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Colorimetric As (V) detection based on S-layer functionalized gold nanoparticles
Author’s Accepted Manuscript Colorimetric As (V) detection based on S-layer functionalized gold Nanoparticles Mathias Lakatos, Sabine Matys, Johannes Raff, Wolfgang Pompe www.elsevier.com/locate/talanta
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S0039-9140(15)30039-4 http://dx.doi.org/10.1016/j.talanta.2015.05.082 TAL15674
To appear in: Talanta Received date: 31 January 2015 Revised date: 27 May 2015 Accepted date: 30 May 2015 Cite this article as: Mathias Lakatos, Sabine Matys, Johannes Raff and Wolfgang Pompe, Colorimetric As (V) detection based on S-layer functionalized gold Nanoparticles, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.05.082 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Colorimetric As (V) Detection Based on S-Layer Functionalized Gold Nanoparticles Mathias Lakatos*, Sabine Matys‡, Johannes Raff‡†, Wolfgang Pompe* *Dresden University of Technology, Institute of Materials Science, Max Bergmann Center of Biomaterials, Budapester Str. 27, 01069 Dresden, Germany ‡ Helmholtz-Zentrum Dresden-Rossendorf, Helmholtz Institute Freiberg for Resource Technology, Halsbruecker Str. 34, 09599 Freiberg Germany †, Helmholtz-Zentrum Dresden-Rossendorf, Institute of Resource Ecology, Bautzner Landstraße 400, 01328 Dresden, Germany
Abstract Herein, we present simple and rapid colorimetric and UV/VIS spectroscopic methods for detecting anionic arsenic (V) complexes in aqueous media. The methods exploit the aggregation of S-layer-functionalized spherical gold nanoparticles of sizes between 20 and 50 nm in the presence of arsenic species. The gold nanoparticles were functionalized with oligomers of the S-layer protein of Lysinibacillus sphaericus JG-A12. The aggregation of the nanoparticles results in a color change from burgundy-red for widely dispersed nanoparticles to blue for aggregated nanoparticles. A detailed signal analysis was achieved by measuring the shift of the particle plasmon resonance signal with UV/VIS spectroscopy. To further improve signal sensitivity, the influence of larger nanoparticles was tested. In the case of 50 nm gold nanoparticles, a concentration of the anionic arsenic (V) complex lower than 24 ppb was detectable.
KEYWORDS: arsenic, colorimetric, nanoparticle, S-layer, detection Corresponding author: tel.: +4935147940298; fax: +4935147940299; e-mail: [email protected]
1. Introduction Under natural conditions, many bacteria and almost all archaea species have a protein layer as its outermost cell structure, commonly known as surface layer (S-layer). The S-layers are composed of identical (glyco-) protein subunits. They form highlyordered two-dimensional crystalline structures, with well-defined pore size of 2 – 8 nm, lattice constants ranging from 10 – 40 nm, and with a thickness of 3 – 40 nm [1-3]. S-layer proteins can reassemble on different substrates or in suspension [4-7]. These characteristics make S-layers uniquely attractive for nano-biotechnological and biomimetic applications, particularly when they are combined with nanoparticles [8,9]. S-layers can feature a significant amount of regularly-arranged functional groups on their surface, such as -OH, -COOH or -NH2, which form specific binding motifs for particular ions or small molecules. Importantly, there are strong indications that bacteria develop S-layers with specific binding motifs depending on their particular habitat. For example, several S-layer proteins of bacteria found in industrial uranium or arsenic-loaded mining areas are known to be capable of binding five times more arsenic in comparison to commercial sorbents [10]. From studies such as this, it has been suggested that among these bacteria, the S-layer of L. sphaericus JG-A12, which was isolated from a uranium mining waste pile in Johanngeorgenstadt (Saxony, Germany), could be a favored candidate for the enhanced binding of anionic arsenic complexes. For example, combining the S-layer protein of L. sphaericus JG-A12 with the plasmonic properties of gold nanoparticles (AuNP) could result in a promising candidate for use as a biosensor with enhanced sensitivity for detecting heavy metals and metalloids in aqueous media. The principle of detection relies on the formation of particle aggregates induced by specific binding of the target molecule, as has been demonstrated by Mirkin and coworkers in their study of DNA-functionalized AuNPs [11]. This aggregation can be measured via colorimetry, UV/VIS spectroscopy, or dynamic light scattering (DLS) [12-14]. In particular, colorimetric detection of the particle aggregation enables the realization of a cost-effective, customer-friendly and easy-to-handle biosensor system because it is based on a simple color shift that occurs when the particles aggregate, which can be detected by the naked eye. For spherical AuNPs this shift is visible as a change in solution color from red to purple/blue. Other commercially-available colorimetric detection methods for arsenic normally possess a detection limit of 1 ppb to 30 ppb [15,16]. Although lower detection limits have been described for arsenic (III) [14], most methods that enhance detection sensitivity usually require expensive instrumentation and are both time consuming and personnel-intensive. Hence, a biosensor system featuring both high sensitivity and lower technical
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measurement requirements in comparison to commercially-available colorimetric detection systems is an attractive scientific and technical goal. This study, therefore, describes the advantages of using S-layers with specific binding motifs for the design of such a system. Specifically, we investigated the arsenic-induced aggregation of spherical AuNPs functionalized with assemblies of various sizes of S-layer proteins of L. sphaericus JG-A12. The functionalization of AuNPs with these proteins was carried out simply and quickly via adsorption, after which we utilized colorimetric measurements and UV/VIS spectroscopy to detect the presence of As (V), which is a dominant species in water.
