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Research Cite this article: Politi J, Spadavecchia J, Fiorentino G, Antonucci I, De Stefano L. 2016 Arsenate reductase from Thermus thermophilus conjugated to polyethylene glycol-stabilized gold nanospheres allow trace sensing and speciation of arsenic ions. J. R. Soc. Interface 13: 20160629. http://dx.doi.org/10.1098/rsif.2016.0629
Arsenate reductase from Thermus thermophilus conjugated to polyethylene glycol-stabilized gold nanospheres allow trace sensing and speciation of arsenic ions Jane Politi1, Jolanda Spadavecchia2,3, Gabriella Fiorentino4, Immacolata Antonucci4 and Luca De Stefano1 1 Institute for Microelectronics and Microsystems, Unit of Naples-National Research Council, via P. Castellino 111, 80127 Naples, Italy 2 Sorbonne Universite´s, UPMC Univ Paris VI, Laboratoire de Re´activite´ de Surface, 4 place Jussieu, 75005 Paris, France 3 CNRS, UMR 7244, CSPBAT, Laboratoire de Chimie, Structures et Proprie´te´s de Biomateriaux et d’Agents Therapeutiques Universite´ Paris 13, Sorbonne Paris Cite´, Bobigny, France CNRS, Paris, France 4 Department of Biology, University of Naples ‘Federico II’, Via Cynthia, 80126 Naples, Italy
LD, 0000-0002-9442-4175
Received: 8 August 2016 Accepted: 7 September 2016
Subject Category: Life Sciences – Physics interface Subject Areas: nanotechnology, environmental science, biochemistry
Water sources pollution by arsenic ions is a serious environmental problem all around the world. Arsenate reductase enzyme (TtArsC) from Thermus thermophilus extremophile bacterium, naturally binds arsenic ions, As(V) and As (III), in aqueous solutions. In this research, TtArsC enzyme adsorption onto hybrid polyethylene glycol-stabilized gold nanoparticles (AuNPs) was studied at different pH values as an innovative nanobiosystem for metal concentration monitoring. Characterizations were performed by UV/Vis and circular dichroism spectroscopies, TEM images and in terms of surface charge changes. The molecular interaction between arsenic ions and the TtArsC-AuNPs nanobiosystem was also monitored at all pH values considered by UV/Vis spectroscopy. Tests performed revealed high sensitivities and limits of detection equal to 10 + 3 M212 and 7.7 + 0.3 M212 for As(III) and As(V), respectively.
Keywords: enzyme, gold nanoparticles, arsenic pollution
1. Introduction Author for correspondence: Luca De Stefano e-mail: [email protected]
Arsenic is a naturally occurring metal; it is among the 20 most common substances in the earth’s crust and is a component of more than 200 minerals [1,2]. Arsenic and its compounds are extremely mobile in the environment. Ageing of rocks by weather conditions converts arsenic sulfides to arsenic trioxide, which enters the arsenic cycle as dust or by dissolution in rivers and groundwater [3,4]. Water sources pollution by arsenic is a serious environmental problem all around the world, as it causes a lot of adverse health effects to humans at every level. Detection and quantification of arsenic ions concentration is thus a hot topic because water management requires optimization of quality assessment [5–7]. Biosensors and, in particular, nanoparticle-enhanced enzyme biosensors have recently emerged as powerful devices for qualitative and quantitative analysis of a variety of analytes for biomedical applications, drugs testing and environmental monitoring. Protein and enzymes can selectively recognize their natural ligands with very high specificity even in complex matrices such as the environmental ones [8]. In this field, there is also a general interest in biomolecules purified from thermophilic organisms due their biotechnological advantages [9]. Enzymes and proteins isolated from thermophilic microorganisms exhibit high stability in conditions that are usually adverse to proteins, such as high temperatures (up to 1008C), heavy concentration of detergents, extreme pH values, ionic strength and chaotropic agents [10–12]. In
& 2016 The Author(s) Published by the Royal Society. All rights reserved.
centrifuged twice at 6000 r.p.m. for 20 min in order to remove excess protein and then the pellets were re-dispersed in 1 ml of MilliQ water. The resultant colloidal solution was sonicated for 30 min at 48C. In order to monitor the adsorption of TtArsC onto AuNPs at different pH values, the pellets were re-dispersed in sodium acetate 20 mM at pH 5, trisodium phosphate 20 mM at pH 6, 7 and 8, glycine 20 mM at pH 9. The adsorption was characterized by UV/Vis spectrophotometry, transmission electron microscopy, circular dichroism (CD) and Z-potential measurements as described below.
Interaction between TtArsC-AuNPs and As (V)/As (III) solutions was performed adding 0.05 ml of heavy metal ions solution at 10212 M, 10210 M, 1028 M, 1026 M, 2.1 1026 M, 8.5 1026 M, 35 1026 M, 85 1026 M to 1 ml of TtArsC-AuNPs solution. After 10 min, the resulting mixtures were analysed by UV –Vis spectrophotometry.
2. Material and methods 2.1. Chemicals Tetrachloroauric acid (HAuCl4), sodium borohydride (NaBH4) and ethanol (C2H5OH), polyethylene glycol 600 Diacid (PEG), Tris – HCl (C4H11NO3), sodium acetate (CH3COONa), trisodium phosphate (Na3PO4), glycine (C2H5NO2), potassium metarsenite (NaAsO2), potassium arsenate (KH2AsO4) were purchased from Sigma Aldrich. All chemicals were used as supplied without further purification.
2.2. Purification and preparation of TtArsC enzyme Recombinant TtArsC (TtArsC: protein arsenate reductase from the Gram-negative bacterium T. thermophilus HB27) was purified up to homogeneity using the purification procedure described in a previous paper [16], basically consisting of thermo-precipitation of the Escherichia coli cell extract followed by anion exchange and gel filtration chromatography. Fractions containing purified TtArsC were pooled, dialysed 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 at a final 1.6 mM concentration.
2.3. Synthesis of PEG-stabilized-Au nanospheres In previous work [13], we have synthesized gold nanoparticles adding dicarboxylic PEG as surfactant in the mixture reaction. Briefly, 20 ml of chloroauric acid (HAuCl4) aqueous solution (2.5 1024 M) was added to 0.25 ml of dicarboxylic PEG and mixed by magnetic stirring for 10 min at room temperature. To this solution, 600 ml of aqueous 0.01 M NaBH4 was added drop by drop. The formation of the PEG-stabilized AuNPs (in the following simply cited as AuNPs) was observed as an instantaneous colour change of the solution from pale yellow to bright red after addition of the reducing agent. The as-prepared AuNPs solution was centrifuged at 15 000 r.p.m. for 30 min for three times and then the supernatant was discarded and the residue was suspended again in an equivalent amount of buffer solution. This last step was repeated twice, in order to remove excess dicarboxylic PEG.
2.4. Adsorption of TtArsC onto AuNPs TtArsC-AuNPs adsorption was achieved using the following procedure: 1 ml of AuNPs was added into separate tubes containing 0.05 ml of TtArsC (1 mg ml21 in 15 mM Tris – HCl, pH 7) for 24 h. The TtArsC-AuNPs suspension was then
2.6. UV –Vis measurements Absorption spectra were recorded using a Cary 100 (Agilent) spectrophotometer in the 200 –800 nm range. The spectra were recorded after 30 min from synthesis of nanoparticles, then from 2 min to 24 h after enzyme adsorption and finally after 10 min of interaction with heavy metals solutions. In order to evaluate TtArsC binding efficiency onto AuNPs, the TtArsCAuNPs were centrifuged for 30 min at 48C and the supernatant optical density (OD) was evaluated at 280 nm, 24 h after colloidal solution formation. Solutions of TtArsC-AuNPs, As (III) at 8.5 mM þ TtArsC-AuNPs, and As (V) at 8.5 mM þ TtArsCAuNPs at pH values from 5 to 9 were also characterized after 10 min of interaction.
2.7. Transmission electron microscopy Transmission electron microscopy (TEM) measurements were recorded on a FEI TEcnai G2 Spirit BioTWIN microscope operating at an accelerating voltage of 120 kV. The TEM graphs were taken after separating the surfactant from the metal particles by centrifugation; 1 ml of the sample was centrifuged for 20 min at a speed of 14 000 r.p.m. min21. The upper part of the colourless solution was removed and the solid part was re-dispersed in 1 ml of water. A droplet (10 ml) of the colloidal solution (TtArsC-AuNPs, As (III) at 8.5 mM þ TtArsC-AuNPs, As (V) at 8.5 mM þ TtArsC-AuNPs) was deposited on a microscope grid, dried at room temperature and analysed.
