Gold nanoparticles (GNP) induced redox modulation in organoselenium compounds: distinction between cyclic vs. linear structures

Pavitra V. Kumarab, Beena G. Singh*a, Anand Ballalc, Vimal K. Jaind, Michio Iwaokae and K. Indira Priyadarsini*ab
aRadiation & Photochemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai – 400 085, India. E-mail: beenam@barc.gov.in; kindira@barc.gov.in
bHomi Bhabha National Institute, Anushaktinagar, Mumbai – 400 094, India
cMolecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai – 400 085, India
dChemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai – 400 085, India
eDepartment of Chemistry, School of Science, Tokai University, Hiratsuka-shi, Kanagawa 259-1292, Japan

Received 10th June 2016 , Accepted 13th July 2016

First published on 13th July 2016


Abstract

Selenium, like other chalcogens, shows a high affinity for gold nanoparticles (GNP). The binding interactions between the selenium centre and GNP can greatly influence its important physico-chemical properties, including electron transfer ability. To know if the chemical structure has any influence on these properties, in the present paper, binding and electron transfer reactions of two simple water soluble organoselenium compounds (SeC), i.e. a linear compound, (bis(2-ethanol)selenide) (SeEOH) and a cyclic compound, DL-trans-3,4-dihydroxy-1-selenolane (DHS) with GNP have been investigated. The binding with GNP of four different sizes (5–58 nm) was characterized by UV-visible spectroscopy, dynamic light scattering (DLS), zeta (ζ) potential, transmission electron microscopy (TEM) and surface enhanced Raman spectroscopy (SERS). Although both the compounds bind GNP through selenium atoms, they differ in orientation on the GNP surface. In DHS, only the selenium atom interacts, while in SeEOH, along with selenium atoms, alkyl groups also interact with the GNP surface. Stronger Se–GNP interaction and an increase in the electrophilicity of DHS as compared to SeEOH, was confirmed by their relative electron transfer reactivity with the ABTS˙ radical. Pulse radiolysis studies suggested that both the compounds on reaction with the hydroxyl (˙OH) radical produced similar selenium centered dimer radical cations ([double bond splayed left]Se∴Se[double bond splayed right])+, either in the presence or in absence of GNP, but significantly increased the yield of selenoxide in the presence of GNP, which is known to influence their antioxidant ability. Thus our results confirm that GNP can be used to modulate the electron transfer ability of selenium compounds.


Introduction

Gold nanoparticles (GNP) are largely explored for application in biology and medicine due to their unique properties which include easy preparation, higher stability and surface functionalization that allows a variety of low molecular weight drugs to be encapsulated through non-covalent interactions or chemical conjugation.1–5 A wide range of ligands containing thiols, amines and carboxylate functional groups have been studied as substrates for the surface modification of GNP.6–9 Particularly, organothiol functionalized GNP have been explored as sensors to identify and quantify proteins and intracellular thiols in living cells.10–12 The optical and chemical properties of these nanocomposites can be tuned by the strength of ligand-GNP interaction which in turn depends on the size of GNP and structure of the ligand.13 Although the interaction between ligands having heteroatom like nitrogen, phosphorus and sulfur with GNP for surface passivation have been well explored, such studies with selenium compounds are scantly carried out.14–16 Selenium is softer and more polarizable as compared to sulfur and therefore the surface passivation can be easily achieved. Selenides like diphenyldiselenide, didodecyldiselenide, etc. have been reported to bind GNP.17,18 The binding of diselenides to GNP surface takes place via cleavage of Se–Se bond resulting in the formation of Au–selenolate bond similar to organic disulfides.19–21 Yee, et al. showed that packing density of aliphatic chains in alkyl-selenolates on the GNP surface is higher compared to the thiol counterpart.17 The binding of alkyl selenides to GNP was found to alter their redox behaviour.22 Earlier we have reported that the binding of a cyclic organoselenium compound, DHS (DL-trans-3,4-dihydroxy-1-selenolane) with GNP affects the one-electron transfer reaction of DHS.23 DHS is a water soluble cyclic selenolane and exhibits glutathione peroxidase (GPx) like catalytic activity, where it reduces hydroperoxides in the presence of thiols, through the intermediacy of selenoxide. This cyclic compound DHS, when compared with its linear counterpart SeEOH (bis(2-ethanol)selenide) exhibits higher GPx like activity, attributed to elevated highest occupied molecular orbital (HOMO) level in DHS and higher yield of selenoxide.24 In the present manuscript, we attempted to address, how the two structural isomers DHS and SeEOH differ in their binding to GNP and whether such binding can influence their redox properties, thereby affecting the selenoxide formation. The structures of the compounds are given in Scheme 1.
image file: c6ra15106j-s1.tif
Scheme 1 Structure of organoselenium compounds (SeC).