2. Materials and Methods 2.1 Preparation of S-layer suspensions The S-layer proteins used in the studies were extracted from L. sphaericus JG-A12, a soil bacterium. Aggregate suspensions of Slayer proteins were prepared according to Raff and coworkers [17], with some minor experimental modifications. Briefly, the bacteria were grown overnight in a nutrient broth (3 g L-1 meat extract (Merck KGaA, Darmstadt, Germany), 5 g L-1 Bacto peptone (Becton, Dickinson and Company, Sparks, U.S.A.) at 30 °C. After harvesting and washing with a standard buffer (50 mM Tris, 1 mM MgCl2, 3 mM NaN3, pH 7.5), they were disintegrated by a high-shear fluid processor (M-110L Microfluidizer Processor, Microfluidics, Newton, MA). The plasma membrane was removed using 1% Triton x 100. To digest the peptidoglycane, the cell wall fragments were incubated with 2 mg ml-1 lysozyme (Merck KGaA, Darmstadt, Germany) overnight at 30 °C. Isolated S-layers were centrifuged at 35,000 x g for 20 – 30 minutes. After discarding the supernatant, the pellet was resuspended in a standard buffer and then centrifuged again. The S-layer proteins were then fractionated from any impurities by gently stirring the centrifuged sample, after which the S-layer was resuspended in the supernatant. This additional step was required because impurities tend to be heavier and form harder pellets in comparison to the S-layer. This multi-step process resulted in S-layer proteins with a purity level of at least 95 %. Atomic force microscopy (Nanoscope IIIa, Digital Instruments, Veeco Metrology Group, Santa Barbara, CA) was utilized to verify its lattice formation properties, which serves as a valid indicator of functionality. S-layer oligomer solutions were prepared by adding 0.1 M HCl until a final pH of 3.5 was reached, followed by centrifugation of the suspension at 40,000 x g for 60 minutes to remove residual aggregates. Monomer solutions were obtained by dissolving the aggregates in 6 M guanidine hydrochloride, followed by centrifugation at 40,000 x g for 60 minutes and dialysis against 100 mM ethylenediaminetetraacetic acid (EDTA) for one hour. Then, the concentrated EDTA was replaced with 1 mM EDTA and the dialysis continued for two days. In the final step, the diluted EDTA was replaced by water for one day to remove trace residues of EDTA. The extracted S-layer proteins were stored at 4 °C. Protein concentrations were calculated using the Micro Lowry Kit (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany).
2.2 Preparation and functionalization of AuNPs Spherical gold nanoparticles were prepared according to a method described by Frens [18]. For the preparation of 20 nm AuNPs, 2.5 ml of a 1% trisodium citrate solution were added to 10 ml 0.1% chloroauric acid (HAuCl4 3H2O) under continuous stirring at 70 °C. Within a few minutes the color changed from colorless to dark ruby red, indicating the formation of AuNPs. Stirring and heating at 70 °C was continued until no further color change was observed. For 50 nm AuNPs 1 ml of a 1% trisodium citrate solution was added to 10 ml 0.1% chloroauric acid. The functionalization of AuNPs with S-layer proteins was done using an adsorption process. In general, 10 ml AuNPs solution with an optical density of 1 was incubated with 5 ml of a 2 mg ml -1 S-layer protein solution of each of the three above-mentioned configurations (monomers, oligomers, assemblates). The mixture was left undisturbed overnight to achieve a saturation of the particle coverage with protein. To separate the unbound S-layer from the S-layer protein-functionalized AuNPs, the suspension was centrifuged for 30 minutes at 3,000 x g. Then, the supernatant was discarded and the sediment of AuNPs functionalized with JGA12 S-layer proteins was resuspended in distilled water.
2.3 Particle characterization Varian Cary 100 UV/VIS spectrophotometer (Varian Inc., Canterbury, Australia) was used to measure the absorption of the colloidal AuNPs solutions. The size, shape and distribution of the AuNPs were determined via scanning electron microscopy (SEM, LEO 982 Gemini, LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) and transmission electron microscopy (TEM, Zeiss Libra 200, Zeiss Oberkochen, Germany).
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The size distribution of the S-layer configurations, AuNPs, and S-layer coated AuNPs was determined using dynamic light scattering in a Zetasizer nano ZS (Malvern Instruments Ltd., Herrenberg, Germany). The same device was also used for zeta potential measurements.
3. Results and Discussion The functionality of a nanoparticles-based colorimetric sensor depends on several parameters. First of all, the target-induced aggregation has to be connected with a detectable change of the plasmon signal. Therefore, the average particle distance should not exceed a critical value in the range of the particle size [19]. Secondly, the aggregation has to be caused by specific binding of the selected target at the functionalized AuNPs. In order to fulfill these two conditions, the AuNPs must be conjugated with small units of the S-layer presenting a specific binding motif. In order to verify this hypothesis, nanoparticles were functionalized with S-layer building units of different sizes: protein monomers, the smallest identical subunits of the protein lattice, small oligomers consisting of a few ordered monomers (hydrodynamic diameter of about 20 to 30 nm), and larger S-layer patches (about 3-4 µm in size). Figure 1 3. 1 Characterization of the S-layer When S-layer proteins are utilized as modifying molecules they usually exist as sheets featuring a square lattice symmetry, which were prepared according to a standard protocol. The sheets, typically between 1 to 10 µm2 in size [20], can be degraded into their subunits by either adding chaotropic agents such as guanidinium hydrochloride (GHCl), or by lowering the pH (Figure 1a). By lowering the pH with 0.1 M hydrochloric acid, we were able to achieve proteins possessing a hydrodynamic diameter of 20 to 30 nm. Considering that the molecular weight of 126 kDa is for the monomer [21], these proteins should be attributed to small nucleation units, hereafter referred to as oligomers. Characterizing the particle size of the S-layer subunits with dynamic light scattering revealed the existence of particles with hydrodynamic diameters of 10 to 20 nm after disintegration with 6 M GHCl (Figure 1b). It is known from other experiments that thusly-prepared S-layer proteins are monomers with a filamentous shape.