2.8. Z-potential measurements The Z-potential measurements of TtArsC-AuNPs, As (III) at 8.5 mM þ TtArsC-AuNPs, As (V) at 8.5 mM þ TtArsC-AuNPs solutions at pH values from 5 to 9 were performed by a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with a He – Ne laser (633 nm, fixed scattering angle of 1738, 258C).
2.9. Circular dichroism measurements The TtArsC-AuNPs, As(III) at 8.5 mM þ TtArsC-AuNPs, As(V) at 8.5 mM þ TtArsC-AuNPs solutions at pH values from 5 to 9 were characterized as follows. Far UV CD spectra were recorded on a Jasco J815 spectropolarimeter equipped with a Peltier thermostatic cell holder, in a quartz cell (0.1 cm light path) from 190 to 250 nm [17]. The temperature was kept at 208C and the sample compartment was continuously flushed with nitrogen gas. Furthermore, all the spectra recorded had corresponding high voltage applied to the photomultiplier below 700 V. Final spectra were obtained by averaging three scans, using a bandwidth of 1 nm, a step width of 0.5 nm and 4 s averaging per
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2.5. Heavy metals interaction monitoring
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our recent paper, we adsorbed a novel chromosomal arsenate reductase (TtArsC) able to reduce the substrate As(V) to As(III), on homemade hybrid gold nanoparticles (NPs) [13], producing a biomolecular probe that can screen for the presence of arsenic ions in water [14]. Thermus thermophilus HB27 is an extremophile bacterium that evolved in arsenic-rich geothermal environments: survival mechanisms of this organism include oxidization and reduction of arsenic ions and extrusion from living cells [15]. The unique features of TtArsC enzyme allowed the speciation of pentavalent arsenic, As(V), and trivalent arsenic, As(III), by a naked-eye assay [14]. In this work, we investigated how conformational changes in the enzyme secondary structure, when adsorbed onto gold nanoparticles, enhanced the monitoring of arsenic ions in water solution at very low concentration.
3. Results and discussions 3.1. Ttarsc adsorption onto AuNPs
3.2. TtArsC-AuNP interaction with arsenic ions From our previous studies it was experimentally evident that interaction of TtArsC-AuNPs with arsenic ions resulted in considerable aggregation of nanocomplexes and thus a shift towards small wavelengths of the LSP. Our hypothesis was that arsenic ions directly interacted with the cysteine residues of the enzyme (with trivalent arsenic ions being more reactive than pentavalent), thus inducing protein aggregation. This mechanism has been well documented in living cells and was strictly related to heavy metal toxicity [20]. The phenomenon of arsenic-induced protein aggregation could be strengthened in the TtArsC-AuNPs nanocomplex which indeed acted as a platform for enzyme clustering. In order to better characterize the TtArsC-AuNPs nanobiosystem as a sensitive bioprobe for arsenic ions monitoring, LSP shifts were measured at different arsenite and arsenate (ions aqueous solutions) concentrations from 10212 to 8.5 1025 M. Figure 3a shows TtArsC-AuNPs LSP intensity absorbance decrease during arsenite ion interaction due to formation of
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In our previous paper [14], we proved that the one-pot synthetized PEG-stabilized AuNPs were really hybrid polymer– metal nanoaggregates forming a stable colloidal solution of about 10 nm in size, which could be effectively bioconjugated with the TtArsC enzyme. In that context, we defined TtArsC-AuNPs as a nanobiocomplex/nanobiosystem that preserved the ability to recognize arsenic ions with high affinity. We used it as an effective system for naked-eye assay speciation of As(III) and As(V). In this work, we focused our studies on a deeper characterization of TtArsC adsorption onto PEG-stabilized AuNPs, with two main aims: (i) a better comprehension of the interactions among enzyme, AuNPs and arsenic ions, and (ii) a full exploitation of this hybrid nanosystems as a working tool for monitoring arsenic contamination in drinking water. First of all, we estimated the amount of TtArsC enzyme adsorbed onto AuNPs by measuring the OD of enzyme solution before nanoparticles coupling and comparing this value with that of the supernatant after bioconjugation to AuNPs. We could estimate, in this way, an adsorption efficiency of 87.5%. A rough stoichiometric relationship of 400 enzyme molecules per gold nanoparticle was evaluated by calculating the volume of a single AuNP (approx. 5 10219 cm3) and assuming that all gold used in solution (approx. 39 mg) was converted in NPs (number of AuNPs about 4 1012, using gold density of 19.32 g cm23). We used 5 mg of TtArsC (16957.49 Da) corresponding to a number of enzymes equal to 2 1015, so that the enzymes/NPs ratio was 0.5 103 and assuming the adsorption efficiency valid for each NP, we could estimate the ratio above reported (400 : 1). On the other hand, one NP had a surface of about 300 nm2, and considering TtArsC occupying an area of about 1 nm2, the ratio 300 : 1 obtained was of the same order of that calculated by considering the masses. Z-potential characterization evidenced a radical sign change in nanoparticles surface charge (figure 1a), from 227 + 2 mV of AuNPs to 25 + 2 mV in the case of TtArsCAuNPs. Both values were quite high, indicating that the two colloidal solutions were very stable. The result related to TtArsC-AuNPs Z-potential was somehow unexpected: as TtArsC enzyme had a theoretical isoelectric point of 6.5 [15,16], its net charge at neutral pH of solution should be slightly negative, so that the Z-potential of the nanobiosystem TtArsC-AuNPs was supposed to be negative, too [18]. The high positive value measured (þ25 mV) could be explained only by a structural re-arrangement of the enzyme during the bioconjugation process to the gold surface. As enzyme self-assembling onto nanoparticles is a key point in the development of efficient biosensors, it became necessary to thoroughly investigate TtArsC enzyme interaction with AuNPs by CD (reported in figure 1b). CD spectra recorded by TtArsC and TtArsC-AuNPs solutions showed that only light rearrangement of enzyme structure occurred when comparing the enzyme in solution and the TtArsC-AuNPs,
suggesting that TtArsC in the complex retained its main structural properties and that in the absence of arsenic ions there was not any aggregation nor relevant changes in the secondary structure. The absolute values of the negative peaks at 208 nm and 220 nm, which are related to the a-helical content in the enzyme structure, were higher in the TtArsC-AuNPs nanobiocomplex, revealing enzyme structural changes [19], which could be interpreted as increase of the a-helical component due to enzyme-gold surface proximity. In particular, in order to estimate changes in the secondary structure of TtArsCAuNPs compared with TtArsC, we analysed spectra using the program K2D in Dichroweb. The analysis performed with the native enzyme reported in [16] was 28% of a-helical and 20% b content. These values refereed to the TtArsC-AuNP complex changed to 37% and 26%, respectively. The enzyme self-assembling around gold nanoparticles was also investigated at different pH values in order to understand its role in the bioconjugation process. Z-potential values and CD spectra were recorded at different pH values ranging from 5 to 9, and results are reported in figure 2. Surface charge quantification revealed a positive value for each pH tested, with a minimum of 21 + 1 mV at pH 5 and a maximum of 26 + 1 mV at pH 6, suggesting that in the whole range the colloidal solution of TtArsC-AuNPs is stable (figure 2a). CD measurements and photographic imaging characterization (figure 2b and c, respectively) evidenced a different enzyme rearrangement for each pH tested, but it was hard to say what precisely occurred as a consequence of pH changes. Major changes in the CD spectra due to variation in the enzyme secondary structure were observed at pH 8 and 9. Table 1 reports the percentage of secondary structure elements obtained by deconvolution of the spectra, using CDPRo software, and clearly shows that at these pH values there was a structural change towards disordered structures, which in turn could determine a different way in the interaction with gold nanoparticles. Similarly, the slight colour differences of each sample (see figure 2c) indicated that the pH value could only cause small modifications of enzyme structure after the self-assembling around AuNPs, and prevented further large aggregations of nanoparticles; in this latter case, a strong shift of local surface plasmon (LSP) resonance, i.e. the colour of the solution, would have been observed.
