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Interaction of Thermus thermophilus, ArsC
enzyme and gold nanoparticles naked-eye
assays speciation between As(III) and As...
besides being more toxic, arsenite is the most mobile and
common form of arsenic found in anaerobic contaminated
aquifers ...
device was set up by focusing the external beam, using an
optimal incident angle of 80°, on the sample using a mirror.
Pri...
(3) stabilizes the colloidal solution through electrostatic
interactions between the carboxylic acid groups and the gold
s...
Figure 2(A) (left graph) reports the LSP bands of PEG-
AuNPs before and after the adsorption of enzyme molecules
at equal ...
surfaces. Figure 4 reports, for comparison, a set of PM-
IRRAS data from planar gold substrates where TtArsC-
AuNPs (black...
attributed this macroscopic evidence of biomolecular inter-
action to the nanoparticle clustering process; this is also
co...
chain molecules. This aggregation–dispersion process leads
to the colorimetric changes in the nanoparticle solution.
Aggre...
[4] Macy J M, Santini J M, Pauling B V, O’Neill A H and Sly L I
2000 Two new arsenate/sulfate-reducing bacteria:
mechanism...
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Nanotechnology J.S (1)

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  • 1. This content has been downloaded from IOPscience. Please scroll down to see the full text. Download details: IP Address: 81.194.42.231 This content was downloaded on 06/10/2015 at 10:32 Please note that terms and conditions apply. Interaction of Thermus thermophilus, ArsC enzyme and gold nanoparticles naked-eye assays speciation between As(III) and As(V) View the table of contents for this issue, or go to the journal homepage for more 2015 Nanotechnology 26 435703 (http://iopscience.iop.org/0957-4484/26/43/435703) Home Search Collections Journals About Contact us My IOPscience
  • 2. Interaction of Thermus thermophilus, ArsC enzyme and gold nanoparticles naked-eye assays speciation between As(III) and As(V) Jane Politi1,2 , Jolanda Spadavecchia3,4 , Gabriella Fiorentino5 , Immacolata Antonucci5 , Sandra Casale3 and Luca De Stefano1 1 Institute for Microelectronics and Microsystems, Unit of Naples-National Research Council Via P. Castellino 111, 80127, Italy 2 Department of Chemical Sciences, University of Naples ‘Federico II’, Via Cynthia, 80126 Naples Italy 3 Sorbonne Universités, UPMC Univ Paris VI, Laboratoire de Réactivité de Surface, 4 place Jussieu, F-75005 Paris, France 4 CNRS, UMR 7244, CSPBAT, Laboratoire de Chimie, Structures et Propriétés de Biomateriaux et d’Agents Therapeutiques Université Paris 13, Sorbonne Paris Cité, Bobigny, France CNRS, Paris, France 5 Department of Biology, University of Naples ‘Federico II’, Via Cynthia, 80126 Naples, Italy E-mail: jane.politi@na.imm.cnr.it and luca.destefano@na.imm.cnr.it Received 29 June 2015, revised 22 August 2015 Accepted for publication 7 September 2015 Published 5 October 2015 Abstract The thermophilic bacterium Thermus thermophilus HB27 encodes chromosomal arsenate reductase (TtArsC), the enzyme responsible for resistance to the harmful effects of arsenic. We report on adsorption of TtArsC onto gold nanoparticles for naked-eye monitoring of biomolecular interaction between the enzyme and arsenic species. Synthesis of hybrid biological–metallic nanoparticles has been characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV–vis), dynamic light scattering (DLS) and phase modulated infrared reflection absorption (PM-IRRAS) spectroscopies. Molecular interactions have been monitored by UV–vis and Fourier transform-surface plasmon resonance (FT-SPR). Due to the nanoparticles’ aggregation on exposure to metal salts, pentavalent and trivalent arsenic solutions can be clearly distinguished by naked-eye assay, even at 85 μM concentration. Moreover, the assay shows partial selectivity against other heavy metals. S Online supplementary data available from stacks.iop.org/NANO/26/435703/mmedia Keywords: arsenate reductase, gold nanoparticles, biorecognition, naked eye assay (Some figures may appear in colour only in the online journal) 1. Introduction Thermus thermophilus HB27 is an extremophile organism living in arsenic-rich geothermal environments: this bacter- ium has developed the ability to both oxidize and reduce arsenic, thus playing an important role in its speciation and bioavailability [1, 2]. Arsenate reduction mechanisms, apparently