Results and discussion

(1) Absorption spectroscopic studies

Absorption spectrum of 7 nM GNP1 (5 nm), GNP2 (15 nm), GNP3 (25 nm) and GNP4 (58 nm) showed surface plasmon resonance (SPR) maximum at 518 nm, 522 nm, 525 nm and 527 nm, respectively (Fig. 1(a) and S1–3(a)). In presence of SeEOH, formation of new red shifted absorption band was observed for GNP2, GNP3 and GNP4. GNP1 did not show formation of new band, but caused broadening of the original absorption band (Fig. S1–3 in ESI).
image file: c6ra15106j-f1.tif
Fig. 1 Absorption spectra of 7 nM GNP2 in absence (a) and in presence of (b) 10 μM, (c) 25 μM, (d) 50 μM, (e) 75 μM and (f) 100 μM SeEOH. Inset shows double reciprocal plot for GNP absorbance at 520 nm as a function of SeEOH concentration.

Fig. 1 shows the representative absorption spectral changes for binding of SeEOH to GNP2, where addition of different concentration of SeEOH (5 μM–0.1 mM) to a fixed concentration of GNP2 (7 nM) resulted in decrease in the absorbance at 522 nm with monotonic evolution of a new band at 650–680 nm. Beyond 100 μM SeEOH, no further change in absorbance was observed. Earlier we reported that DHS causes similar spectral shifts on binding to GNP. The differential spectral shift, i.e. change in the absorption maximum, as a result of binding to GNP and the FWHM (full width at half maximum) was found to be lower for SeEOH as compared to that for DHS indicating higher polydispersity in the latter case. The binding constant (K) between GNP and SeC was calculated by using modified Benesi–Hildebrand equation (eqn (1)):25

 
image file: c6ra15106j-t1.tif(1)
here, n, [small script l], ε, [SeC]0 and [GNP]0 represent number of binding sites on GNP surface, optical path length, molar extinction coefficient and initial concentration of SeC and GNP (number of moles of particles per litre volume), respectively.

The double reciprocal plot of ΔA as a function of [SeC]0 gave a straight line and the ratio of the intercept and slope corresponds to the product of number of binding sites (n) and binding constant (K) (inset Fig. 1). Estimating the value of n according to the method described in ref. 26, the K values were calculated and are listed in Table 1.26 From the results, it can be inferred that under identical condition, the K value for SeEOH is higher than that for DHS by an order of magnitude.

Table 1 Physico-chemical properties of different DHS–GNP composites
S. no Number of binding sites (n) Binding constant (K, M−1) × 10−2 Hydrodynamic diameter (nm)
SeEOH–GNP DHS–GNP GNP SeEOH–GNP DHS–GNP
GNP1 1.9 × 104 5.2 ± 0.1 2.38 ± 0.09
GNP2 5.2 × 105 5.9 ± 0.2 0.97 ± 0.02 15 ± 3 26 ± 5 34 ± 4
GNP3 2.4 × 106 3.8 ± 0.1 0.38 ± 0.05 25 ± 3 69 ± 5 79 ± 6
GNP4 3.0 × 107 0.40 ± 0.02 0.09 ± 0.01 58 ± 5 140 ± 10 195 ± 8


(2) Characterization by dynamic light scattering (DLS) and transmission electron microscopy (TEM)

DLS measurements were carried out to estimate the effect of binding of SeEOH on the size of GNP. Due to overlapping absorption by GNP1 with the excitation wavelength of the instrument, it was difficult to monitor the changes in size, however significant differences were noticed for GNP2, GNP3 and GNP4. Fig. 2 shows the exponential decay of correlation function (G1(q,t)) for 7 nM GNP2 in absence and presence of 0.1 mM SeEOH and DHS respectively.
image file: c6ra15106j-f2.tif
Fig. 2 Plot of correlation function G1(q,t) as a function of time for 7 nM GNP2 in absence (a) and in presence of (b) 0.1 mM SeEOH and (c) 0.1 mM DHS.

Fitting the data to a monomodal distribution, the hydrodynamic diameter of GNP2 was estimated to be (15 ± 3) nm, which in the presence of 0.1 mM SeEOH and DHS increased to (26 ± 5) and (34 ± 4) nm, respectively. Similarly, hydrodynamic diameter of GNP3, GNP4 and their SeC-composites were estimated and the values are listed in Table 1.