3.2 Characterization of the functionalized AuNPs Figure 2 shows micrographs of AuNPs functionalized with JG-A12 S-layer proteins of different assembly size. Coverage was observed for both monomers and oligomers, which confirms the extensive adsorption of the S-layer protein at the AuNPs site; in contrast, adsorption of S-layer sheets of micrometer size leads to thicker rough protein layers. Corresponding measurements of the S-layer-conjugated nanoparticles with DLS confirmed that the effective particle size tended to increase with the increasing size of the protein units. For example, modifying 20 nm AuNPs with monomers and oligomers resulted in the formation of particles with hydrodynamic diameters of 40 nm and 60 nm, respectively. These results can be attributed to the real structure of the S-layer protein in water. The DLS measurements suggest that the diffusion behavior of the proteins generally depends on their molecular weight, as well as the interactions between water molecules and ions on the surface of the particles. Conversely, TEM investigations revealed much smaller protein shell structures due to a degree of protein shrinkage under vacuum conditions (Figure 2). Figure 2 Zeta potential analysis revealed that the charge on the surface of AuNPs changed from negative to positive as a result of functionalization with S-layer subunits, as shown in Figure 3. The oligomer units provided the highest positive potential on the nanoparticles. Based on these results, one can conclude that there is an excess of positive charge domains on the S-layer oligomer surfaces, which could serve as preferential binding motifs for anionic arsenic complexes. Figure 3 3.3 Colorimetric measurements and UV/VIS spectroscopy Prior to interacting with an analyte, functionalized AuNPs (20 nm in diameter) are reddish in color and possess a strong absorption band located at 520 nm. Despite the fact that AuNPs were successfully functionalized with all the three types of protein assemblies, only the oligomer-stabilized AuNPs showed a significant change in the plasmon signal upon interacting with arsenic complexes. Figure 4 summarizes the concentration dependence of arsenic-induced signal changes. Figure 4
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By increasing the arsenic concentration from 0.17 µM to 170 mM, a distinct color change of the colloidal solution from red to blue was detectable at a concentration of 0.17 mM. The corresponding UV/VIS spectra showed a broadening and a strong shift of the plasmon absorption peak to a higher wavelength ( =550 nm), which can be attributed to particle agglomeration. It should also be noted that in the full range of 17 µM to 8 mM, a continuous small shift of the plasmon signal was detectable. The outstanding characteristic of the system is the significant change in the plasmon signal in a sharply defined concentration range, which is typical for the respective S-layer and the target ion, and indicates specific interactions between the protein and the target molecule. In this regard, any changes in the plasmon signal in the presence of phosphate as a competing ion complex were not detectable (data not shown). The experiments have been also repeated with larger spherical AuNPs of 50 nm in diameter (Figure 5). Figure 5 By increasing the particle size, the upper detection limit was lowered from 0.17 mM to 1.7 µM. Strong color changes from red to blue occurred at analyte concentrations from 1.7 µM to 17 µM due to particle agglomeration. This means that the 50 nm AuNPs have an effect of lowering the detection limit by 10- to 100-fold compared to smaller nanoparticles. This can be attributed to a decrease of the nanoparticle concentration with increasing particle size. The particle concentration of the 50 nm nanoparticles in solution at the same optical density of 1 is one order of magnitude less than for the 20 nm nanoparticles. Furthermore, the plasmon coupling efficiency is improved with increasing particle size. Larger particles produce a detectable signal even with smaller aggregates, which widens the detection range. Unfortunately, that benefit competes with the increased instability of the sensing suspension caused by the sedimentation tendency of larger AuNPs. In addition, an increase in particle size leads also to an expansion of the plasmon band due to excitation of multipolschwingungen and increased damping [22], which complicates the signal interpretation for particle agglomeration of very large nanoparticles. As discussed earlier in this section, the nanoparticle concentration itself also has an influence on the final detectable analyte concentration. Generally, a reduction of the AuNPs concentration results in a decrease of the analyte concentration that is necessary for particle agglomeration. In this study the nanoparticle concentration was optimized for a colorimetric signal based on a simple color change from red to blue (OD=1). In general, the interactions between S-layer-functionalized AuNPs and arsenic (V) can be attributed to a series of relevant functional groups such as NH2, NH, OH, CO, COOH, SH, and PO4, which are commonly found on the surface of S-layer proteins [23-26]. Some examples of the binding of platinum, palladium, uranium and gold complexes to S-layer have been published in the literature [17,27,28]. Frequently, the binding mechanisms are highly complex since multiple functional groups of the amino acids are involved [17]. As is known, only a few metal ions (e.g., the above-mentioned examples) were bound by the JG-A12 S-layer on the surface, resulting in an agglomeration of S-layer coated AuNPs. Most other metal ions in environmental samples (e.g., Na+, K+, Mg2+, Ca2+, Fe3+, Co2+, Ni2+, Cu2+ and Zn2+) were bound in another mode or did not show any significant interaction at all. Interestingly, the fact that S-layers of different species vary in their binding behavior towards different metals affords the opportunity to identify selective binders. Therefore, selecting the appropriate S-layer coating will facilitate the production of metalselective detection systems. In general, producing deeper insights into the complex nature of specific or unspecific interactions between S-layer proteins and particular ions of interest remains a challenging task. In the study presented herein, only the typical binding of arsenic complexes on SH-groups [29] can be excluded, because sequencing analysis revealed that the S-layer structure of JG-A12 has no sulfur groups [27]. Hence, it is more likely that the interaction of anionic arsenic complexes results from NH 2 or NH. As mentioned above, the special arrangement of functional groups plays an important role in the interaction with arsenic (V). Regarding the mechanism of particle agglomeration, the results of zeta potential measurements at pH 3.5 to 4 of the S-layer JG-A12 predominantly show a positive charge, which corresponds to findings for S-layer-functionalized AuNPs. From the literature it is known that under these conditions the arsenic (V) forms a negative complex with a monovalent charge [30]. In this context, the results indicate a non-specific interaction of particle agglomeration of the S-layer-functionalized AuNPs with increasing analyte concentration. A simplified schematic drawing of the corresponding relationship is shown in Figure 6. Figure 6 Initially, the S-layer-functionalized AuNPs exist as a fine dispersion, stabilized by repulsive Coulomb interactions. The addition of the anionic analyte leads to a decrease in the repulsive forces. Concomitantly, the probability for agglomeration of the nanoparticles rises. By further increasing the amount of the negatively-charged arsenic complex, the positive charge of the S-layer-functionalized AuNPs diminishes cumulatively, resulting in particle agglomeration at the point where attractive forces exceed electrostatic repulsion [31,32]. These attractive forces may result from hydrogen bonds, as well as hydrophobic interactions at the S-layer surface. The plasmon coupling and the resulting intensity of the color shift depend on the size of the emerging aggregates and their packing density. For a high analyte concentration, the charge value of the nanoparticles can change from positive to negative. In such a case, particle aggregation would also be prevented, and no plasmon coupling would occur. This phenomenon is visible in the UV/VIS spectra in Figure 4. To develop a signal analysis algorithm toward an automated sensor application, we looked at the shift of the plasmon resonance signal for three fixed wavelengths (see vertical dashed lines Figure 5). Figure 7 shows a transformed plot of this plasmon signal that
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depends on the analyte concentration for 50 nm AuNPs. In considering these curves as wavelength-dependent references, it might be possible to estimate the analyte concentration of an unknown sample (for specified reaction time and nanoparticle size, in this case 50 nm). Depending on the concentration of this unknown solution, the results of a dilution series will follow the plotted curves.