due to convergent evolution or originating in a common ancestor and then transferred to [3, 4], can be individuated into three families: the first family has been typified as arsC glutathione–glutaredoxin dependent (arsC- GSH/Grx) and was identified in enteric bacteria (e.g. Escherichia coli); the second one is known as arsC tioredoxin dependent (arsC-Trx) and was found in Gram-positive bac- teria (e.g. Staphylococcus). The last family, which includes the ars2 gene, was amplified from Saccharomyces cerevisiae [5]. Microbial activities play critical roles in the geochemical cycling of arsenic because they can promote or inhibit its release from sediment material, mainly by redox reactions [6– 8]. The reduction of pentavalent arsenate, As (V), to trivalent arsenite, As (III), is the major reaction causing the release of arsenic from the mineral surfaces into groundwater; in fact, Nanotechnology Nanotechnology 26 (2015) 435703 (9pp) doi:10.1088/0957-4484/26/43/435703 0957-4484/15/435703+09$33.00 © 2015 IOP Publishing Ltd Printed in the UK1
  • 3. besides being more toxic, arsenite is the most mobile and common form of arsenic found in anaerobic contaminated aquifers [9]. There is a worldwide demand to sense and quantify arsenic pollution, both natural and anthropogenic, in fresh water using low-cost and easy-to-use devices, especially in developing countries. Nanostructured materials claim a range of exciting phy- sical and chemical properties, which make them fundamental building blocks for the next generation of instruments and devices. In particular, gold nanoparticles (AuNPs) are among the most-used nano-objects, and are exploited in many applications ranging from medical to environment monitor- ing. The most popular method for preparing AuNPs in water uses citrate to reduce HAuCl4 under boiling conditions [10]. Therefore, several approaches have been developed to reduce Au (III) salts in water using different ligands as colloid par- ticle stabilizers [11]. Stabilizers, usually surfactant molecules, protect particles by avoiding aggregation mechanisms and controlling their physio-chemical properties [12, 13], but these molecules are mostly toxic. Dangerous organic mole- cules could be substituted by some biocompatible molecules, such as polyethylene glycol (PEG), in order to prepare bio- compatible PEG-stabilized AuNPs [14, 15]. Recently, great advances have been made in the use of gold nanoparticles for signaling applications, owing to their stability, chemical reactivity, non-toxic nature, strong absorption and scattering properties, and electrostatic charges that allow strong inter- actions with proteins and enzymes [16, 17]. For instance, biomolecule- and/or biopolymer-conjugated AuNPs are lar- gely used as biomarkers or biodelivery vehicles, as well as for cosmetics and as anti-aging components for skin protec- tion [18, 19]. In the following study, we report our results on the adsorption of TtArsC enzyme onto PEG-stabilized AuNPs (PEG-AuNPs) for monitoring its interaction with pentavalent arsenic ions (As (V)) and trivalent arsenic ions (As (III)). Both the adsorption of enzyme onto PEG-AuNPs and its interaction with As (V) and As (III) salts can be followed easily by the naked eye, since solutions completely change their colors. UV–vis spectroscopy, polarization modulation infrared reflection/adsorption (PM-IRRAS) spectroscopy, dynamic light scattering (DLS) and Fourier transform-surface plasmon resonance (FT-SPR) were used as the main char- acterization techniques. 2. Experimental 2.1. Chemicals Tetrachloroauric acid (HAuCl4), sodium borohydride (NaBH4), polyethylene glycol 600 diacid (PEG diacid), β-Mercap- toethylamine (cysteamine), 1, 4-phenylenediisothiocyanate (PDC), ethanol (C2H5OH), pyridine, dimethylformamide (DMF), 15 mM Tris-HCl, potassium metarsenite (NaAsO2), potassium arsenate (KH2AsO4), cadmium ions solution, lead (II) methanesulfonate (C2H6O6PbS2) and mercury (II) nitrate solution (HgN2O6) were purchased from Sigma Aldrich. All chemicals were used without any further purification. 2.2. Purification and preparation of TtArsC enzyme Recombinant TtArsC (TtArsC: protein arsenate reductase from the Gram-negative bacterium Thermus thermophilus HB27) was purified to homogeneity using the purification procedure already described, basically consisting of a thermo- precipitation of the Escherichia coli cell extract followed by anion exchange and gel filtration chromatography [20]. Fractions containing purified TtArsC were pooled, dialyzed against 15 mM Tris-HCl, 1 mM DTT, pH 7.5 and lyophilized in aliquots of 1 mg using a freeze dryer (HetoPowerDry PL6000, Thermo Scientific). Protein aliquots for nanoparticle interaction were prepared by resuspension of the protein in 1 ml of 15 mM Tris-HCl, pH 7.5. 