The observed hydrodynamic diameter of SeC bound to GNP increases with increasing SeC concentration. At any given concentration, the size of SeEOH–GNP composites was lower than that for DHS–GNP composites. Since the hydrodynamic diameter obtained from DLS measurements is not a true representation of the actual size of the nanoparticles, TEM analysis was carried out. The TEM images revealed the average size of GNP1, 2, and 3 to be (5 ± 2), (12 ± 3), and (21 ± 4) nm, respectively. Fig. 3 shows the TEM images and size distribution of GNP1 and GNP3 in absence and presence of SeEOH and DHS. Addition of 1 mM SeEOH to the GNP caused aggregation without changing the shape of the GNP and the sizes were (5 ± 3), (17 ± 5) and (26 ± 5) nm for GNP1, GNP2 and GNP3, respectively. In the presence of 1 mM DHS, the size of GNP 1, 2 and 3 was found to be (7 ± 3), (20 ± 6) and (27 ± 8) nm, respectively. TEM images did not suggest any particle growth due to the binding of these compounds with GNP within a range of standard deviation, but indicate decrease in the interparticle distance leading to aggregation.


image file: c6ra15106j-f3.tif
Fig. 3 Size distribution analysis of TEM images of (A) GNP1, (B) GNP1–SeEOH (1 mM), (C) GNP1–DHS (1 mM), (D) GNP3, (E) GNP3–SeEOH (1 mM) and (F) GNP3–DHS (1 mM) composites. Insets show the corresponding TEM images.

(3) Zeta (ζ)-potential measurements

The stability of colloids is governed by the charge on the surface of the particles that can be measured in terms of ζ-potential values. The GNP prepared in this work has net negative surface charge due to the capping by the citrate ions. Generally binding of any ligand may replace the citrate ion which leads to lowering in surface charge of the GNP and thereby reducing the ζ-potential values. The average ζ-potential values for GNP2, SeEOH(0.1 mM)–GNP2 and DHS(0.1 mM)–GNP2 composites were (−39.5 ± 2.9), (−27.6 ± 0.8) and (−31.6 ± 1.5) mV, respectively. Similar results were obtained for SeEOH and DHS composites of GNP1, GNP3 and GNP4 as listed in Table 2. For all sizes of GNP the decrease in ζ-potential due to binding of SeEOH was found to be higher than that of DHS.
Table 2 Physico-chemical properties of different DHS–GNP composites
GNP sample ζ-potential (mV)
GNP SeEOH–GNP DHS–GNP
GNP1 −46.1 ± 0.5 −30.1 ± 0.5 −39.1 ± 0.8
GNP2 −39.5 ± 2.9 −27.6 ± 0.8 −31.6 ± 1.5
GNP3 −38.2 ± 1.7 −24.2 ± 1.6 −28.6 ± 1.2
GNP4 −34.5 ± 0.9 −21.6 ± 2.4 −25.3 ± 0.4


(4) Raman and surface enhanced Raman spectroscopy (SERS) studies

The standard Raman spectrum of neat SeEOH was recorded in the region 200–3000 cm−1 and the bands were assigned by quantum chemical calculation (Fig. S4 in ESI) and also by comparing them with the reported Raman spectrum of its sulfur analogue, bis(2-ethanol)sulfide (SEOH).27 The band assignments are given in Table 3. The sharp Raman peaks observed in the spectrum of neat SeEOH at 344 cm−1 and 449 cm−1 arise due to Se-deformation band while peaks at 562 cm−1 and 668 cm−1 correspond to C–Se–C stretching mode. Along with these, a few more bands at higher wave numbers corresponding to the vibration modes of the –CH2 were observed and are listed in Table 3. To understand binding of SeEOH and DHS to GNP, SERS was used. In this technique, depending upon the orientation of the ligand on electron rich GNP surface, Raman intensity of selected vibration modes of the ligand are enhanced, from which the nature of interaction can be explicitly understood. Fig. 4 shows the SERS spectra of 7 nM GNP2, GNP3 and GNP4 treated with 0.1 mM SeEOH in the frequency range 300–2000 cm−1. No SERS peaks were observed for SeEOH–GNP1 composites as the SPR of this composite lies in the wavelength range of 520–550 nm. For SeEOH–GNP2 two small but broad peaks were observed at 551 cm−1 and 672 cm−1 corresponding to C–Se–C stretching, symmetric and asymmetric modes, respectively. For GNP3 and GNP4, along with the above peaks, a new sharp peak in the region 1000–1050 cm−1 and other small overlapping peaks from 1100–1500 cm−1 corresponding to different CH2 bending modes were observed. Comparison of these data with the earlier reported SERS data of DHS–GNP composites, it is clear that the two compounds bind to GNP differently.23 For DHS–GNP composites two-major peaks (636 cm−1 and 1234 cm−1) due to C–Se–C stretching and –CH2 bending were reported.
Table 3 Assignment of Raman peaks of SeEOH and SERS peaks of SeEOH–GNP composites
Raman (SEOH),27 cm−1 Raman peak (SeEOH), cm−1 SERS (SeEOH–GNP)
Theory Experimental Peak position (cm−1) Assignment
510.9 466 449   S/Se-deformation
529.9 536 562 551 C–Se–C stretching
698.1 771 668 672 C–Se–C stretching
1015.2 984 1003 999 CH2 bending (rocking)
1059.9 1039, 1057 1055 1023, 1047 CH2 bending (scissor)
1127.8 1132 1124 1112 CH2 bending (rocking)
1198.4 1192 1186 1170 CH2 bending (rocking)
1298.8 1263 1276 1251 CH2 bending (twisting)
1332.6 1366 1321 1322 CH2 bending (wagging)
1502.0 1440 1467 1455 CH2 bending (twisting)
1558.0 1568 1563 1557 CH2 bending (wagging)