Figure 7 Interestingly, this method could also be used to further increase the detection limit. As a result of this signal transformation, it may be possible to further reduce the minimum concentration (detection limit) of the anionic arsenic complex when measuring signal change in the case of a 50 nm AuNPs sample, which is estimated to reach a final value of 50 nM (7 ppb). However, in order to actually quantify this promising principle for experimental use, at least two tasks have to be solved. First, it will be necessary to clarify all calibration and linearity details in order to quantitate the concentration of the analyte. Second, cross-reactions with other anions or the effects of complex media must be studied in more detail. In considering the findings discussed herein, we suggest that small oligomers of S-layer proteins represent promising candidates for the design of colorimetric and UV/VIS spectrophotometric sensors for the detection of toxic metal complexes. Furthermore, the sensitivity of such sensors based on oligomer-functionalized AuNPs can be increased with increasing particle size up to few tens of ppb.
4. Conclusions The results documented in this paper confirm that it is possible to detect anionic arsenic (V) species with S-layer-functionalized AuNPs. The developed colorimetric method possesses a high potential to be extended to the detection of various metals and metalloids by simply modifying the S-layer functionalization. The oligomer units of the native S-layer of L. sphaericus JG-A12 were the most suitable S-layer component for particle stabilization and high sensitivity against arsenic (V). Depending on nanoparticle size, it was possible to reduce the detection limit of the anionic arsenic complex H 2AsO4- down to a concentration of 1.7 µM, which corresponds to a value of 240 ppb. These changes were visible to the naked eye because of a color change from red to blue in colloidal solution. Furthermore, as confirmed by UV/VIS measurements, an enhancement of the arsenic (V) sensitivity up to 24 ppb has been realized, which demonstrates the overall applicability of bacterial surface proteins for metal/metalloid sensing applications with AuNPs. This system can also be readily applied to several other ions or ion complexes of multivalent metal species by using S-layers with another metal selectivity. Indeed, further extensive investigations are in progress, especially targeting the specificity of the system in the presence of competing ions and different species. Moreover, the variability of naturally-occurring S-layer proteins and the opportunity to modify them biochemically or genetically offer multifaceted prospects for the design and development of customized metal-sensing devices.
AUTHOR INFORMATION Corresponding Author * [email protected].
Author Contributions ‡These authors contributed equally.
ACKNOWLEDGMENT We thank B. Katzschner for the preparation of S-layers and A. Caspari for the DLS and zeta potential measurements. The work was partly supported by grant 03WKP08 from the Bundesministerium für Bildung und Forschung (BMBF). .
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Figure captions Figure 1. Subunits of different size S-layer proteins extracted from Lysinibacillus sphaericus JG-A12: (a) schematic drawing, and (b) the distribution of dynamic light scattering intensity for S-layer particles of different assembly size. Figure 2. TEM images showing (a) pure AuNPs and AuNPs functionalized with different configurations of the S-layer protein of L. sphaericus JGA-12: (b) monomers, (c) oligomers, and (d) sheets, no protein shell was detectable (scale bar 20 nm). Figure 3. Zeta potential of AuNPs, S-layer subunits and conjugates of both. Figure 4. Interaction of S-layer-functionalized AuNPs (20 nm in diameter) with Na2HAsO4: (a) colorimetric assay, and (b) UV/VIS measurements. Figure 5. Interaction of S-layer-functionalized AuNPs (50 nm in diameter) with Na2HAsO4: (a) colorimetric assay, and (b) UV/VIS measurements. Figure 6. Interaction modes of S-layer-functionalized AuNPs for different concentrations of As (V) complexes. Figure 7. Relative changes in intensity depending on the concentration of anionic arsenic complex As (V) for 50 nm AuNPs.
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Highlights - A general method for AuNP functionalization with s-layer proteins is proposed. - A colorimetric assay based on AuNP, covered with s-layer protein is presented. - The high specific binding affinity of JG-A12 s-layer proteins to arsenic (V) is proven. - Detection limits of 24 ppb for arsenic (V) is reached using UV/VIS spectroscopy
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Graphical Abstract S-layer-functionalized spherical gold nanoparticles are used for detection of arsenic species by colorimetric method. The aggregation of the nanoparticles results into color change from wine red for widely dispersed to blue for aggregated nanoparticles. Additionally, UV/VIS spectroscopy allows an increase of the detection sensitivity up to 24 ppb.