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point. The spectra were then corrected for background signal using a reference solution without the protein. Deconvolutions of CD spectra were obtained using the web-based program Dichroweb (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) or CdPro (http://lamar.colostate.edu/~sreeram/CDPro/).
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Figure 1. Z-potential evaluation highlights how surface charge of TtArsC changes when bioconjugated to AuNPs (TtArsC-AuNPs) (a); CD measurements reveals slight TtArsC enzyme rearrangement onto AuNPs surface (b). (Online version in colour.)
large aggregates enhanced by the presence of As(III) ions. The absorbance ratio A0/A (where A0 refers to TtArsC-AuNPs absorbance at 560 nm before addition of arsenic ions, and A the absorbance after addition of arsenic ions), as a function of arsenite concentration reported in figure 3b, was fitted by OriginLab SoftwareTM using the Hill equation: A0 ½Asn n , ð3:1Þ y¼ A max k þ ½Asn where (A0/A)max is the saturation point; k is the affinity constant; [As] is As ion concentration; n is the Hill coefficient [21]. The fit (R 2 ¼ 0.99) gave a Hill coefficient equal to 1, suggesting the absence of cooperativity in the interaction with As(III); therefore, the Hill equation was formally equivalent to a Langmuir equation [22]. Furthermore, the software estimated a system affinity constant k equal to 21 + 4 nM (2.7 + 0.5 ppb). The sensitivity, calculated in the range of linearity of the system response
(R 2 ¼ 0.90), was equal to 21 + 3 arb. units nM21 (4 + 0.3 arb. units ppb21), while the limit of detection (LOD), defined as three times the ratio between the signal without arsenic ions and the sensitivity, was equal to 10 + 3 pM (0.13 + 0.04 ppb). The same measurements were performed in order to characterize the interaction between TtArsC-AuNPs nanobiocomplex and As(V); data are reported in figure 4. In this case, figure 4a shows TtArsC-AuNPs LSP intensity absorbance decrease during interaction with arsenate ions, which was ascribed to the same aggregation phenomenon. The fit of the absorbance ratio is reported in figure 4b, obtained again by the Hill equation (3.1), using OriginLab SoftwareTM . In this case, the Hill coefficient was found to be equal to 3, indicating a cooperative effect in As(V) binding to TtArsC-AuNPs. The system affinity constant was equal to 26 + 1 nM (4.7 + 0.2 ppb) (R 2 ¼ 0.99), sensitivity 5.9 + 0.7 arb. units nM21 (1.1 + 0.1 arb. 21 units ppb ) (calculated in the range of linearity of system
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response, R 2 ¼ 0.96) and LOD 7.7 + 0.3 pM (0.14 + 0.01 ppb). These analytical results pointed out that the TtArsC-AuNPs nanobiosystem had well-improved enzyme properties in comparison with the free enzyme in solution, as it demonstrated an enhanced arsenic binding capability. The enzyme in solution
uses arsenate as substrate and thioredoxin reductase, thioredoxin and NADPH as the reducing system; its kinetic parameters have been calculated and reported in a recent paper [16]. Addition of arsenate up to 1 mM did not determine enzyme aggregation. In this paper, we were interested in
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evaluating sensing capabilities; as we observed a strong increase in substrate recognition by the nanocomplex in comparison with the native enzyme, we did not check activity.
Furthermore, as the World Health Organization (WHO) recommended a limit value of arsenic in drinking water of 10 ppb (Fact sheet N8372, December 2012), the proposed TtArsC-AuNPs nanobiosystem could represent a very valuable tool for monitoring even traces of arsenic, due to high affinity constants and very low LOD values found. In figure 5, digital images of TtArsC-AuNPs mixtures in Eppendorf tubes at low concentrations of arsenic ions are reported, which clearly demonstrated naked-eye assay in monitoring the presence of this heavy metal in solution. In order to better analyse the effect of arsenic ions on the nanocomplex, TEM images were registered after addition of arsenic ion solutions at concentrations of 8.5 mM to TtArsCAuNPs, a value that was in the middle of the linear range of the sensing response curve. Images, reported in figure 6, confirmed that after interaction with both ions, the
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Figure 7. Trend of surface charge (Z-potential values) changes after 24 h as function of different pH values in the absence and on exposure to arsenic ions (a); naked-eye evidence of TtArsC-AuNPs solutions colour change at different pH values in the presence of As(III) (b) and As(V) (c). (Online version in colour.) (b)
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Figure 6. TEM images of TtArsC-AuNPs nanocomplex after bioconjugation of enzyme and PEG-stabilized gold nanoparticles (a); image of a cluster of TtArsC-AuNPs on exposure to As(III) ions at 8.5 mM (b); image of a cluster of TtArsC-AuNPs on exposure to As(V) at 8.5 mM (c).
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metal nanoparticles in aqueous solution. The Shulze – Hardy rule, which states that increment of ion concentration and/or addition of ions into well-dispersed solution induces rapid and controlled coagulation of metal nanoclusters, could properly justify our findings [23].
4. Conclusion
Authors’ contributions. All authors equally contributed to experimental work and to manuscript preparation.
Competing interests. We declare we have no competing interests. Funding. No funding has been received for this article. Acknowledgements. The authors wish to thank Dr M. Pirozzi of the Bioimaging Facility at Institute of Protein Biochemistry in Napoli, Italy for TEM characterizations and Dr E. Pedone of the Institute of Biostructures and Bioimages in Napoli for helpful discussions.
References 1.
2.
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5.
Smedley PL, Kinniburgh DG. 2002 A review of the source, behaviour and distribution of arsenic in natural waters. Appl. Geochem. 17, 517 –568. (doi:10.1016/S0883-2927(02)00018-5) Moore JW, Ramamoorthy S. 2012 Heavy metals in natural waters: applied monitoring and impact assessment. Berlin, Germany: Springer Science & Business Media. Song J, Zhao FJ, McGrath SP, Luo YM. 2006 Influence of soil properties and aging on arsenic phytotoxicity. Environ. Toxicol. Chem. 25, 1663–1670. (doi:10.1897/05-480R2.1) Casentini B, Pettine M, Millero FJ. 2010 Release of arsenic from volcanic rocks through interactions with inorganic anions and organic ligands. Aquat. Geochem. 16, 373–393. (doi:10.1007/s10498-010-9090-3) Mandal BK, Suzuki KT. 2002 Arsenic round the world: a review. Talanta 58, 201–235. (doi:10. 1016/S0039-9140(02)00268-0)
Espino DP, Tamames J, de Lorenzo V, Ca´novas D. 2009 Microbial responses to environmental arsenic. Biometals 22, 117–130. (doi:10.1007/s10534-0089195-y) 7. Chapman DV (ed). 1996 Water quality assessments: a guide to the use of biota, sediments and water in environmental monitoring. London, UK: F & FN Spon. 8. Karimi A, Othman A, Andreescu S. 2016 Portable enzyme-paper biosensors based on redox-active CeO 2 nanoparticles. Methods Enzymol. 571, 177 –195. (doi:10.1016/bs.mie.2016.03.006) 9. Contursi P, Fusco S, Limauro D, Fiorentino G. 2013 Host and viral transcriptional regulators in Sulfolobus: an overview. Extremophiles 17, 881 –895. (doi:10.1007/s00792-013-0586-9) 10. De Stefano L, Vitale A, Rea I, Staiano M, Rotiroti L, Labella T, D’Auria S. 2008 Enzymes and proteins from extremophiles as hyperstable probes in 6.