image file: c6ra15106j-f4.tif
Fig. 4 (a) Raman spectrum of pure SeEOH. SERS spectra of 0.1 mM SeEOH in presence of 7 nM (b) GNP1, (c) GNP, (d) GNP and (e) GNP4.

However, in the case of SeEOH–GNP, along with these peaks (i.e. at 551 cm−1 and 1047 cm−1) several other peaks were also observed indicating interaction of alkyl side chain with GNP surface. Due to such interaction, it is expected that in SeEOH–GNP composites, the alkyl chain attached to selenium atom may remain parallel to the GNP surface. This kind of alkyl chain interaction appears to be dominating for larger GNP probably due to the fact that the surface curvature decreases with increase in GNP size. This assumption was further supported by the shift in peak maximum of SeC–GNP composites relative to SeC Raman peaks.

As listed in Table 4 the shift in peak maxima for C–Se–C stretching is more for DHS–GNP composites (636 cm−1) as compared to SeEOH–GNP composites (551 cm−1), whereas reverse trend is observed for CH2 bending peaks (DHS – 1234 cm−1 and SeEOH – 1023 cm−1), indicating role of alkyl chain in binding of SeEOH with GNP.

Table 4 SERS analysis for SeEOH–GNP and DHS–GNP composites
Compounds Shift in peak position in absence and presence of GNP
  GNP2 GNP3 GNP4
SeEOH C–Se–C stretching (551 cm−1) 9.7 9.7 11.8
CH2 bending (1023 cm−1) 9 32.1
DHS C–Se–C stretching (636 cm−1) 20 16 18
CH2 bending (1234 cm−1) 1.8 1.8 0.6


(5) Reaction with 2,2′-azino-bis-ethylsulfonate (ABTS˙) radical

ABTS˙ radical is a stable radical and its neutralization reaction is used as a tool to measure the electron transfer ability of an antioxidant molecule.28 Nucleophilic selenium compounds react with ABTS˙ radical through one electron transfer reaction. In our earlier study, it was reported that DHS when bound to GNP reacts with ABTS˙ radical much faster than in the free form.23 Similar experiments were performed with SeEOH and the decay of ABTS˙ radical in presence of SeC–GNP composites was monitored at 820 nm (Fig. 5).
image file: c6ra15106j-f5.tif
Fig. 5 Absorbance-time plot showing the decay of 30 μM ABTS˙ (a) and in presence of (b) 7 nM GNP1, (c) 1 mM SeEOH, (d) 1 mM SeEOH–GNP4, (e) 1 mM SeEOH–GNP3, (f) 1 mM SeEOH–GNP2 and (g) 1 mM SeEOH–GNP1. Inset shows linear fit for the variation of observed decay rate constant as function of (diameter)−1 of GNP.

For these studies the concentration of ABTS˙ radical, GNP and SeEOH were fixed at 30 μM, 7 nM and 1 mM respectively. ABTS˙ radical in the absence of SeC or GNP showed a very slow decay with a first order rate constant of (3.7 ± 0.3) × 10−6 s−1. On addition of 1 mM SeEOH or 7 nM GNP(1–4) only marginal changes in the decay of ABTS˙ were observed (Table 5). However on addition of 1 mM SeEOH–GNP1/2/3/4 to ABTS˙ radical solution, the absorbance at 820 nm decreased with simultaneous increase in the absorbance at 340 nm (due to formation of ABTS2−) (Fig. S5 in ESI). Also, the rate constant values were found to increase with decrease in GNP size in a linear fashion (inset of Fig. 5). The observed rate constants (k) for the decay of ABTS˙ radical in the presence of SeEOH–GNP composite is listed in Table 5. Comparing these values with the earlier reported results for DHS–GNP composites, SeEOH–GNP composites appear to have low reactivity. However as reported earlier, since DHS itself showed significant reactivity with ABTS˙ radical as compared to SeEOH, the relative increase in the reactivity with ABTS˙ radical can be considered to be significantly higher for SeEOH than that for DHS. No attempt has been made to evaluate the bimolecular rate constant with GNP bound SeEOH or DHS.