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PII: DOI: Reference:
S0039-9140(15)30039-4 http://dx.doi.org/10.1016/j.talanta.2015.05.082 TAL15674
To appear in: Talanta Received date: 31 January 2015 Revised date: 27 May 2015 Accepted date: 30 May 2015 Cite this article as: Mathias Lakatos, Sabine Matys, Johannes Raff and Wolfgang Pompe, Colorimetric As (V) detection based on S-layer functionalized gold Nanoparticles, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.05.082 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Colorimetric As (V) Detection Based on S-Layer Functionalized Gold Nanoparticles Mathias Lakatos*, Sabine Matys‡, Johannes Raff‡†, Wolfgang Pompe* *Dresden University of Technology, Institute of Materials Science, Max Bergmann Center of Biomaterials, Budapester Str. 27, 01069 Dresden, Germany ‡ Helmholtz-Zentrum Dresden-Rossendorf, Helmholtz Institute Freiberg for Resource Technology, Halsbruecker Str. 34, 09599 Freiberg Germany †, Helmholtz-Zentrum Dresden-Rossendorf, Institute of Resource Ecology, Bautzner Landstraße 400, 01328 Dresden, Germany
Abstract Herein, we present simple and rapid colorimetric and UV/VIS spectroscopic methods for detecting anionic arsenic (V) complexes in aqueous media. The methods exploit the aggregation of S-layer-functionalized spherical gold nanoparticles of sizes between 20 and 50 nm in the presence of arsenic species. The gold nanoparticles were functionalized with oligomers of the S-layer protein of Lysinibacillus sphaericus JG-A12. The aggregation of the nanoparticles results in a color change from burgundy-red for widely dispersed nanoparticles to blue for aggregated nanoparticles. A detailed signal analysis was achieved by measuring the shift of the particle plasmon resonance signal with UV/VIS spectroscopy. To further improve signal sensitivity, the influence of larger nanoparticles was tested. In the case of 50 nm gold nanoparticles, a concentration of the anionic arsenic (V) complex lower than 24 ppb was detectable.
KEYWORDS: arsenic, colorimetric, nanoparticle, S-layer, detection Corresponding author: tel.: +4935147940298; fax: +4935147940299; e-mail: [email protected]
1. Introduction Under natural conditions, many bacteria and almost all archaea species have a protein layer as its outermost cell structure, commonly known as surface layer (S-layer). The S-layers are composed of identical (glyco-) protein subunits. They form highlyordered two-dimensional crystalline structures, with well-defined pore size of 2 – 8 nm, lattice constants ranging from 10 – 40 nm, and with a thickness of 3 – 40 nm [1-3]. S-layer proteins can reassemble on different substrates or in suspension [4-7]. These characteristics make S-layers uniquely attractive for nano-biotechnological and biomimetic applications, particularly when they are combined with nanoparticles [8,9]. S-layers can feature a significant amount of regularly-arranged functional groups on their surface, such as -OH, -COOH or -NH2, which form specific binding motifs for particular ions or small molecules. Importantly, there are strong indications that bacteria develop S-layers with specific binding motifs depending on their particular habitat. For example, several S-layer proteins of bacteria found in industrial uranium or arsenic-loaded mining areas are known to be capable of binding five times more arsenic in comparison to commercial sorbents [10]. From studies such as this, it has been suggested that among these bacteria, the S-layer of L. sphaericus JG-A12, which was isolated from a uranium mining waste pile in Johanngeorgenstadt (Saxony, Germany), could be a favored candidate for the enhanced binding of anionic arsenic complexes. For example, combining the S-layer protein of L. sphaericus JG-A12 with the plasmonic properties of gold nanoparticles (AuNP) could result in a promising candidate for use as a biosensor with enhanced sensitivity for detecting heavy metals and metalloids in aqueous media. The principle of detection relies on the formation of particle aggregates induced by specific binding of the target molecule, as has been demonstrated by Mirkin and coworkers in their study of DNA-functionalized AuNPs [11]. This aggregation can be measured via colorimetry, UV/VIS spectroscopy, or dynamic light scattering (DLS) [12-14]. In particular, colorimetric detection of the particle aggregation enables the realization of a cost-effective, customer-friendly and easy-to-handle biosensor system because it is based on a simple color shift that occurs when the particles aggregate, which can be detected by the naked eye. For spherical AuNPs this shift is visible as a change in solution color from red to purple/blue. Other commercially-available colorimetric detection methods for arsenic normally possess a detection limit of 1 ppb to 30 ppb [15,16]. Although lower detection limits have been described for arsenic (III) [14], most methods that enhance detection sensitivity usually require expensive instrumentation and are both time consuming and personnel-intensive. Hence, a biosensor system featuring both high sensitivity and lower technical
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measurement requirements in comparison to commercially-available colorimetric detection systems is an attractive scientific and technical goal. This study, therefore, describes the advantages of using S-layers with specific binding motifs for the design of such a system. Specifically, we investigated the arsenic-induced aggregation of spherical AuNPs functionalized with assemblies of various sizes of S-layer proteins of L. sphaericus JG-A12. The functionalization of AuNPs with these proteins was carried out simply and quickly via adsorption, after which we utilized colorimetric measurements and UV/VIS spectroscopy to detect the presence of As (V), which is a dominant species in water.
