaks measured before and after glutathione addi-
tion. Fig. 9a shows that a higher ligand adsorption oc-
curs for AuNP_citr_abl in accordance with a higher 
reactivity of the nanoparticles surface. Besides, after 
glutathione addition, free-zeatin signal increases only 
for AuNP_citr_abl, due to the ligand-exchange reaction 
(Fig. 9b). 
 A. ZOPPI, J.R. MOLINA-CONTRERAS ET AL. PROC. NAP 4, 02NNSA02 (2015) 
 
 
02NNSA02-4 
 
 
Fig. 7 – UV-vis spectra of AuNP_citr_abl, before and after the 
addition of 10-5 M zeatin and 10-3 M glutathione. 
 
 
Fig. 8 – SERS spectra taken on zeatin@AuNP_citr_abl, before 
and after addition of glutathione (10-5 M zeatin, 10-3 M gluta-
thione). 
 
Fig. 9 – Comparison of the free-zeatin absorption peak (meas-
ured in the supernatant solution) before (a) and after (b) addi-
tion of 10-3 M glutathione to AuNP_abl and AuNP_citr_abl 
colloidal suspensions with 10-5 M zeatin. 
 
Coming to the conclusions of this work, we found 
that glutathione-induced release of zeatin occurred 
solely on citrate-laser-ablated nanoparticles.  At this 
stage we are not able to explain other questions, such 
as why AuNP_chem behaved differently. This aspect is 
set aside for future developments. 
 
 
REFERENCES 
 
1. W. Russ Algar, D.E. Prasuhn, M.H. Stewart, T.L. Jennings, 
J.B. Blanco-Canosa, P.E. Dawson, I.L. Medintz, Bioconju-
gate Chem. 22, 825 (2011). 
2. R.A. Sperling, W.J. Parak, Phil. Trans. R. Soc. A 368, 1333 
(2010). 
3. Y. Wang, B. Yan, L. Chen, Chem. Rev. 113, 1391 (2013). 
4. C. Kim, E.C. Cho, J. Chen, K.H. Song, L. Au, C. Favazza, 
Q. Zhang, C.M. Cobley, F. Gao, Y. Xia, L.V. Wang, ACS 
Nano 4 No8, 4559 (2010). 
5. S.E.J. Bell, N.M.S. Sirimuthu, J. Am. Chem. Soc. 128, 
15580 (2006). 
6. J.S. Suh, M. Moskovits, J. Am. Chem. Soc. 108, 4711 (1986). 
7. D. Zalabák, H. Pospíšilová, M. Šmehilová, K. Mrízová, 
I. Frébort, P. Galuszka, Biotechnol. Adv. 31, 97 (2013). 
8. M. Pagliai, S. Caporali, M. Muniz-Miranda, G. Pratesi, 
V. Schettino, J. Phys. Chem. Lett. 3, 242 (2012). 
9. J. Kundu, O. Neumann, B.G. Janesko, D. Zhang, S. Lal, 
A. Barhoumi, G.E. Scuseria, N.J. Halas, J. Phys. Chem. C 
113, 14390 (2009). 
10. M. Muniz-Miranda, C. Gellini, M. Pagliai, M. Innocenti, 
P.R. Salvi, V. Schettino, J. Phys. Chem. C 114, 13730 (2010). 
11. G. Noctor, A. Mhamdi, S. Chaouch, Y. Han, J. Neukermans, 
B. Marquez-Garcia, G. Queval, C.H. Foyer, Plant Cell Envi-
ron. 35, 454 (2012). 
12. K. Ock, W.I. Jeon, E.O. Ganbold, M. Kim, J. Park, J.H. Seo, 
K. Cho, S.W. Joo, S.Y. Lee, Anal. Chem. 84, 2172 (2012). 
13. I. Kokinaa, V. Gerbredersb, E. Sledevskisb, A. Bulanovsb, 
J. Biotechnol. 165, 127 (2013). 
14. G. Zhai, K.S. Walters, D.W. Peate, P.J.J. Alvarez, 
J. L. Schnoor, Environ. Sci. Technol. Lett. 1, 146 (2014). 
15. J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, 
A. Plech, J. Phys. Chem. B 110, 15700 (2006). 
16. M. Muniz-Miranda, C. Gellini, E. Giorgetti, G. Margheri, 
P. Marsili, L. Lascialfari, L. Becucci, S. Trigari, 
F. Giammanco, Thin Solid Films 543, 118 (2013). 
17. I.S. Lim, D. Mott, W. Ip, P.N. Njoki, Y. Pan, S. Zhou, 
C.J. Zhong, Langmuir 24, 8857 (2008). 
 


English

https://essuir.sumdu.edu.ua/bitstream/123456789/42489/1/Zoppi.pdf

Glutathione-Induced Release of Zeatin From Functionalized Gold Nanovectors

Abstract

Similar works

Full text

Available Versions

Electronic Sumy State University Institutional Repository

SocioEconomic Challenges Journal (Sumy State University)