Table 5 Rate constants (k) for the reduction of ABTS˙ by 1 mM SeC–GNP (7 nM) composites
S. no (kABTS˙+GNP), s−1 (kABTS˙+DHS), s−1 (kABTS˙+SeEOH), s−1
In absence of GNP 3.7 ± 0.3 × 10−6 0.97 ± 0.05 × 10−3 7.96 ± 0.08 × 10−5
GNP1 2.1 ± 0.1 × 10−4 1.08 ± 0.07 × 10−2 5.39 ± 0.08 × 10−3
GNP2 1.1 ± 0.1 × 10−4 3.52 ± 0.05 × 10−3 2.12 ± 0.05 × 10−3
GNP3 0.95 ± 0.05 × 10−4 2.94 ± 0.09 × 10−3 1.85 ± 0.09 × 10−3
GNP4 0.67 ± 0.03 × 10−4 2.39 ± 0.08 × 10−3 1.37 ± 0.07 × 10−3


(6) Reaction of hydroxyl (˙OH) radical

Like ABTS˙ radicals, ˙OH radicals are oxidizing radicals and participate in electron transfer reactions with SeC. However due to the short life time of these radicals the reactions must be followed using pulse radiolysis. Both DHS and SeEOH on reaction with ˙OH radicals produced selenium centered dimer radical cations (([double bond splayed left]Se∴Se[double bond splayed right])+) absorbing at 480 nm.29 Formation of such species can take place by the one-electron oxidation of SeC by ˙OH radical followed by reaction with parent SeC molecule. As seen in Fig. 6 the transient absorption spectra obtained on reaction of ˙OH radical with SeEOH–GNP and DHS–GNP nanocomposites exhibit similar features with absorption maximum at ∼480 nm, assigned to ([double bond splayed left]Se∴Se[double bond splayed right])+ radical. The ([double bond splayed left]Se∴Se[double bond splayed right])+ radical derived from SeEOH decayed by second order kinetics with 2k/εl value of (1.05 ± 0.08) × 106 s−1 and (6.03 ± 0.05) × 105 s−1 in the absence and presence of GNP1 respectively. On the other hand, no significant change was observed in the decay pattern of ([double bond splayed left]Se∴Se[double bond splayed right])+ radical of DHS in absence and in presence of GNP1. This observation indicated that the ([double bond splayed left]Se∴Se[double bond splayed right])+ radical of the linear analogue is stabilized to a larger extent as compared to the cyclic form.
image file: c6ra15106j-f6.tif
Fig. 6 Transient absorption spectrum obtained on reaction of ˙OH radical with 250 μM SeEOH (A)/DHS (B) in absence (a) and presence of 7 nM GNP1 (b) at pH 7. Inset (c) shows the absorption-time plot at 480 nm. Inset (d) shows the plot of reciprocal of absorbance at 480 nm due to dimer radical cation ([double bond splayed left]Se∴Se[double bond splayed right])+ in absence (□) and presence of 7 nM GNP1 (○). The slope of the linear fit of the plot gives the 2k/εl for ([double bond splayed left]Se∴Se[double bond splayed right])+ radical decay.

As reported earlier, the ([double bond splayed left]Se∴Se[double bond splayed right])+ radical undergoes radical–radical reactions to produce selenoxide and more the stability of the radical, larger will be the radiation chemical yield (G-value) of the selenoxide. It has been well established that the selenoxide quantitatively reacts with dithiothretol (DTT) to give oxidised dithiothretol (DTTox).30 Therefore the radiolytic yield of selenoxide was estimated by monitoring the amount of DTTox by the above reaction using high performance liquid chromatography (HPLC) in the absence and presence of GNP1 (Fig. S6 in ESI) at different absorbed doses (80 Gy to 120 Gy). The amount of selenoxide estimated was found to increase linearly with the absorbed dose and the slope of this plot corresponds to the G-value of the selenoxide.