2.3. Synthesis of PEG-stabilized Au nanospheres (PEG- AuNPs) Li et al [10] have previously reported an easy method of synthesizing AuNPs from concentrated chloroauric acid solutions by adding sodium hydroxide as a reducer agent, citrate molecules as a stabilizer of colloidal solution. We modified this protocol using PEG-diacid as stabilizer mole- cules by the one-step method, using it inside the mixture reaction for AuNPs solution in spite of citrate molecules [21]. Briefly, 25 ml of chloroauric acid (HAuCl4) aqueous solution (2.5×10−4 M) was added to 0.25 ml of PEG-diacid under stirring for 10 min at room temperature. After that, 20 ml of aqueous 0.01 M NaBH4 was added at once. The formation of the PEG-AuNPs solution was observed by an instantaneous color change of the pale yellow solution to typical red/rose solution after addition of the NaBH4 reducing agent. The PEG-AuNPs solution, prepared as described above, was centrifuged at 15 000 rpm for 26 min three times; then the supernatant was discarded while the residue was resuspended in an equivalent amount of buffer solution (PBS pH: 7). These procedures were repeated twice in order to remove the excess PEG-diacid. 2.4. Adsorption of TtArsC onto PEG-AuNPs The enzyme TtArsC was adsorbed on PEG-AuNPs by using the following procedure: 1 ml of PEG-AuNPs was added into separate tubes containing 0.05 ml of TtArsC (1 mg ml−1 in 15 mM TrisHCl, pH 7). The resulting suspension of hybrid nanoparticles, reported in the following as TtArsC-AuNPs, was centrifuged twice at 6000 rpm for 20 min to remove excess protein, and then the pellets were re-dispersed in 1 ml MilliQ water. This colloidal solution was sonicated for 5 min and then stirred for 1 h at room temperature. 2.5. PM-IRRAS characterization Polarization modulation infrared reflection absorption spec- troscopy (PM-IRRAS) spectra were recorded on a commer- cial Thermo Nexus spectrometer (Les Ulis, France). The 2 Nanotechnology 26 (2015) 435703 J Politi et al
  • 4. device was set up by focusing the external beam, using an optimal incident angle of 80°, on the sample using a mirror. Prior to this, a ZnSe grid polarizer and a ZnSe photo-elastic modulator were placed on the sample, and the incident beam was tuned between p- and s-polarizations (HINDS Instru- ments, PEM 90, modulation frequency=37 kHz). Finally, the light reflected by the sample was focused onto a nitrogen- cooled MCT detector. The presented spectra result from the sum of 128 scans recorded at 8 cm−1 resolution. Each spec- trum reported represents the average of at least three mea- surements. The glass substrates (11×11 mm2 ), coated by a 5 nm thick layer of chromium and a 200 nm thick layer of gold, were purchased from Arrandee (Werther, Germany). The gold-coated substrates were annealed on a butane flame to ensure a good crystallinity of the gold top layer and rinsed in a bath of absolute ethanol for 15 min before use. Chemistry procedures based on a self-assembling monolayer of β-mercaptoethylamine (cysteamine) and a crosslinker have been described previously [22]. Briefly, the freshly cleaned gold substrates were immersed in an unstirred 10 mM ethanol solution of cysteamine at room temperature, in the dark, for 6 h. The gold substrates were then washed with ethanol and ultrapure water (Milli-Q, Millipore, France) to remove the excess thiols. The amino surface was treated following two strategies represented in scheme 1. Scheme S1 (A) shows that the amino surface was treated using 0.2% (w/v) of 1, 4-phenylenediisothio-cyanate (PDC) solution in a solution of 10% pyridine/90% dimethylformamide (DMF) for 2 h at room temperature. Then, the samples were suc- cessively washed in DMF and in ethanol and dried under a stream of nitrogen, leaving an isothiocyanate-derivatized surface. TtArsC was then chemically adsorbed to the iso- thiocyanate-covered slides by exposing the entire surface to the TtArsC solution for 40 min and then thoroughly rinsed twice in buffer and once in milliQ water. Scheme S1(B) shows how the amino surface was treated by EDC/NHS (80 mg/20 mg) and PEG-AuNPs modified with TtArsC solutions for 1 h and then rinsed with phosphate buffer solution and MilliQ water three times for 5 min. The resulting samples were used for PM-IRRAS investigations. 