nanotechnology: the use of D-trehalose/D-maltosebinding protein from the hyperthermophilic archaeon Thermococcus litoralis for sugars monitoring. Extremophiles 12, 69– 73. (doi:10. 1007/s00792-006-0058-6) 11. Fiorentino G, Del Giudice I, Bartolucci S, Durante L, Martino L, Del Vecchio P. 2011 Identification and physicochemical characterization of BldR2 from Sulfolobus solfataricus, a novel archaeal member of the MarR transcription factor family. Biochemistry 50, 6607–6621. (doi:10.1021/bi200187j) 12. Politi J, Spadavecchia J, Iodice M, De Stefano L. 2015 Oligopeptide –heavy metal interaction monitoring by hybrid gold nanoparticle based assay. Analyst 140, 149 –155. (doi:10.1039/ C4AN01491J) 13. Spadavecchia J, Apchain E, Albe´ric M, Fontan E, Reiche I. 2014 One-step synthesis of collagen hybrid gold nanoparticles and formation on Egyptian-like
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A nanobiosystem, composed of TtArsC, derived from T. thermophilus extremophile bacterium, bioconjugated with AuNPs was obtained and characterized in terms of LSP, surface charge, CD spectra and TEM imaging at pH values from 5 to 9. This work highlighted that the TtArsC-AuNPs nanobiosystem was able to efficiently interact with As(III) and As(V) ion solutions at different pH values from 5 to 9, even though CD spectra highlighted slight enzyme destructuration. The experimental findings demonstrated that the enzyme bioconjugated in the nanocomplex underwent effective structural modifications, and preserved its stability in a pH range from 5 to 8. Structural rearrangements enhanced the recognition of both As(V) and As(III) in the same range of pH values, and also at pH 9, a threshold at which the enzyme secondary structure is compromised. The results also confirmed that monitoring of arsenic could be performed by a colorimetric assay with very high sensitivity (detection limits below those suggested by the WHO). This work encourages the use of hybrid biological-AuNPs to boost specific substrate recognition by enzymes in many medical and environmental biosensing applications.
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nanosystem, though retaining sensing capability, promoted the formation of aggregates with irregular shapes. In real conditions, the influence of pH changes on sensor device efficiency during environmental monitoring must be considered, as extreme pH values in water are strictly correlated to their pollution level. To this aim, TtArsC-AuNP nanobiosystem stability/aggregation and sensing performances were further studied as function of pH, using arsenic ions solutions at concentrations of 8.5 mM and measuring surface charge change after 24 h from interaction (figure 7) and LSP shift (figure 8). The nanoaggregate surface charge (figure 7a) was between 18 and 27 mV for all the pH values tested and for both As(III) and As(V), but with a strong charge decrease at pH 7 in the case of 8.5 mM As(V) ions solutions (10 + 3 mV). However, the colour of all solutions (after 24 h from interactions) revealed a low grade of precipitation (figure 7b,c), according to surface charge experimental evidence. LSP change evidenced different spectra for each pH tested (figure 8a– e) in either the absence or the presence of arsenic ions with a major difference at pH 9. Nevertheless, the TtArsC-AuNPs nanobiosystem was able to monitor the interaction at pH 5– 6–7–8 –9 as proved by the evident LSP decrease upon As(III) or As(V) interaction. Furthermore, the absorbance ratio (figure 8f ) assumed values from 1.4 ( pH 9) to 2.6 arb. units ( pH7). The monitoring at pH values 5–6 –7 seemed to be more efficient because the absorbance ratio values oscillate from a minimum of 1.6 to a maximum of 2.6 arb. units, while at pH values 8–9 the absorbance ratio ranged from 1.5 to 1.4 arb. units. These data suggested that the TtArsC-AuNPs nanocomplex could be more stable under these pH values, accordingly to CD analysis (figure 2b), which showed spectra representative of enzyme with higher a-helical content at pH 5– 6–7 than at pH values 8–9, where changes in secondary structure were very evident. It is worth noting that the absorbance ratio values of 8.5 mM As(III) and As(V) in aqueous solutions were 1.6 and 1.2 arb. units, respectively. In this experimental frame, it was clear that pH changes tuned the competition between dispersion and ordering of our hybrid biological–
21. Politi J, Rea I, Nici F, Dardano P, Terracciano M, Oliviero G, Borbone N, Piccialli G, De Stefano L. 2016 Nanogravimetric and optical characterizations of thrombin interaction with a self-assembled thiolated aptamer. J. Sens. 2016, 3561863. (doi:10.1155/2016/ 3561863) 22. Politi J, De Stefano L, Rea I, Gravagnuolo AM, Giardina P, Methivier C, Spadavecchia J. 2016 One-pot synthesis of a gold nanoparticle –Vmh2 hydrophobin nanobiocomplex for glucose monitoring. Nanotechnology 27, 195701. (doi:10. 1088/0957-4484/27/19/195701) 23. Shiraishi Y, Arakawa D, Toshima N. 2002 pHdependent color change of colloidal dispersions of gold nanoclusters: effect of stabilizer. Eur. Phys. J. E 8, 377 –383. (doi:10.1140/epje/i200010103-4)
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17. Fiorentino G, Del Giudice I, Petraccone L, Bartolucci S, Del Vecchio P. 2014 Conformational stability and ligand binding properties of BldR, a member of the MarR family, from Sulfolobus solfataricus. Biochim. Biophys. Acta 1844, 1167–1172. (doi:10.1016/j. bbapap.2014.03.011) 18. Verwey EJW, Overbeek JTG. 1999 Theory of the stability of lyophobic colloids. Eindhoven, The Netherlands: Courier Corporation. 19. Johnson WC. 1990 Protein secondary structure and circular dichroism: a practical guide. Proteins Struct. Funct. Bioinformatics 7, 205–214. (doi:10.1002/ prot.340070302) 20. Tama´s MJ, Sharma SK, Ibstedt S, Jacobson T, Christen P. 2014 Heavy metals and metalloids as a cause for protein misfolding and aggregation. Biomolecules 4, 252–267. (doi:10.3390/ biom4010252)
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gold-plated archaeological ivory. Angew. Chem. Int. Ed. 53, 1756 –1789. (doi:10.1002/anie.201403567) 14. Politi J, Spadavecchia J, Fiorentino G, Antonucci I, Casale S, De Stefano L. 2015 Interaction of Thermus thermophiles ArsC enzyme and gold nanoparticles naked-eye assays speciation between As (III) and As (V). Nanotechnology 26, 435703. (doi:10.1088/ 0957-4484/26/43/435703) 15. Ul N. 1971 Isoelectric points and conformation of proteins: I. Effect of urea on the behavior of some proteins in isoelectric focusing. Biochim. Biophys. Acta Protein Struct. 229, 567 –581. (doi:10.1016/ 0005-2795(71)90272-8) 16. Del Giudice I, Limauro D, Pedone E, Bartolucci S, Fiorentino G. 2013 A novel arsenate reductase from the bacterium Thermus thermophilus HB27: its role in arsenic detoxification. Biochim. Biophys. Acta 1834, 2071–2079. (doi:10.1016/j.bbapap.2013.06.007)
Research Cite this article: Politi J, Spadavecchia J, Fiorentino G, Antonucci I, De Stefano L. 2016 Arsenate reductase from Thermus thermophilus conjugated to polyethylene glycol-stabilized gold nanospheres allow trace sensing and speciation of arsenic ions. J. R. Soc. Interface 13: 20160629. http://dx.doi.org/10.1098/rsif.2016.0629
Arsenate reductase from Thermus thermophilus conjugated to polyethylene glycol-stabilized gold nanospheres allow trace sensing and speciation of arsenic ions Jane Politi1, Jolanda Spadavecchia2,3, Gabriella Fiorentino4, Immacolata Antonucci4 and Luca De Stefano1 1 Institute for Microelectronics and Microsystems, Unit of Naples-National Research Council, via P. Castellino 111, 80127 Naples, Italy 2 Sorbonne Universite´s, UPMC Univ Paris VI, Laboratoire de Re´activite´ de Surface, 4 place Jussieu, 75005 Paris, France 3 CNRS, UMR 7244, CSPBAT, Laboratoire de Chimie, Structures et Proprie´te´s de Biomateriaux et d’Agents Therapeutiques Universite´ Paris 13, Sorbonne Paris Cite´, Bobigny, France CNRS, Paris, France 4 Department of Biology, University of Naples ‘Federico II’, Via Cynthia, 80126 Naples, Italy
LD, 0000-0002-9442-4175
Received: 8 August 2016 Accepted: 7 September 2016
Subject Category: Life Sciences – Physics interface Subject Areas: nanotechnology, environmental science, biochemistry
Water sources pollution by arsenic ions is a serious environmental problem all around the world. Arsenate reductase enzyme (TtArsC) from Thermus thermophilus extremophile bacterium, naturally binds arsenic ions, As(V) and As (III), in aqueous solutions. In this research, TtArsC enzyme adsorption onto hybrid polyethylene glycol-stabilized gold nanoparticles (AuNPs) was studied at different pH values as an innovative nanobiosystem for metal concentration monitoring. Characterizations were performed by UV/Vis and circular dichroism spectroscopies, TEM images and in terms of surface charge changes. The molecular interaction between arsenic ions and the TtArsC-AuNPs nanobiosystem was also monitored at all pH values considered by UV/Vis spectroscopy. Tests performed revealed high sensitivities and limits of detection equal to 10 + 3 M212 and 7.7 + 0.3 M212 for As(III) and As(V), respectively.