As shown in the inset of Fig. S6, it can be observed that G-value of selenoxide increases for both the compounds in the presence of GNP. However the extent of increase is higher for SeEOH as compared to DHS. Recently, Sicard-Roselli, et al. have demonstrated that the yield of ˙OH radical (G-value) increases in the presence of GNP which depends on the dose-rate, size and concentration of the nanoparticles.31 Therefore the yield of ˙OH radical in presence of GNP1 was estimated by monitoring fluorescence from 7-hydroxy coumarin (7-HOC) formed due to the reaction of ˙OH radical with coumarin.31 The yield of ˙OH radical in the absence and in presence of GNP1 were (0.6 ± 0.05) and (0.86 ± 0.08) μmol J−1, respectively. Since the yield of ˙OH radical increases in the presence of GNP, the amount of selenoxide formed was normalized with respect to the yield of ˙OH radical. After normalization, the amount of selenoxide was found to be (39.6 ± 6.0)% and (51.7 ± 4.0)% of the total ˙OH radical for SeEOH and SeEOH–GNP composites. Under similar experimental condition and normalisation, the yield of the selenoxide of DHS was estimated to be (51.0 ± 7.0)% and (56.0 ± 5.0)% of the total ˙OH radical in the absence and presence of GNP1 respectively. This clearly shows that, the selenoxide yield increases in SeEOH–GNP than that for DHS–GNP composites.

Discussion

In order to understand how two structural isomers differ in the interaction and physico-chemical properties on binding to GNP, we have compared the results of SeEOH, an alcohol functionalized aliphatic selenoether with its cyclic analogue DHS. Both the compounds on binding to GNP shifted the absorption maximum of GNP from 520 nm to 680–720 nm indicating nanoparticle aggregation.32 The nK values for SeEOH–GNP composites are higher by an order of magnitude than those of DHS–GNP composites (Table 1). As nK value represents the strength of interaction between ligand and GNP, it indicates that SeEOH is more strongly bound to GNP surface as compared to DHS. Binding of SeEOH/DHS will lead to decrease in the surface charge i.e. its ζ-potential. However, the extent of decrease in ζ-potential is higher for SeEOH–GNP composites than that for DHS–GNP composites indicating higher interaction of SeEOH. This could be due to different mode of interaction of SeEOH and DHS. SeEOH being a linear molecule, has ease of bending of alkyl chain on GNP surface, where DHS with a rigid cyclic structure would show resistance towards change in structure. This would also lead to better packing density of SeEOH on GNP surface than DHS. Therefore, SeEOH can passivate the surface energy of the GNP to a greater extent than DHS, resulting in lower aggregation size.

The differential mode of binding in SeEOH and DHS towards GNP was further confirmed by SERS studies. A large enhancement in C–Se–C stretching and CH2 bending modes was observed for both compounds, indicating involvement of Se atom in binding with GNP. The increase in strength of Se–GNP interaction leads to weakening of Se–C bond which is reflected by the shift of C–Se–C stretching peak towards lower wave number. For a given size, the shift in C–Se–C peak is more for DHS–GNP composites (20 cm−1 for GNP2) than that of SeEOH–GNP composites (9.7 cm−1 for GNP2) (Table 4). While the reverse trend is observed for CH2 bending peaks. This difference is negligible at lower GNP size but becomes significant at higher GNP size (GNP3 and GNP4). These results suggest that in SeEOH, the alkyl chain plays an important role in its binding and the orientation of SeEOH when bound to GNP surface. In DHS, such interactions are not feasible due to structural rigidity, therefore major interaction with GNP takes place via selenium atom.