2.6. Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) measurements were recorded using a JEOL JEM 1011 microscope, which operates at an accelerating voltage of 100 KV. The TEM acquisitions were taken after separating the surfactant solution from the metal particles by centrifugation. Specifically, 1 ml of the nanoparticle solution was centrifuged at 14 000 rpm for 20 min. The supernatant was removed while the pellet was re- dispersed in 1 ml of water; then, a liquid droplet (10 μl) of the colloidal solution was deposited and dried on a microscope grid and finally analyzed. 2.7. Dynamic light scattering (DLS) The size measurements were performed by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with a He-Ne laser (633 nm, fixed scattering angle of 173°, room tempera- ture 25 °C). 2.8. UV/Vis measurements The absorption spectra of each sample were recorded using a Jasco V-570 UV/VIS/NIR Spectrophotometer from Jasco Int. Co., Ltd, Tokyo, Japan, in the 200–800 nm range. The spectra were recorded after 30 min from the synthesis of PEG AuNPs, and from 2 min to 24 h after TtArsC enzyme adsorption. Finally, spectra were recorded after 10 min of TtArsC–AuNP interaction with each heavy metal solution. 2.9. FT-SPR Fourier Transform-Surface Plasmon Resonance (FT-SPR) measurements were performed with an SPR 100 module from Thermo, equipped with a flow cell mounted on a goniometer. The setup was inserted in a Thermo-scientific Nexus FT-IR spectrometer, and a near-IR tungsten halogen light source was used. The incidence angle was adjusted at the beginning of each experiment with the minimal reflectivity located at 9000 cm−1 , in order to be in the highest sensitivity region of the InGaAs detector. Gold substrates for FT-SPR measure- ments were prepared at IMM-CNR in Lecce (Italy). 2.10. Heavy metals interaction monitoring The interaction between TtArsC-AuNPs, As (V) and As (III) solutions was followed using UV–vis spectra of the TtArsC- AuNPs solution (50 μl of heavy metal solutions were added to 1 ml TtArsC-AuNPs solution) and the FT-SPR shifts of TtArsC-AuNPs–modified gold substrates using As (V) and As (III) at 750-325-170-85 μM. Furthermore, the interaction between TtArsC-AuNPs, Pb2+ , Cd2+ and Hg2+ solutions was followed using UV–vis spectra (50 μl of heavy metal solu- tions at 170 μM were added to 1 ml TtArsC-AuNPs solution). 3. Results and discussion The interface properties of AuNPs are an interesting topic of study. In particular, the presence of chemical groups at the outer surfaces of AuNPs improves the ability of nanoparticles to interact with biological probes and consequently enhances the interaction of biosensing systems with target analytes. Coating AuNPs with a bifunctional PEG linker carrying two carboxylic groups using a one-step method [15–21] is one useful way to enhance the properties of interfaces: the so- called PEG-diacid can be used as a capping agent, an alter- native approach with respect to the citrate-stabilized synthesis process [23, 24]. Furthermore, particle formation and growth can be tuned by exploiting the amphiphilic character of the PEG-diacid polymer in three steps: (1) reduction and stabi- lization of HAuCl4 is facilitated by dicarboxylic acid-termi- nated PEG to form gold clusters through the exchange of electrons between them; (2) the presence of PEG-diacid molecules on gold surfaces shortens cluster dimensions and 3 Nanotechnology 26 (2015) 435703 J Politi et al
  • 5. (3) stabilizes the colloidal solution through electrostatic interactions between the carboxylic acid groups and the gold surface [15]. Figure 1(A) shows a TEM image of PEG-AuNPs after deposition on a microscope grid. The TEM picture of the PEG-AuNPs reveals fairly regular and monodispersed Au nanospheres. Figure 1(B) shows the histogram of 1623 particles: it can be fitted by a Gaussian curve with a mean size of 7 nm with a standard deviation of 2 nm. PEG-AuNPs were used as nanostructured supports for binding TtArsC enzymes in the realization of an assay for biomolecular interaction. TtArsC enzyme adsorption onto PEG-AuNPs was monitored by the following methods: UV–Vis spec- troscopy in order to monitor Localized Surface Plasmon (LSP) band shift; DLS characterizations in order to observe the aggregation/dispersion behavior of nanoparticles; TEM characterization in order to confirm the aggregation/dis- persion behavior of nanoparticles; and PM-IRRAS char- acterizations in order to evaluate the chemical groups showed at the outer surfaces. Figure 1. TEM images of PEG AuNPs (A) and histogram of nanoparticle size distribution (B). Figure 2. (A) UV–vis spectra of PEG AuNPs during adsorption of TtArsC enzyme as a function of time. (B) Schematic representation of nanoparticle solution change of color during adsorption of TtArsC. 4 Nanotechnology 26 (2015) 435703 J Politi et al