2. Materials and Methods 2.1 Preparation of S-layer suspensions The S-layer proteins used in the studies were extracted from L. sphaericus JG-A12, a soil bacterium. Aggregate suspensions of Slayer proteins were prepared according to Raff and coworkers [17], with some minor experimental modifications. Briefly, the bacteria were grown overnight in a nutrient broth (3 g L-1 meat extract (Merck KGaA, Darmstadt, Germany), 5 g L-1 Bacto peptone (Becton, Dickinson and Company, Sparks, U.S.A.) at 30 °C. After harvesting and washing with a standard buffer (50 mM Tris, 1 mM MgCl2, 3 mM NaN3, pH 7.5), they were disintegrated by a high-shear fluid processor (M-110L Microfluidizer Processor, Microfluidics, Newton, MA). The plasma membrane was removed using 1% Triton x 100. To digest the peptidoglycane, the cell wall fragments were incubated with 2 mg ml-1 lysozyme (Merck KGaA, Darmstadt, Germany) overnight at 30 °C. Isolated S-layers were centrifuged at 35,000 x g for 20 – 30 minutes. After discarding the supernatant, the pellet was resuspended in a standard buffer and then centrifuged again. The S-layer proteins were then fractionated from any impurities by gently stirring the centrifuged sample, after which the S-layer was resuspended in the supernatant. This additional step was required because impurities tend to be heavier and form harder pellets in comparison to the S-layer. This multi-step process resulted in S-layer proteins with a purity level of at least 95 %. Atomic force microscopy (Nanoscope IIIa, Digital Instruments, Veeco Metrology Group, Santa Barbara, CA) was utilized to verify its lattice formation properties, which serves as a valid indicator of functionality. S-layer oligomer solutions were prepared by adding 0.1 M HCl until a final pH of 3.5 was reached, followed by centrifugation of the suspension at 40,000 x g for 60 minutes to remove residual aggregates. Monomer solutions were obtained by dissolving the aggregates in 6 M guanidine hydrochloride, followed by centrifugation at 40,000 x g for 60 minutes and dialysis against 100 mM ethylenediaminetetraacetic acid (EDTA) for one hour. Then, the concentrated EDTA was replaced with 1 mM EDTA and the dialysis continued for two days. In the final step, the diluted EDTA was replaced by water for one day to remove trace residues of EDTA. The extracted S-layer proteins were stored at 4 °C. Protein concentrations were calculated using the Micro Lowry Kit (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany).
2.2 Preparation and functionalization of AuNPs Spherical gold nanoparticles were prepared according to a method described by Frens [18]. For the preparation of 20 nm AuNPs, 2.5 ml of a 1% trisodium citrate solution were added to 10 ml 0.1% chloroauric acid (HAuCl4 3H2O) under continuous stirring at 70 °C. Within a few minutes the color changed from colorless to dark ruby red, indicating the formation of AuNPs. Stirring and heating at 70 °C was continued until no further color change was observed. For 50 nm AuNPs 1 ml of a 1% trisodium citrate solution was added to 10 ml 0.1% chloroauric acid. The functionalization of AuNPs with S-layer proteins was done using an adsorption process. In general, 10 ml AuNPs solution with an optical density of 1 was incubated with 5 ml of a 2 mg ml -1 S-layer protein solution of each of the three above-mentioned configurations (monomers, oligomers, assemblates). The mixture was left undisturbed overnight to achieve a saturation of the particle coverage with protein. To separate the unbound S-layer from the S-layer protein-functionalized AuNPs, the suspension was centrifuged for 30 minutes at 3,000 x g. Then, the supernatant was discarded and the sediment of AuNPs functionalized with JGA12 S-layer proteins was resuspended in distilled water.
2.3 Particle characterization Varian Cary 100 UV/VIS spectrophotometer (Varian Inc., Canterbury, Australia) was used to measure the absorption of the colloidal AuNPs solutions. The size, shape and distribution of the AuNPs were determined via scanning electron microscopy (SEM, LEO 982 Gemini, LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) and transmission electron microscopy (TEM, Zeiss Libra 200, Zeiss Oberkochen, Germany).
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The size distribution of the S-layer configurations, AuNPs, and S-layer coated AuNPs was determined using dynamic light scattering in a Zetasizer nano ZS (Malvern Instruments Ltd., Herrenberg, Germany). The same device was also used for zeta potential measurements.
3. Results and Discussion The functionality of a nanoparticles-based colorimetric sensor depends on several parameters. First of all, the target-induced aggregation has to be connected with a detectable change of the plasmon signal. Therefore, the average particle distance should not exceed a critical value in the range of the particle size [19]. Secondly, the aggregation has to be caused by specific binding of the selected target at the functionalized AuNPs. In order to fulfill these two conditions, the AuNPs must be conjugated with small units of the S-layer presenting a specific binding motif. In order to verify this hypothesis, nanoparticles were functionalized with S-layer building units of different sizes: protein monomers, the smallest identical subunits of the protein lattice, small oligomers consisting of a few ordered monomers (hydrodynamic diameter of about 20 to 30 nm), and larger S-layer patches (about 3-4 µm in size). Figure 1 3. 1 Characterization of the S-layer When S-layer proteins are utilized as modifying molecules they usually exist as sheets featuring a square lattice symmetry, which were prepared according to a standard protocol. The sheets, typically between 1 to 10 µm2 in size [20], can be degraded into their subunits by either adding chaotropic agents such as guanidinium hydrochloride (GHCl), or by lowering the pH (Figure 1a). By lowering the pH with 0.1 M hydrochloric acid, we were able to achieve proteins possessing a hydrodynamic diameter of 20 to 30 nm. Considering that the molecular weight of 126 kDa is for the monomer [21], these proteins should be attributed to small nucleation units, hereafter referred to as oligomers. Characterizing the particle size of the S-layer subunits with dynamic light scattering revealed the existence of particles with hydrodynamic diameters of 10 to 20 nm after disintegration with 6 M GHCl (Figure 1b). It is known from other experiments that thusly-prepared S-layer proteins are monomers with a filamentous shape.