Keywords: enzyme, gold nanoparticles, arsenic pollution
1. Introduction Author for correspondence: Luca De Stefano e-mail: [email protected]
Arsenic is a naturally occurring metal; it is among the 20 most common substances in the earth’s crust and is a component of more than 200 minerals [1,2]. Arsenic and its compounds are extremely mobile in the environment. Ageing of rocks by weather conditions converts arsenic sulfides to arsenic trioxide, which enters the arsenic cycle as dust or by dissolution in rivers and groundwater [3,4]. Water sources pollution by arsenic is a serious environmental problem all around the world, as it causes a lot of adverse health effects to humans at every level. Detection and quantification of arsenic ions concentration is thus a hot topic because water management requires optimization of quality assessment [5–7]. Biosensors and, in particular, nanoparticle-enhanced enzyme biosensors have recently emerged as powerful devices for qualitative and quantitative analysis of a variety of analytes for biomedical applications, drugs testing and environmental monitoring. Protein and enzymes can selectively recognize their natural ligands with very high specificity even in complex matrices such as the environmental ones [8]. In this field, there is also a general interest in biomolecules purified from thermophilic organisms due their biotechnological advantages [9]. Enzymes and proteins isolated from thermophilic microorganisms exhibit high stability in conditions that are usually adverse to proteins, such as high temperatures (up to 1008C), heavy concentration of detergents, extreme pH values, ionic strength and chaotropic agents [10–12]. In
& 2016 The Author(s) Published by the Royal Society. All rights reserved.
centrifuged twice at 6000 r.p.m. for 20 min in order to remove excess protein and then the pellets were re-dispersed in 1 ml of MilliQ water. The resultant colloidal solution was sonicated for 30 min at 48C. In order to monitor the adsorption of TtArsC onto AuNPs at different pH values, the pellets were re-dispersed in sodium acetate 20 mM at pH 5, trisodium phosphate 20 mM at pH 6, 7 and 8, glycine 20 mM at pH 9. The adsorption was characterized by UV/Vis spectrophotometry, transmission electron microscopy, circular dichroism (CD) and Z-potential measurements as described below.
Interaction between TtArsC-AuNPs and As (V)/As (III) solutions was performed adding 0.05 ml of heavy metal ions solution at 10212 M, 10210 M, 1028 M, 1026 M, 2.1 1026 M, 8.5 1026 M, 35 1026 M, 85 1026 M to 1 ml of TtArsC-AuNPs solution. After 10 min, the resulting mixtures were analysed by UV –Vis spectrophotometry.
2. Material and methods 2.1. Chemicals Tetrachloroauric acid (HAuCl4), sodium borohydride (NaBH4) and ethanol (C2H5OH), polyethylene glycol 600 Diacid (PEG), Tris – HCl (C4H11NO3), sodium acetate (CH3COONa), trisodium phosphate (Na3PO4), glycine (C2H5NO2), potassium metarsenite (NaAsO2), potassium arsenate (KH2AsO4) were purchased from Sigma Aldrich. All chemicals were used as supplied without further purification.
2.2. Purification and preparation of TtArsC enzyme Recombinant TtArsC (TtArsC: protein arsenate reductase from the Gram-negative bacterium T. thermophilus HB27) was purified up to homogeneity using the purification procedure described in a previous paper [16], basically consisting of thermo-precipitation of the Escherichia coli cell extract followed by anion exchange and gel filtration chromatography. Fractions containing purified TtArsC were pooled, dialysed 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 at a final 1.6 mM concentration.
2.3. Synthesis of PEG-stabilized-Au nanospheres In previous work [13], we have synthesized gold nanoparticles adding dicarboxylic PEG as surfactant in the mixture reaction. Briefly, 20 ml of chloroauric acid (HAuCl4) aqueous solution (2.5 1024 M) was added to 0.25 ml of dicarboxylic PEG and mixed by magnetic stirring for 10 min at room temperature. To this solution, 600 ml of aqueous 0.01 M NaBH4 was added drop by drop. The formation of the PEG-stabilized AuNPs (in the following simply cited as AuNPs) was observed as an instantaneous colour change of the solution from pale yellow to bright red after addition of the reducing agent. The as-prepared AuNPs solution was centrifuged at 15 000 r.p.m. for 30 min for three times and then the supernatant was discarded and the residue was suspended again in an equivalent amount of buffer solution. This last step was repeated twice, in order to remove excess dicarboxylic PEG.
2.4. Adsorption of TtArsC onto AuNPs TtArsC-AuNPs adsorption was achieved using the following procedure: 1 ml of AuNPs was added into separate tubes containing 0.05 ml of TtArsC (1 mg ml21 in 15 mM Tris – HCl, pH 7) for 24 h. The TtArsC-AuNPs suspension was then
2.6. UV –Vis measurements Absorption spectra were recorded using a Cary 100 (Agilent) spectrophotometer in the 200 –800 nm range. The spectra were recorded after 30 min from synthesis of nanoparticles, then from 2 min to 24 h after enzyme adsorption and finally after 10 min of interaction with heavy metals solutions. In order to evaluate TtArsC binding efficiency onto AuNPs, the TtArsCAuNPs were centrifuged for 30 min at 48C and the supernatant optical density (OD) was evaluated at 280 nm, 24 h after colloidal solution formation. Solutions of TtArsC-AuNPs, As (III) at 8.5 mM þ TtArsC-AuNPs, and As (V) at 8.5 mM þ TtArsCAuNPs at pH values from 5 to 9 were also characterized after 10 min of interaction.
2.7. Transmission electron microscopy Transmission electron microscopy (TEM) measurements were recorded on a FEI TEcnai G2 Spirit BioTWIN microscope operating at an accelerating voltage of 120 kV. The TEM graphs were taken after separating the surfactant from the metal particles by centrifugation; 1 ml of the sample was centrifuged for 20 min at a speed of 14 000 r.p.m. min21. The upper part of the colourless solution was removed and the solid part was re-dispersed in 1 ml of water. A droplet (10 ml) of the colloidal solution (TtArsC-AuNPs, As (III) at 8.5 mM þ TtArsC-AuNPs, As (V) at 8.5 mM þ TtArsC-AuNPs) was deposited on a microscope grid, dried at room temperature and analysed.
2.8. Z-potential measurements The Z-potential measurements of TtArsC-AuNPs, As (III) at 8.5 mM þ TtArsC-AuNPs, As (V) at 8.5 mM þ TtArsC-AuNPs solutions at pH values from 5 to 9 were performed by a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with a He – Ne laser (633 nm, fixed scattering angle of 1738, 258C).
2.9. Circular dichroism measurements The TtArsC-AuNPs, As(III) at 8.5 mM þ TtArsC-AuNPs, As(V) at 8.5 mM þ TtArsC-AuNPs solutions at pH values from 5 to 9 were characterized as follows. Far UV CD spectra were recorded on a Jasco J815 spectropolarimeter equipped with a Peltier thermostatic cell holder, in a quartz cell (0.1 cm light path) from 190 to 250 nm [17]. The temperature was kept at 208C and the sample compartment was continuously flushed with nitrogen gas. Furthermore, all the spectra recorded had corresponding high voltage applied to the photomultiplier below 700 V. Final spectra were obtained by averaging three scans, using a bandwidth of 1 nm, a step width of 0.5 nm and 4 s averaging per
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2.5. Heavy metals interaction monitoring
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our recent paper, we adsorbed a novel chromosomal arsenate reductase (TtArsC) able to reduce the substrate As(V) to As(III), on homemade hybrid gold nanoparticles (NPs) [13], producing a biomolecular probe that can screen for the presence of arsenic ions in water [14]. Thermus thermophilus HB27 is an extremophile bacterium that evolved in arsenic-rich geothermal environments: survival mechanisms of this organism include oxidization and reduction of arsenic ions and extrusion from living cells [15]. The unique features of TtArsC enzyme allowed the speciation of pentavalent arsenic, As(V), and trivalent arsenic, As(III), by a naked-eye assay [14]. In this work, we investigated how conformational changes in the enzyme secondary structure, when adsorbed onto gold nanoparticles, enhanced the monitoring of arsenic ions in water solution at very low concentration.