  • 6. Figure 2(A) (left graph) reports the LSP bands of PEG- AuNPs before and after the adsorption of enzyme molecules at equal concentrations of PEG-AuNPs in aqueous solution (10−4 M) as a function of time. Figure 2(A) (right image) reports the TEM image of PEG-AuNPs after adsorption to TtArsC (TtArsC-AuNPs), revealing an aggregation behavior of nanoparticles, while in figure 2(B) photographic images of the cuvettes containing the correspondent solutions are reported. The PEG-AuNP solution, before enzyme adsorp- tion, shows an absorbance peak at 530 nm with a typical red/ rose color, whereas, after mixing with the enzyme, in two minutes the color started changing and completed the reaction in about 10 min, which corresponded to a shift of the LSP peak at around 640 nm. UV–vis spectra were recorded up to 24 h after TtArsC adsorption, although after 5 h the hybrid biological–metal nano-complex became stable, conferring a characteristic violet color to the solution. We estimated a hydrodynamic diameter of 14±5 nm for PEG-AuNPs (figure 3(A)), while TtArsC-AuNPs have a hydrodynamic diameter of 39±13 nm, which means that the enzyme aggregated three to four PEG-AuNPs on average. A more accurate evaluation of TtArsC adsorption on PEG-AuNPs was confirmed by PM-IRRAS, which is particularly useful to reveal the chemical groups exposed on nanoparticles’ outer Figure 3. (A) Size change after each interaction step. (B) Schematization of aggregation process of PEG AuNPs with TtArsC enzyme and arsenate/arsenite ions. 5 Nanotechnology 26 (2015) 435703 J Politi et al
  • 7. surfaces. Figure 4 reports, for comparison, a set of PM- IRRAS data from planar gold substrates where TtArsC- AuNPs (black line) and TtArsC alone (red line) have been covalently bonded. Figure 4 (black line) shows a peak at 1100 cm−1 attributed to -COOH groups of PEG-diacid, and peaks at 1400 cm−1 and 1450 cm−1 , typical of -COO- stretching vibrations. Peaks at 1660 cm−1 and 1530 cm−1 are also present, representing amide II and I, respectively, which are char- acteristic of all proteins and enzymes. Peak at 1730 cm−1 represent the C=O stretching mode of PEG-AuNPs immo- bilized onto a gold surface, thus endorsing an effective functionalization of the surface. The presence of a strong stretching band at 1100 cm−1 together with peaks at 1450 cm−1 and 1730 cm−1 suggests the stabilization of the AuNPs with PEG molecules. Figure 4 (red line) shows a peak at 1100 cm−1 repre- senting stretching of aliphatic ethers, a peak at 1240 cm−1 characteristic of C=S stretching of the PDC crosslinker, and the amide I and II peaks at 1530 cm−1 and 1660 cm−1 , respectively. The intensities of these peaks are higher with respect to the precedent case, due to rearrangement of TtArsC on AuNPs, where PEG-diacid functional groups interacting with the enzyme can partially mask the amide bonds. In this paper, we investigate a versatile chemistry modification that uses homobifunctional crosslinker PDC in order to achieve covalent binding of TtArsC before and after interaction with PEG-AuNPs. The isothiocyanate group present in PDC crosslinker generally act as electrophiles with a carbon atom as the electrophilic center. Electrophilic substitutions with the amino group of cysteamine lead to a stable ligand with a crosslinker that allows covalent binding of TtArsC and TtArsC-AuNPs. Since the TtArsC enzyme is specialized in binding and transforming arsenic compounds, FT-SPR measurements were used to monitor the interaction between TtArsC-AuNPs and arsenate (As(V))/arsenite (As(III)) ions; four different concentrations were used for each salt and the details of the results are reported in figure 5. Figure S2 represents a typical shift of surface plasmon resonance from 9000 cm−1 before interaction (black line in left graph) to 8600 cm−1 after ion detection (red line in left graph), and the shift of peak position