3.2 Characterization of the functionalized AuNPs Figure 2 shows micrographs of AuNPs functionalized with JG-A12 S-layer proteins of different assembly size. Coverage was observed for both monomers and oligomers, which confirms the extensive adsorption of the S-layer protein at the AuNPs site; in contrast, adsorption of S-layer sheets of micrometer size leads to thicker rough protein layers. Corresponding measurements of the S-layer-conjugated nanoparticles with DLS confirmed that the effective particle size tended to increase with the increasing size of the protein units. For example, modifying 20 nm AuNPs with monomers and oligomers resulted in the formation of particles with hydrodynamic diameters of 40 nm and 60 nm, respectively. These results can be attributed to the real structure of the S-layer protein in water. The DLS measurements suggest that the diffusion behavior of the proteins generally depends on their molecular weight, as well as the interactions between water molecules and ions on the surface of the particles. Conversely, TEM investigations revealed much smaller protein shell structures due to a degree of protein shrinkage under vacuum conditions (Figure 2). Figure 2 Zeta potential analysis revealed that the charge on the surface of AuNPs changed from negative to positive as a result of functionalization with S-layer subunits, as shown in Figure 3. The oligomer units provided the highest positive potential on the nanoparticles. Based on these results, one can conclude that there is an excess of positive charge domains on the S-layer oligomer surfaces, which could serve as preferential binding motifs for anionic arsenic complexes. Figure 3 3.3 Colorimetric measurements and UV/VIS spectroscopy Prior to interacting with an analyte, functionalized AuNPs (20 nm in diameter) are reddish in color and possess a strong absorption band located at 520 nm. Despite the fact that AuNPs were successfully functionalized with all the three types of protein assemblies, only the oligomer-stabilized AuNPs showed a significant change in the plasmon signal upon interacting with arsenic complexes. Figure 4 summarizes the concentration dependence of arsenic-induced signal changes. Figure 4
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By increasing the arsenic concentration from 0.17 µM to 170 mM, a distinct color change of the colloidal solution from red to blue was detectable at a concentration of 0.17 mM. The corresponding UV/VIS spectra showed a broadening and a strong shift of the plasmon absorption peak to a higher wavelength ( =550 nm), which can be attributed to particle agglomeration. It should also be noted that in the full range of 17 µM to 8 mM, a continuous small shift of the plasmon signal was detectable. The outstanding characteristic of the system is the significant change in the plasmon signal in a sharply defined concentration range, which is typical for the respective S-layer and the target ion, and indicates specific interactions between the protein and the target molecule. In this regard, any changes in the plasmon signal in the presence of phosphate as a competing ion complex were not detectable (data not shown). The experiments have been also repeated with larger spherical AuNPs of 50 nm in diameter (Figure 5). Figure 5 By increasing the particle size, the upper detection limit was lowered from 0.17 mM to 1.7 µM. Strong color changes from red to blue occurred at analyte concentrations from 1.7 µM to 17 µM due to particle agglomeration. This means that the 50 nm AuNPs have an effect of lowering the detection limit by 10- to 100-fold compared to smaller nanoparticles. This can be attributed to a decrease of the nanoparticle concentration with increasing particle size. The particle concentration of the 50 nm nanoparticles in solution at the same optical density of 1 is one order of magnitude less than for the 20 nm nanoparticles. Furthermore, the plasmon coupling efficiency is improved with increasing particle size. Larger particles produce a detectable signal even with smaller aggregates, which widens the detection range. Unfortunately, that benefit competes with the increased instability of the sensing suspension caused by the sedimentation tendency of larger AuNPs. In addition, an increase in particle size leads also to an expansion of the plasmon band due to excitation of multipolschwingungen and increased damping [22], which complicates the signal interpretation for particle agglomeration of very large nanoparticles. As discussed earlier in this section, the nanoparticle concentration itself also has an influence on the final detectable analyte concentration. Generally, a reduction of the AuNPs concentration results in a decrease of the analyte concentration that is necessary for particle agglomeration. In this study the nanoparticle concentration was optimized for a colorimetric signal based on a simple color change from red to blue (OD=1). In general, the interactions between S-layer-functionalized AuNPs and arsenic (V) can be attributed to a series of relevant functional groups such as NH2, NH, OH, CO, COOH, SH, and PO4, which are commonly found on the surface of S-layer proteins [23-26]. Some examples of the binding of platinum, palladium, uranium and gold complexes to S-layer have been published in the literature [17,27,28]. Frequently, the binding mechanisms are highly complex since multiple functional groups of the amino acids are involved [17]. As is known, only a few metal ions (e.g., the above-mentioned examples) were bound by the JG-A12 S-layer on the surface, resulting in an agglomeration of S-layer coated AuNPs. Most other metal ions in environmental samples (e.g., Na+, K+, Mg2+, Ca2+, Fe3+, Co2+, Ni2+, Cu2+ and Zn2+) were bound in another mode or did not show any significant interaction at all. Interestingly, the fact that S-layers of different species vary in their binding behavior towards different metals affords the opportunity to identify selective binders. Therefore, selecting the appropriate S-layer coating will facilitate the production of metalselective detection systems. In general, producing deeper insights into the complex nature of specific or unspecific interactions between S-layer proteins and particular ions of interest remains a challenging task. In the study presented herein, only the typical binding of arsenic complexes on SH-groups [29] can be excluded, because sequencing analysis revealed that the S-layer structure of JG-A12 has no sulfur groups [27]. Hence, it is more likely that the interaction of anionic arsenic complexes results from NH 2 or NH. As mentioned above, the special arrangement of functional groups plays an important role in the interaction with arsenic (V). Regarding the mechanism of particle agglomeration, the results of zeta potential measurements at pH 3.5 to 4 of the S-layer JG-A12 predominantly show a positive charge, which corresponds to findings for S-layer-functionalized AuNPs. From the literature it is known that under these conditions the arsenic (V) forms a negative complex with a monovalent charge [30]. In this context, the results indicate a non-specific interaction of particle agglomeration of the S-layer-functionalized AuNPs with increasing analyte concentration. A simplified schematic drawing of the corresponding relationship is shown in Figure 6. Figure 6 Initially, the S-layer-functionalized AuNPs exist as a fine dispersion, stabilized by repulsive Coulomb interactions. The addition of the anionic analyte leads to a decrease in the repulsive forces. Concomitantly, the probability for agglomeration of the nanoparticles rises. By further increasing the amount of the negatively-charged arsenic complex, the positive charge of the S-layer-functionalized AuNPs diminishes cumulatively, resulting in particle agglomeration at the point where attractive forces exceed electrostatic repulsion [31,32]. These attractive forces may result from hydrogen bonds, as well as hydrophobic interactions at the S-layer surface. The plasmon coupling and the resulting intensity of the color shift depend on the size of the emerging aggregates and their packing density. For a high analyte concentration, the charge value of the nanoparticles can change from positive to negative. In such a case, particle aggregation would also be prevented, and no plasmon coupling would occur. This phenomenon is visible in the UV/VIS spectra in Figure 4. To develop a signal analysis algorithm toward an automated sensor application, we looked at the shift of the plasmon resonance signal for three fixed wavelengths (see vertical dashed lines Figure 5). Figure 7 shows a transformed plot of this plasmon signal that
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depends on the analyte concentration for 50 nm AuNPs. In considering these curves as wavelength-dependent references, it might be possible to estimate the analyte concentration of an unknown sample (for specified reaction time and nanoparticle size, in this case 50 nm). Depending on the concentration of this unknown solution, the results of a dilution series will follow the plotted curves.