Such binding is expected to differentially modulate the redox properties and rate of electron transfer reaction of SeEOH and DHS. This was assessed by monitoring the rate of electron transfer reaction between SeEOH in both free and GNP bound form with ABTS˙ radical (Fig. 5). The observed rate constants indicated that the reducing ability of SeEOH/DHS increased in presence of GNP and the reactivity increased with decrease in the GNP particle size (Table 5). The increase in the reducing ability of the GNP–SeC composites can be due to increase in the stability of the selenium centred radical formed on the GNP surface. Further, pulse radiolysis studies have shown that the ([double bond splayed left]Se∴Se[double bond splayed right])+ radical formed on reaction with ˙OH radical is more stable in SeEOH–GNP composites as compared to DHS–GNP composites (Fig. 6(A and B)). ˙OH radical reacts with SeEOH/DHS to form one-electron oxidised product which decays by two major pathways: (i) deprotonation to form α-C centred radical and (ii) association with parent SeEOH molecule to form ([double bond splayed left]Se∴Se[double bond splayed right])+ radical.33 The former pathway is assisted by overlapping of σ orbital of α C–H bond with the p-orbital of Se atom. As indicated by SERS studies interaction of alkyl chain of SeEOH will lead to bending of CH2 moiety towards GNP surface and in turn will lower overlapping between σ orbital of α-CH2 and p-orbital of Se atom and deactivate the deprotonation pathway. Thus the binding of SeEOH with GNP will lead to preferential formation of ([double bond splayed left]Se∴Se[double bond splayed right])+ radical which on disproportionation and sequential hydrolysis forms selenoxide.33 This was confirmed by the fact that there is increase in selenoxide formation in presence of GNP and the increase is much more pronounced in case of SeEOH than that for DHS. Formation of selenoxide is very important as it can regenerate parent SeC molecule in the presence of thiols. In absence of GNP, the selenium centred radical cation in SeEOH undergoes radiolytic degradation via deprotonation forming formaldehyde as one of the product. This pathway competes with dimer radical formation and results in low radiation chemical yield of its corresponding selenoxide. The deprotonation pathway is suppressed on binding of SeEOH with GNP which leads to increase in the yield of selenoxide. However, in DHS such degradation pathways are not observed, therefore no difference in radiation chemical yield of its selenoxide is observed in absence or presence of GNP. This differential increase in selenoxide is expected to influence their antioxidant activity significantly on binding to GNP surface. Thus our result demonstrates that small structural variations in selenium compounds can drastically change their redox properties that may have significance in their overall biological activity. Also such conjugation of selenium compound with GNP will result in the decrease of the radiosensitization effect of GNP and also decrease the toxicity of SeEOH due to suppression of oxidative degradation.

Experimental

Potassium tetrachloroaurate (KAuCl4), trisodium citrate (Na3-citrate), sodium borohydride (NaBH4), monosodium hydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), potassium persulfate (K2S2O8) were purchased from Sisco research laboratory (SRL). ABTS2−, DTT and DTTox of purity >98% were purchased from Sigma. Carbon coated copper grid were procured from Tedpella. Selenium compounds (SeC) were synthesized according to the reported method.34,35

GNP of different sizes (GNP1–5 nm, GNP2–15 nm, GNP3–25 nm and GNP4–58 nm) were prepared by modified Turkevich method and separated from unreacted reactants by centrifugation as reported in our earlier work.23,36 Concentration of gold atoms in the GNP samples was estimated using a continuum source flame atomic absorption spectrometer (Contra AA 300, Analytik Jena, Germany), as reported earlier.23 The yield of the GNP was estimated to be 95 ± 5, 43 ± 5, 32 ± 3 and 30 ± 3% for GNP1, GNP2, GNP3 and GNP4 respectively. Absorption spectra were recorded on JASCO V-630 UV-visible spectrophotometer. For this, 7 nM GNP was incubated with varying concentration of SeEOH (0–1 mM) for 1 minute at pH 7 and corresponding absorption spectra were recorded.

ABTS˙ radicals was prepared by oxidation of aqueous 1 mM ABTS2− using 0.5 mM K2S2O8. The concentration of ABTS˙ radical was estimated by using its absorbance at 415 nm (ε415 nm = 3.6 × 104 M−1 cm−l).28 The kinetics of the reaction of ABTS˙ radical was studied by following the decrease in absorbance of ABTS˙ radical at 820 nm as a function of time, to avoid any interference from GNP.

The effect of binding of SeC with GNP on the particle size was studied by DLS technique.37 The measurements were done on Malvern-4800 Zetasizer instrument with He–Ne laser (632.8 nm) at room temperature and the scattered light was collected at an angle of 130 degree. The dynamic information of the particle can be derived from auto correlation function G1(q,t) where q is wave vector and t is time in seconds. The auto correlation data were fitted to single exponential decay to give translational diffusion constant, from which the hydrodynamic size was calculated using Stoke–Einstein equation.

TEM images were taken on Libra-120 plus TEM (Carl Zeiss, Germany) operated at 120 kV as the accelerating voltage. The GNP samples were prepared by room temperature evaporation of 10 μL GNP solution on the carbon coated copper grids.

The ζ-potential measurements were performed on Zetasizer Nano (Malvern Instruments, Ltd.). A sample cell with two gold electrodes is used to measure the ζ-potential using Henry equation.38 To estimate the change in ζ-potential due to binding of SeC with GNP, the concentration of GNP was fixed at 2.5 nM and that of SeEOH or DHS was varied from 50 μM to 1 mM. A voltage of 200 V was applied to induce electrophoresis of GNP solution taken in sample cell with gold electrodes.