3. Results and discussions 3.1. Ttarsc adsorption onto AuNPs
3.2. TtArsC-AuNP interaction with arsenic ions From our previous studies it was experimentally evident that interaction of TtArsC-AuNPs with arsenic ions resulted in considerable aggregation of nanocomplexes and thus a shift towards small wavelengths of the LSP. Our hypothesis was that arsenic ions directly interacted with the cysteine residues of the enzyme (with trivalent arsenic ions being more reactive than pentavalent), thus inducing protein aggregation. This mechanism has been well documented in living cells and was strictly related to heavy metal toxicity [20]. The phenomenon of arsenic-induced protein aggregation could be strengthened in the TtArsC-AuNPs nanocomplex which indeed acted as a platform for enzyme clustering. In order to better characterize the TtArsC-AuNPs nanobiosystem as a sensitive bioprobe for arsenic ions monitoring, LSP shifts were measured at different arsenite and arsenate (ions aqueous solutions) concentrations from 10212 to 8.5 1025 M. Figure 3a shows TtArsC-AuNPs LSP intensity absorbance decrease during arsenite ion interaction due to formation of
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In our previous paper [14], we proved that the one-pot synthetized PEG-stabilized AuNPs were really hybrid polymer– metal nanoaggregates forming a stable colloidal solution of about 10 nm in size, which could be effectively bioconjugated with the TtArsC enzyme. In that context, we defined TtArsC-AuNPs as a nanobiocomplex/nanobiosystem that preserved the ability to recognize arsenic ions with high affinity. We used it as an effective system for naked-eye assay speciation of As(III) and As(V). In this work, we focused our studies on a deeper characterization of TtArsC adsorption onto PEG-stabilized AuNPs, with two main aims: (i) a better comprehension of the interactions among enzyme, AuNPs and arsenic ions, and (ii) a full exploitation of this hybrid nanosystems as a working tool for monitoring arsenic contamination in drinking water. First of all, we estimated the amount of TtArsC enzyme adsorbed onto AuNPs by measuring the OD of enzyme solution before nanoparticles coupling and comparing this value with that of the supernatant after bioconjugation to AuNPs. We could estimate, in this way, an adsorption efficiency of 87.5%. A rough stoichiometric relationship of 400 enzyme molecules per gold nanoparticle was evaluated by calculating the volume of a single AuNP (approx. 5 10219 cm3) and assuming that all gold used in solution (approx. 39 mg) was converted in NPs (number of AuNPs about 4 1012, using gold density of 19.32 g cm23). We used 5 mg of TtArsC (16957.49 Da) corresponding to a number of enzymes equal to 2 1015, so that the enzymes/NPs ratio was 0.5 103 and assuming the adsorption efficiency valid for each NP, we could estimate the ratio above reported (400 : 1). On the other hand, one NP had a surface of about 300 nm2, and considering TtArsC occupying an area of about 1 nm2, the ratio 300 : 1 obtained was of the same order of that calculated by considering the masses. Z-potential characterization evidenced a radical sign change in nanoparticles surface charge (figure 1a), from 227 + 2 mV of AuNPs to 25 + 2 mV in the case of TtArsCAuNPs. Both values were quite high, indicating that the two colloidal solutions were very stable. The result related to TtArsC-AuNPs Z-potential was somehow unexpected: as TtArsC enzyme had a theoretical isoelectric point of 6.5 [15,16], its net charge at neutral pH of solution should be slightly negative, so that the Z-potential of the nanobiosystem TtArsC-AuNPs was supposed to be negative, too [18]. The high positive value measured (þ25 mV) could be explained only by a structural re-arrangement of the enzyme during the bioconjugation process to the gold surface. As enzyme self-assembling onto nanoparticles is a key point in the development of efficient biosensors, it became necessary to thoroughly investigate TtArsC enzyme interaction with AuNPs by CD (reported in figure 1b). CD spectra recorded by TtArsC and TtArsC-AuNPs solutions showed that only light rearrangement of enzyme structure occurred when comparing the enzyme in solution and the TtArsC-AuNPs,
suggesting that TtArsC in the complex retained its main structural properties and that in the absence of arsenic ions there was not any aggregation nor relevant changes in the secondary structure. The absolute values of the negative peaks at 208 nm and 220 nm, which are related to the a-helical content in the enzyme structure, were higher in the TtArsC-AuNPs nanobiocomplex, revealing enzyme structural changes [19], which could be interpreted as increase of the a-helical component due to enzyme-gold surface proximity. In particular, in order to estimate changes in the secondary structure of TtArsCAuNPs compared with TtArsC, we analysed spectra using the program K2D in Dichroweb. The analysis performed with the native enzyme reported in [16] was 28% of a-helical and 20% b content. These values refereed to the TtArsC-AuNP complex changed to 37% and 26%, respectively. The enzyme self-assembling around gold nanoparticles was also investigated at different pH values in order to understand its role in the bioconjugation process. Z-potential values and CD spectra were recorded at different pH values ranging from 5 to 9, and results are reported in figure 2. Surface charge quantification revealed a positive value for each pH tested, with a minimum of 21 + 1 mV at pH 5 and a maximum of 26 + 1 mV at pH 6, suggesting that in the whole range the colloidal solution of TtArsC-AuNPs is stable (figure 2a). CD measurements and photographic imaging characterization (figure 2b and c, respectively) evidenced a different enzyme rearrangement for each pH tested, but it was hard to say what precisely occurred as a consequence of pH changes. Major changes in the CD spectra due to variation in the enzyme secondary structure were observed at pH 8 and 9. Table 1 reports the percentage of secondary structure elements obtained by deconvolution of the spectra, using CDPRo software, and clearly shows that at these pH values there was a structural change towards disordered structures, which in turn could determine a different way in the interaction with gold nanoparticles. Similarly, the slight colour differences of each sample (see figure 2c) indicated that the pH value could only cause small modifications of enzyme structure after the self-assembling around AuNPs, and prevented further large aggregations of nanoparticles; in this latter case, a strong shift of local surface plasmon (LSP) resonance, i.e. the colour of the solution, would have been observed.
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point. The spectra were then corrected for background signal using a reference solution without the protein. Deconvolutions of CD spectra were obtained using the web-based program Dichroweb (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) or CdPro (http://lamar.colostate.edu/~sreeram/CDPro/).