Figure 7 Interestingly, this method could also be used to further increase the detection limit. As a result of this signal transformation, it may be possible to further reduce the minimum concentration (detection limit) of the anionic arsenic complex when measuring signal change in the case of a 50 nm AuNPs sample, which is estimated to reach a final value of 50 nM (7 ppb). However, in order to actually quantify this promising principle for experimental use, at least two tasks have to be solved. First, it will be necessary to clarify all calibration and linearity details in order to quantitate the concentration of the analyte. Second, cross-reactions with other anions or the effects of complex media must be studied in more detail. In considering the findings discussed herein, we suggest that small oligomers of S-layer proteins represent promising candidates for the design of colorimetric and UV/VIS spectrophotometric sensors for the detection of toxic metal complexes. Furthermore, the sensitivity of such sensors based on oligomer-functionalized AuNPs can be increased with increasing particle size up to few tens of ppb.
4. Conclusions The results documented in this paper confirm that it is possible to detect anionic arsenic (V) species with S-layer-functionalized AuNPs. The developed colorimetric method possesses a high potential to be extended to the detection of various metals and metalloids by simply modifying the S-layer functionalization. The oligomer units of the native S-layer of L. sphaericus JG-A12 were the most suitable S-layer component for particle stabilization and high sensitivity against arsenic (V). Depending on nanoparticle size, it was possible to reduce the detection limit of the anionic arsenic complex H 2AsO4- down to a concentration of 1.7 µM, which corresponds to a value of 240 ppb. These changes were visible to the naked eye because of a color change from red to blue in colloidal solution. Furthermore, as confirmed by UV/VIS measurements, an enhancement of the arsenic (V) sensitivity up to 24 ppb has been realized, which demonstrates the overall applicability of bacterial surface proteins for metal/metalloid sensing applications with AuNPs. This system can also be readily applied to several other ions or ion complexes of multivalent metal species by using S-layers with another metal selectivity. Indeed, further extensive investigations are in progress, especially targeting the specificity of the system in the presence of competing ions and different species. Moreover, the variability of naturally-occurring S-layer proteins and the opportunity to modify them biochemically or genetically offer multifaceted prospects for the design and development of customized metal-sensing devices.
AUTHOR INFORMATION Corresponding Author * [email protected].
Author Contributions ‡These authors contributed equally.
ACKNOWLEDGMENT We thank B. Katzschner for the preparation of S-layers and A. Caspari for the DLS and zeta potential measurements. The work was partly supported by grant 03WKP08 from the Bundesministerium für Bildung und Forschung (BMBF). .
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Figure captions Figure 1. Subunits of different size S-layer proteins extracted from Lysinibacillus sphaericus JG-A12: (a) schematic drawing, and (b) the distribution of dynamic light scattering intensity for S-layer particles of different assembly size. Figure 2. TEM images showing (a) pure AuNPs and AuNPs functionalized with different configurations of the S-layer protein of L. sphaericus JGA-12: (b) monomers, (c) oligomers, and (d) sheets, no protein shell was detectable (scale bar 20 nm). Figure 3. Zeta potential of AuNPs, S-layer subunits and conjugates of both. Figure 4. Interaction of S-layer-functionalized AuNPs (20 nm in diameter) with Na2HAsO4: (a) colorimetric assay, and (b) UV/VIS measurements. Figure 5. Interaction of S-layer-functionalized AuNPs (50 nm in diameter) with Na2HAsO4: (a) colorimetric assay, and (b) UV/VIS measurements. Figure 6. Interaction modes of S-layer-functionalized AuNPs for different concentrations of As (V) complexes. Figure 7. Relative changes in intensity depending on the concentration of anionic arsenic complex As (V) for 50 nm AuNPs.
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Highlights - A general method for AuNP functionalization with s-layer proteins is proposed. - A colorimetric assay based on AuNP, covered with s-layer protein is presented. - The high specific binding affinity of JG-A12 s-layer proteins to arsenic (V) is proven. - Detection limits of 24 ppb for arsenic (V) is reached using UV/VIS spectroscopy
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Graphical Abstract S-layer-functionalized spherical gold nanoparticles are used for detection of arsenic species by colorimetric method. The aggregation of the nanoparticles results into color change from wine red for widely dispersed to blue for aggregated nanoparticles. Additionally, UV/VIS spectroscopy allows an increase of the detection sensitivity up to 24 ppb.
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