as a function of time during the binding cycle followed by rinsing (right graph). Figure 5 clearly shows that the interaction between TtArsC-AuNPs and arsenate/arsenite ions is concentration dependent (panels A and C); in both panels, each point reported represents the value of plasmon stabilization after interaction with arsenate/arsenite ions as a function of different concentration. The linear regression parameters obtained by OriginLab Software™ for both arsenite/arsenate ion interaction monitoring are reported in tables S1 and S2 in supplementary data. Moreover, the absolute position of the plasmon absorbance peak changes as a function of different concentrations for both arsenate and arsenite (panels B and D, respectively). Experimental data points in figures 5(B) and (D) were fitted using OriginLab Software™ by Michaelis- Mentens dose-response exponential equation: y x A ye 1x C 0( ) ( )( )*= +/ where A represents the amplitude and C a growth constant. The first derivative of equation (1) is y x A C e 2x C1 ( ) ( ) ( )( )*= / / By equation (2), the sensitivity of the nanosystem in ion biorecognition can be obtained as y1 (xM) where xM is the middle point of each data set: S 1.6 0.2 cm MAsV 1 1m=  - - S 2.82 0.02 cm MAsIII 1 1m=  - - where SAsV is the sensitivity of the system against arsenate and SAsIII is the sensitivity of the system against arsenite. The estimated sensitivities reveal that the nanobiocomplexes have a higher sensitivity for As (III) ions with respect to As (V) ions, even if the natural substrates of TtArsC enzyme are arsenate ions. As is already known [19], the TtArsC enzyme has a redox system, able to link the reduction of arsenate to the consumption of dihydronicotinamide adenine dinucleotide phosphate, the so-called NADPH, by using the thioredoxin reductase/thioredoxin (Tr/Trx) system for the redox recycling with a catalytic mechanism that involves the thiol group of the N-terminal cysteine residue (Cys7). In view of our results, we can deduce that these amino acid residues, essential in redox reactions, are partially or totally masked, due to the adsorption of the enzyme onto the PEG-AuNPs’ surface. Further investigations on biorecognition at 85 μM con- centrations of both arsenite and arsenate have been performed by UV–vis measurements (see curves in figure 6). The LSP bands and the images reported also showed that, at the lowest concentration tested, a change of LSP band position and of the color of the solutions is clearly observable, thus con- firming the biomolecular interaction quantified by FT-SPR measurements. Furthermore, the photographic images repor- ted on the left of figure 6 highlight how the solution color change is a function of the arsenic ions’ oxidation state: the solution of TtArsC-AuNPs became violet/pink on exposure to As(V), while in the case of As(III) it turned to blue. In both cases, it was clearly visible to the naked eye. Again, we Figure 4. PM IRRAS spectra of TtArsC immobilized on cysteamine modified gold substrates (red line) and TtArsC-AuNPs on cystea- mine-modified gold substrates (black line). 6 Nanotechnology 26 (2015) 435703 J Politi et al
  • 8. attributed this macroscopic evidence of biomolecular inter- action to the nanoparticle clustering process; this is also confirmed by the DLS data in figure 3(A), specifically the fourth (red) and fifth (green) curves. We sketched the interaction mechanism in the scheme reported in figure 3(B). Enzyme biosensing was achieved using gold nanoparticles [25]. These peptides lead to the assembly of nanoparticles due to their crosslinking by long- Figure 5. Trasmittance of SPR trend by increasing concentration of As V (A) and As III (C) solutions; shift of SPR trasmittance as a function of increasing concentration (85–170–325–750 μM) of As V (B) and As III (D) solutions. Figure 6. UV–vis spectra (vertically shifted for clarity) of TtArsC-AuNPs before and after interaction with arsenate and arsenite ions (right graph). Images of TtArsC-AuNPs color change after interaction with arsenate and arsenite ions (left scheme). 7 Nanotechnology 26 (2015) 435703 J Politi et al