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large aggregates enhanced by the presence of As(III) ions. The absorbance ratio A0/A (where A0 refers to TtArsC-AuNPs absorbance at 560 nm before addition of arsenic ions, and A the absorbance after addition of arsenic ions), as a function of arsenite concentration reported in figure 3b, was fitted by OriginLab SoftwareTM using the Hill equation: A0 ½Asn n , ð3:1Þ y¼ A max k þ ½Asn where (A0/A)max is the saturation point; k is the affinity constant; [As] is As ion concentration; n is the Hill coefficient [21]. The fit (R 2 ¼ 0.99) gave a Hill coefficient equal to 1, suggesting the absence of cooperativity in the interaction with As(III); therefore, the Hill equation was formally equivalent to a Langmuir equation [22]. Furthermore, the software estimated a system affinity constant k equal to 21 + 4 nM (2.7 + 0.5 ppb). The sensitivity, calculated in the range of linearity of the system response
(R 2 ¼ 0.90), was equal to 21 + 3 arb. units nM21 (4 + 0.3 arb. units ppb21), while the limit of detection (LOD), defined as three times the ratio between the signal without arsenic ions and the sensitivity, was equal to 10 + 3 pM (0.13 + 0.04 ppb). The same measurements were performed in order to characterize the interaction between TtArsC-AuNPs nanobiocomplex and As(V); data are reported in figure 4. In this case, figure 4a shows TtArsC-AuNPs LSP intensity absorbance decrease during interaction with arsenate ions, which was ascribed to the same aggregation phenomenon. The fit of the absorbance ratio is reported in figure 4b, obtained again by the Hill equation (3.1), using OriginLab SoftwareTM . In this case, the Hill coefficient was found to be equal to 3, indicating a cooperative effect in As(V) binding to TtArsC-AuNPs. The system affinity constant was equal to 26 + 1 nM (4.7 + 0.2 ppb) (R 2 ¼ 0.99), sensitivity 5.9 + 0.7 arb. units nM21 (1.1 + 0.1 arb. 21 units ppb ) (calculated in the range of linearity of system
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response, R 2 ¼ 0.96) and LOD 7.7 + 0.3 pM (0.14 + 0.01 ppb). These analytical results pointed out that the TtArsC-AuNPs nanobiosystem had well-improved enzyme properties in comparison with the free enzyme in solution, as it demonstrated an enhanced arsenic binding capability. The enzyme in solution
uses arsenate as substrate and thioredoxin reductase, thioredoxin and NADPH as the reducing system; its kinetic parameters have been calculated and reported in a recent paper [16]. Addition of arsenate up to 1 mM did not determine enzyme aggregation. In this paper, we were interested in
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A0/A
2.0
1.8
1.6
1.4 0
20
40
60
80
AsIII (mM)
Figure 3. LSP shift as function of As(III) ions increasing concentration in solution (a); LSP change trend as function of As(III) ions increasing concentration in solution, normalized to LSP values in the absence of As(III) (b). In the inset, signals generated by TtArsC-AuNPs on exposure to small concentrations of As(III) ions. (Online version in colour.) Table 1. Deconvolution of CD spectra data estimated by CdPro software. alpha helix (%)
strand (%)
random (%)
TtArsC-AuNps pH 5 TtArsC-AuNps pH 6
55 47
8 6
37 47
TtArsC-AuNps pH 7 TtArsC-AuNps pH 8
63 36
11 10
26 54
TtArsC-AuNps pH 9
36
6
58
evaluating sensing capabilities; as we observed a strong increase in substrate recognition by the nanocomplex in comparison with the native enzyme, we did not check activity.
Furthermore, as the World Health Organization (WHO) recommended a limit value of arsenic in drinking water of 10 ppb (Fact sheet N8372, December 2012), the proposed TtArsC-AuNPs nanobiosystem could represent a very valuable tool for monitoring even traces of arsenic, due to high affinity constants and very low LOD values found. In figure 5, digital images of TtArsC-AuNPs mixtures in Eppendorf tubes at low concentrations of arsenic ions are reported, which clearly demonstrated naked-eye assay in monitoring the presence of this heavy metal in solution. In order to better analyse the effect of arsenic ions on the nanocomplex, TEM images were registered after addition of arsenic ion solutions at concentrations of 8.5 mM to TtArsCAuNPs, a value that was in the middle of the linear range of the sensing response curve. Images, reported in figure 6, confirmed that after interaction with both ions, the
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3.5 × 10–5 M 8.5 × 10–5 M
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0M 10–12 M
(a)
7 0.4
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0
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600 l (nm)
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experimental data hill fit
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A0/A
3.0
2.5
2.0
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1.0 0
10
20
30
40 50 As V (mM)
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70
80
Figure 4. LSP shift as function of As(V) ions increasing concentration in solution (a); LSP change trend as function of As(V) ions increasing concentration in solution, normalized to LSP value in the absence of As(V) (b). In the inset, signals generated by TtArsC-AuNPs on exposure to small concentrations of As(V). (Online version in colour.) 10–12
10–10
10–8
10–6
2 × 10–6
8.5 × 10–6
10–12
10–10
10–8
10–6
2 × 10–6
8.5 × 10–6
As(III)
As(V)
Figure 5. Digital images of TtArsC-AuNPs mixtures in eppendorfs at low concentration of arsenic ions which clearly demonstrate naked-eye assay in monitoring the presence of this heavy metal in solution. (Online version in colour.)
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0.1
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0M 10–12 M 10–10 M 10–8 M 10–6 M 2.1 × 10–6 M 8.5 × 10–6 M 3.5 × 10–5 M 8.5 × 10–5 M
(a)
(b)
8
(c)
100 nm
200 nm
(a)
surface charge (mV)
(b)
TtArsC-AuNPs Z-pot before sensing TtArsC-AuNPs Z-pot after AsIII 8.5 mM sensing TtArsC-AuNPs Z-pot after AsV 8.5 mM sensing
30 25 20 15 10
(c) 5 5
6
7 pH
8
9
Figure 7. Trend of surface charge (Z-potential values) changes after 24 h as function of different pH values in the absence and on exposure to arsenic ions (a); naked-eye evidence of TtArsC-AuNPs solutions colour change at different pH values in the presence of As(III) (b) and As(V) (c). (Online version in colour.) (b)
(a) pH 5 ON pH 5 AsIII 8.5 mM pH 5 AsV 8.5 mM
1.30
1.04
0.78
0.78
0.78
0.26 0 400
0.52 0.26
500
600 700 wavelength (nm)
0 400
800
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500
600 700 wavelength (nm)
1.12
0.81 0.54
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0.27
500
600 700 wavelength (nm)
800
pH 9 ON pH 9 AsIII 8.5 mM pH 9 AsV 8.5 mM
1.08
0.56
0 400
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600 700 wavelength (nm)
800
(f) 1.35
abs
0.84
0 400
800
(e) pH 8 ON pH 8 AsIII 8.5 mM pH 8 AsV 8.5 mM
1.40
0.52
0 400
2.6
A0/AAs(III)
2.4 A0/A
(d)
abs
abs
1.04
0.52
pH 7 ON pH 7 AsIII 8.5 mM pH 7 AsV 8.5 mM
1.30
1.04
abs
abs
1.30
(c) pH 6 ON pH 6 AsIII 8.5 mM pH 6 AsV 8.5 mM
A0/AAs(V)
2.2 2.0 1.8 1.6 1.4
500
600 700 800 wavelength (nm)
900
5
6
7 pH
8
9
Figure 8. TtArsC-AuNPs LSP signal changes at different pH values (ON stands for overnight incubation) in the absence and on exposure to arsenic ions As(III) and As(V) at 8.5 mM (from 5 (a) to 9 (e)); LSP values as function of pH values in the absence and on exposure to arsenic ions normalized on LSP values in the absence of arsenic ions (f ). (Online version in colour.)
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Figure 6. TEM images of TtArsC-AuNPs nanocomplex after bioconjugation of enzyme and PEG-stabilized gold nanoparticles (a); image of a cluster of TtArsC-AuNPs on exposure to As(III) ions at 8.5 mM (b); image of a cluster of TtArsC-AuNPs on exposure to As(V) at 8.5 mM (c).
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50 nm
metal nanoparticles in aqueous solution. The Shulze – Hardy rule, which states that increment of ion concentration and/or addition of ions into well-dispersed solution induces rapid and controlled coagulation of metal nanoclusters, could properly justify our findings [23].
4. Conclusion
Authors’ contributions. All authors equally contributed to experimental work and to manuscript preparation.
Competing interests. We declare we have no competing interests. Funding. No funding has been received for this article. Acknowledgements. The authors wish to thank Dr M. Pirozzi of the Bioimaging Facility at Institute of Protein Biochemistry in Napoli, Italy for TEM characterizations and Dr E. Pedone of the Institute of Biostructures and Bioimages in Napoli for helpful discussions.
References 1.
2.
3.
4.
5.