Raman spectra were recorded using a micro-Raman spectrometer (SEKI Technotron) using excitation at 532 nm (power ∼20 mW at sample position) and a 10× objective lens (Olympus). The scattered light was collected by the same objective lens and passed through an edge filter (LPO3-532RU-25) to filter Rayleigh and anti-Stokes scattered signals. The surface enhanced Raman spectra were recorded at room temperature using the 660 nm line from a 50 mW DPSS laser (Ignis 660-500) M s−1 Laser Quantum Ltd. England. The sample solutions were exposed to laser in a standard 1 × 1 cm cuvette for 200 s and the Raman scattered light was collected at 90° scattering geometry and detected using a CCD (Synapse, Horiba JobinYvon) based monochromator (Triax550, Horiba JobinYvon, France) together with an edge filter, covering a spectral range of 200–1700 cm−1. Calibration of the spectrometer was carried out using benzene: CCl4 mixture (1[thin space (1/6-em)]:[thin space (1/6-em)]1 v/v) and indene.

Pulse radiolysis studies were performed on 7 MeV linac electron accelerator with 100 ns pulse width coupled with absorption detector.39 Average dose of 9 Gy per pulse as estimated by thiocyanate dosimeter (aerated aqueous solution of 10 mM KSCN) was used for all experiments.40 Radiolysis of water at neutral pH generates both oxidizing radical ˙OH radical and reducing radical like hydrated electron (eaq), along with H atom, and hydronium ion (H3O+). The reaction with ˙OH radical was carried out in N2O saturated solution, where eaq generated during water radiolysis is quantitatively converted to ˙OH radical (G˙OH = 0.6 μmol J−1).41

Steady state radiolysis: or this N2O saturated aqueous solution containing 1 mM SeC and SeC–GNP composite containing 7 nM GNP1 was radiolyzed under steady state condition by γ-radiation using 60Co source (dose rate = 13.4 Gy min−1). The sample was radiolyzed at different doses and the maximum dose was set in such a way that not more that 10% of primary radical with respect to the SeC were generated. After radiolysis the samples were subjected to HPLC analysis to detect and quantify the selenoxide formed. For this 49 μL of the radiolyzed sample was incubated with 2 μL of DTT (50 mM) for 5 minutes and 20 μL of this reaction mixture was injected to Prontosil 120-5-C18 reverse phase column and eluted using 5[thin space (1/6-em)]:[thin space (1/6-em)]95 acetonitrile[thin space (1/6-em)]:[thin space (1/6-em)]water (v/v) containing 0.1% TFA under isocratic method. The DTTox formed was detected using absorption detector at 240 nm and quantified by using standard DTTox calibration plot. HPLC chromatogram showed SeEOH, DHS, DTT and DTTox peaks at 4.3, 5.4, 8.3 and 14.4 min, respectively. The yield of ˙OH radical was calculated by monitoring the fluorescence of 7-HOC formed due to the reaction of ˙OH radical with coumarin. The fluorescence measurements were done on Syngene hybrid micro plate reader with excitation and emission wavelength as 326 nm and 455 nm respectively.31

Quantum chemical calculations: the geometry of SeEOH was optimized in vacuo using Density Functional Theory (DFT) at B3LYP (Becke non local model and Lee–Yang–Parr non local correlation functional)/6-31+G(d,p) level adopting the GAMESS suite of programs on a PC-based LINUX cluster platform.42 The resulted geometry was further optimized in water using solvent density model (SMD). The optimized structures were verified as global minima structure by performing the frequency calculation. Raman spectra of optimized geometry was calculated at B3LYP/6-31+G(d,p) level and visualization of different Raman modes was done using MacMolPlt v7.6.1.

Conclusions

The study presents comparative binding of two structural isomers, SeEOH and DHS with GNP and impact of this binding on their electron transfer reactions. SeEOH and DHS show different mode of interaction with GNP arising due to their structural difference which differentially modulate their reaction with oxidants like ABTS˙ and ˙OH radical. These results demonstrate that GNP can be used to differentially modulate the redox properties of simple water soluble selenium compounds. This study would be of great significance in actual biological studies where GNP is being explored as a radio sensitizer. Changing the structure of the binding ligand can be used to fine tune their activity of interest either as radioprotectors or radio sensitizers.

Acknowledgements

The authors express their sincere thanks to Dr P. P. Phadnis, Chemistry Division, BARC for providing bis(2-ethanol)selenide. The authors are thankful to Drs Nandita Maiti and Apurva Guleria for taking the SERS and Raman spectra. The authors also thank Dr Harshala Parab, Analytical Chemistry Division, BARC and the Computer Center, BARC, for help in AAS experiments and for providing ANUPAM parallel computing facility, respectively. Ms. Pavitra Kumar acknowledges CSIR for the senior research fellowship.

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra15106j

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