  • 9. chain molecules. This aggregation–dispersion process leads to the colorimetric changes in the nanoparticle solution. Aggregation of gold nanoparticles leads to red shift in the plasmon band due to the electric dipole–dipole interaction, which in turn leads to a coupling between the plasmon oscillations of different particles [26]. The color of the gold nanoparticle solution turns from red to blue/purple due to red shift in the plasmon band. Aggregated or assembled nanoparticles display red shift in the plasmon band when compared to the isolated gold nanoparticles. This phe- nomenon is attributed to the coupling between the dipole modes of plasmons of different particles. As the inter- particle distance is decreased, more red shift in the plasmon band is observed due to an increase in the extent of coupling. In order to verify the interference of other heavy metal ions that are not natural substrates for the enzyme in the recognition of arsenic ions, UV–vis measurements have been performed in the presence of single ion species (Cd2+ , Pb2+ and Hg2+ ) at a concentration of 170 μM. Figure 7 shows that in the presence of such heavy metals, there are not relevant changes in LSP position or intensity, indicating that the assay is highly specific against arsenic compounds. In order to verify whether the nanobiocomplex was also selective for As (III) and As (V), we measured the UV–vis spectra in the presence of a mixture of heavy metals. The results are reported in figure S3. We found that, despite the apparent insensitivity to other heavy metal ions that can be conjectured by data in figure 6, on exposure to a complex mix, the LSP in UV–vis spectra is quite different from the reference ones, i.e. the spectra obtained for As (III) and As(V) alone. This behavior demonstrates a lack of selectivity and prevents the use of the assay for a quantitative measurement of arsenic ions in a complex mixture. Nevertheless, since the solution always changes color in the presence of As(III) and As(V), the assay can be simply and usefully used in fast and cheap screening of water quality. 4. Conclusions In this work, we used a novel chromosomal arsenate reduc- tase (TtArsC) as biomolecular probe to screen for the presence of arsenic in water. Using optical, label-free techniques, we have characterized the interaction between TtArsC and arsenic ions, quantitatively evaluating interaction and biorecognition with pentavalent arsenic, As(V), and trivalent arsenic, As(III). The novel and original nanobiocomplexes demonstrated stability and the capacity to strongly bind the toxic ions. Experimental data demonstrated relevant signal changes, i.e. variation of the FT-SPR peak position (about 200 cm−1 also at low concentrations). TtArsC-AuNPs showed greater sen- sitivity for arsenite ions, as opposed to what happens in nat- ure, where arsenate ions are the main substrate of TtArsC enzymes. On these bases, TtArsC-AuNP nanobiocomplexes were found to be able to interact with arsenite ions solutions, veering to blue solutions, and arsenate ions solutions, veering to violet/pink solutions, at all concentrations tested. These phenomena were confirmed quantitatively by LSP shifts in UV–vis spectra, and DLS characterization reveals that the nanobiocomplex aggregates in the presence of arsenic ions. Finally, LSP band study in the presence of metal ions that are not enzyme substrates (Cd2+ , Pb2+ and Hg2+ ) indicated that the biorecognition is highly specific but not completely selective. A straightforward application in fast and low-cost screening of water can be envisaged. Acknowledgments This manuscript was compiled using contributions from all authors. All of the authors have given approval to the final version of the manuscript. This work was partially supported by national research program PON MONICA 01_01525. Supplementary data Scheme of immobilization on gold support for PM-IRRAS analysis; working principle of FT-SPR; further investigation of LSP band in UV–vis spectrum and naked-eye response of the TtArsC-AuNPs in the presence of complex mixtures of heavy metals are reported. References [1] Espino D P, Tamames J, de Lorenzo V and Cánovas D 2009 Microbial responses to environmental arsenic Biometals 22 117–30 [2] Gihring T M and Banfield J F 2001 Arsenite oxidation and arsenate respiration by a new Thermus isolate Microbiol. Lett. 204 335–40 [3] Jackson C R and Dugas S L 2003 Phylogenetic analysis of bacterial and archaealarsC gene sequences suggests an ancient, common origin for arsenate reductase BMC Evol. Biol. 3 18 Figure 7. UV–vis spectra of TtArsC adsorbed on PEG AuNPs after interaction with cadmium, mercury and lead ions. 8 Nanotechnology 26 (2015) 435703 J Politi et al
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