Smedley PL, Kinniburgh DG. 2002 A review of the source, behaviour and distribution of arsenic in natural waters. Appl. Geochem. 17, 517 –568. (doi:10.1016/S0883-2927(02)00018-5) Moore JW, Ramamoorthy S. 2012 Heavy metals in natural waters: applied monitoring and impact assessment. Berlin, Germany: Springer Science & Business Media. Song J, Zhao FJ, McGrath SP, Luo YM. 2006 Influence of soil properties and aging on arsenic phytotoxicity. Environ. Toxicol. Chem. 25, 1663–1670. (doi:10.1897/05-480R2.1) Casentini B, Pettine M, Millero FJ. 2010 Release of arsenic from volcanic rocks through interactions with inorganic anions and organic ligands. Aquat. Geochem. 16, 373–393. (doi:10.1007/s10498-010-9090-3) Mandal BK, Suzuki KT. 2002 Arsenic round the world: a review. Talanta 58, 201–235. (doi:10. 1016/S0039-9140(02)00268-0)
Espino DP, Tamames J, de Lorenzo V, Ca´novas D. 2009 Microbial responses to environmental arsenic. Biometals 22, 117–130. (doi:10.1007/s10534-0089195-y) 7. Chapman DV (ed). 1996 Water quality assessments: a guide to the use of biota, sediments and water in environmental monitoring. London, UK: F & FN Spon. 8. Karimi A, Othman A, Andreescu S. 2016 Portable enzyme-paper biosensors based on redox-active CeO 2 nanoparticles. Methods Enzymol. 571, 177 –195. (doi:10.1016/bs.mie.2016.03.006) 9. Contursi P, Fusco S, Limauro D, Fiorentino G. 2013 Host and viral transcriptional regulators in Sulfolobus: an overview. Extremophiles 17, 881 –895. (doi:10.1007/s00792-013-0586-9) 10. De Stefano L, Vitale A, Rea I, Staiano M, Rotiroti L, Labella T, D’Auria S. 2008 Enzymes and proteins from extremophiles as hyperstable probes in 6.
nanotechnology: the use of D-trehalose/D-maltosebinding protein from the hyperthermophilic archaeon Thermococcus litoralis for sugars monitoring. Extremophiles 12, 69– 73. (doi:10. 1007/s00792-006-0058-6) 11. Fiorentino G, Del Giudice I, Bartolucci S, Durante L, Martino L, Del Vecchio P. 2011 Identification and physicochemical characterization of BldR2 from Sulfolobus solfataricus, a novel archaeal member of the MarR transcription factor family. Biochemistry 50, 6607–6621. (doi:10.1021/bi200187j) 12. Politi J, Spadavecchia J, Iodice M, De Stefano L. 2015 Oligopeptide –heavy metal interaction monitoring by hybrid gold nanoparticle based assay. Analyst 140, 149 –155. (doi:10.1039/ C4AN01491J) 13. Spadavecchia J, Apchain E, Albe´ric M, Fontan E, Reiche I. 2014 One-step synthesis of collagen hybrid gold nanoparticles and formation on Egyptian-like
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A nanobiosystem, composed of TtArsC, derived from T. thermophilus extremophile bacterium, bioconjugated with AuNPs was obtained and characterized in terms of LSP, surface charge, CD spectra and TEM imaging at pH values from 5 to 9. This work highlighted that the TtArsC-AuNPs nanobiosystem was able to efficiently interact with As(III) and As(V) ion solutions at different pH values from 5 to 9, even though CD spectra highlighted slight enzyme destructuration. The experimental findings demonstrated that the enzyme bioconjugated in the nanocomplex underwent effective structural modifications, and preserved its stability in a pH range from 5 to 8. Structural rearrangements enhanced the recognition of both As(V) and As(III) in the same range of pH values, and also at pH 9, a threshold at which the enzyme secondary structure is compromised. The results also confirmed that monitoring of arsenic could be performed by a colorimetric assay with very high sensitivity (detection limits below those suggested by the WHO). This work encourages the use of hybrid biological-AuNPs to boost specific substrate recognition by enzymes in many medical and environmental biosensing applications.
9
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nanosystem, though retaining sensing capability, promoted the formation of aggregates with irregular shapes. In real conditions, the influence of pH changes on sensor device efficiency during environmental monitoring must be considered, as extreme pH values in water are strictly correlated to their pollution level. To this aim, TtArsC-AuNP nanobiosystem stability/aggregation and sensing performances were further studied as function of pH, using arsenic ions solutions at concentrations of 8.5 mM and measuring surface charge change after 24 h from interaction (figure 7) and LSP shift (figure 8). The nanoaggregate surface charge (figure 7a) was between 18 and 27 mV for all the pH values tested and for both As(III) and As(V), but with a strong charge decrease at pH 7 in the case of 8.5 mM As(V) ions solutions (10 + 3 mV). However, the colour of all solutions (after 24 h from interactions) revealed a low grade of precipitation (figure 7b,c), according to surface charge experimental evidence. LSP change evidenced different spectra for each pH tested (figure 8a– e) in either the absence or the presence of arsenic ions with a major difference at pH 9. Nevertheless, the TtArsC-AuNPs nanobiosystem was able to monitor the interaction at pH 5– 6–7–8 –9 as proved by the evident LSP decrease upon As(III) or As(V) interaction. Furthermore, the absorbance ratio (figure 8f ) assumed values from 1.4 ( pH 9) to 2.6 arb. units ( pH7). The monitoring at pH values 5–6 –7 seemed to be more efficient because the absorbance ratio values oscillate from a minimum of 1.6 to a maximum of 2.6 arb. units, while at pH values 8–9 the absorbance ratio ranged from 1.5 to 1.4 arb. units. These data suggested that the TtArsC-AuNPs nanocomplex could be more stable under these pH values, accordingly to CD analysis (figure 2b), which showed spectra representative of enzyme with higher a-helical content at pH 5– 6–7 than at pH values 8–9, where changes in secondary structure were very evident. It is worth noting that the absorbance ratio values of 8.5 mM As(III) and As(V) in aqueous solutions were 1.6 and 1.2 arb. units, respectively. In this experimental frame, it was clear that pH changes tuned the competition between dispersion and ordering of our hybrid biological–
21. Politi J, Rea I, Nici F, Dardano P, Terracciano M, Oliviero G, Borbone N, Piccialli G, De Stefano L. 2016 Nanogravimetric and optical characterizations of thrombin interaction with a self-assembled thiolated aptamer. J. Sens. 2016, 3561863. (doi:10.1155/2016/ 3561863) 22. Politi J, De Stefano L, Rea I, Gravagnuolo AM, Giardina P, Methivier C, Spadavecchia J. 2016 One-pot synthesis of a gold nanoparticle –Vmh2 hydrophobin nanobiocomplex for glucose monitoring. Nanotechnology 27, 195701. (doi:10. 1088/0957-4484/27/19/195701) 23. Shiraishi Y, Arakawa D, Toshima N. 2002 pHdependent color change of colloidal dispersions of gold nanoclusters: effect of stabilizer. Eur. Phys. J. E 8, 377 –383. (doi:10.1140/epje/i200010103-4)
10
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17. Fiorentino G, Del Giudice I, Petraccone L, Bartolucci S, Del Vecchio P. 2014 Conformational stability and ligand binding properties of BldR, a member of the MarR family, from Sulfolobus solfataricus. Biochim. Biophys. Acta 1844, 1167–1172. (doi:10.1016/j. bbapap.2014.03.011) 18. Verwey EJW, Overbeek JTG. 1999 Theory of the stability of lyophobic colloids. Eindhoven, The Netherlands: Courier Corporation. 19. Johnson WC. 1990 Protein secondary structure and circular dichroism: a practical guide. Proteins Struct. Funct. Bioinformatics 7, 205–214. (doi:10.1002/ prot.340070302) 20. Tama´s MJ, Sharma SK, Ibstedt S, Jacobson T, Christen P. 2014 Heavy metals and metalloids as a cause for protein misfolding and aggregation. Biomolecules 4, 252–267. (doi:10.3390/ biom4010252)
rsif.royalsocietypublishing.org
gold-plated archaeological ivory. Angew. Chem. Int. Ed. 53, 1756 –1789. (doi:10.1002/anie.201403567) 14. Politi J, Spadavecchia J, Fiorentino G, Antonucci I, Casale S, De Stefano L. 2015 Interaction of Thermus thermophiles ArsC enzyme and gold nanoparticles naked-eye assays speciation between As (III) and As (V). Nanotechnology 26, 435703. (doi:10.1088/ 0957-4484/26/43/435703) 15. Ul N. 1971 Isoelectric points and conformation of proteins: I. Effect of urea on the behavior of some proteins in isoelectric focusing. Biochim. Biophys. Acta Protein Struct. 229, 567 –581. (doi:10.1016/ 0005-2795(71)90272-8) 16. Del Giudice I, Limauro D, Pedone E, Bartolucci S, Fiorentino G. 2013 A novel arsenate reductase from the bacterium Thermus thermophilus HB27: its role in arsenic detoxification. Biochim. Biophys. Acta 1834, 2071–2079. (doi:10.1016/j.bbapap.2013.06.007)
