Parts per Trillion Detection of Ni(II) Ions by Nanoparticle-Enhanced

Oct 15, 2012 - The ability to detect Ni(II) ions as low as 50 parts per trillion (ppt) is a remarkable improvement compared to .... Iris R. Bell , Gar...
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Parts per Trillion Detection of Ni(II) Ions by Nanoparticle-Enhanced Surface Plasmon Resonance Eum Ji Kim,† Bong Hyun Chung,‡ and Hye Jin Lee*,† †

Department of Chemistry and Green-Nano Materials Research Center, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu-city, 702-701, Republic of Korea ‡ BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, 125, Gwahak-ro, Yuseong-gu, Daejeon, 305-806, Republic of Korea S Supporting Information *

ABSTRACT: This paper demonstrates the development of a novel nanoparticle-enhanced surface plasmon resonance (SPR) sensing platform for the selective and sensitive in situ detection of nickel(II) ions at concentrations as low as 50 parts per trillion (211 pM). An enhancement in selectivity was achieved by designing a surface sandwich assay involving two different ligands each selective toward nickel(II) ions, namely, N-[5-(3′-maleimidopropylamido)-1-carboxypentyl]iminodiacetic acid (NTA) and polyhistidine. Maleimido-modified NTA was first immobilized on an alkanedithiol-modified gold thin film, followed by the sequential adsorption of Ni(II) ions. Next, polyhistidine-functionalized quasispherical gold nanoparticles, designed to enhance the SPR sensitivity, were specifically adsorbed onto surface Ni(II)−NTA complexes. This process was monitored by real-time SPR. The ability to detect Ni(II) ions as low as 50 parts per trillion (ppt) is a remarkable improvement compared to other optical and colorimetric techniques utilizing nanoparticles and is comparable to what can be achieved by state-of-the-art inductively coupled plasma mass spectrometry (ICP-MS). The improved selectivity for Ni(II) ions by the sandwich assay approach was confirmed by comparing measurements involving other divalent cations such as Zn(II), Pb(II), and Cu(II), some of which individually possess binding affinities toward either the NTA or histidine moieties.

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sensors. This disparity is partly due to the highly toxic effects of species such as Hg(II) ions being well-established and also due to the availability of ligands that enable ions to be targeted with good specificity.22,23 For example, Hung et al.24 demonstrated that good specificity for Hg(II), Ag(II), and Pb(II) cations could be obtained with the strong complexation of these ions affecting the aggregation behavior of alkanethiol-coated gold nanoparticles depending on the choice of monolayer end group. Other approaches include ligand exchange reactions on nanoparticles25 and the preparation of fluorescent gold nanoparticles functionalized with nucleic acid aptamer probes established for both Pb(II)26 and Hg(II) ions.27,28 Colorimetric approaches involving the controlled aggregation of nanoparticles typically have a detection limit in the low-to-mid parts per million (ppm) range. Li and co-workers26 have reported the colorimetric detection of Ni(II) ions using glutathionefunctionalized silver nanoparticles, which had moderate selectivity in the presence of other divalent cations, with a similar approach also reported for Pb(II) ion detection.29 Recently, Krpetić et al.30 used a combination of extinction and surface-enhanced Raman scattering (SERS) measurements to

eavy metal ion detection is important in many areas related to the environmental monitoring of water and soil contamination as well as investigating their toxicological effects. The need for methods capable of accurate and sensitive trace metal analysis continues to drive the emergence of innovative new approaches for detection. For example, Ni(II) ions are recognized as an essential trace element in bacteria, plants, and animals,1,2 and at higher concentrations, nickel(II) ion compounds have been shown to be carcinogenic,3 associated with dermatitis.4,5 Also, contact sensitivity, where the skin develops an allergic reaction, is an increasingly common problem.6 Established techniques for metal ion sensing include atomic absorption spectroscopy,7−10 anodic stripping voltammetry,11−13 fluorescence spectrometry,14−16 and inductively coupled plasma optical emission spectroscopy (ICP-OES),17−19 but the current “gold standard” for multiplexed heavy metal ion sensing is inductively coupled plasma mass spectrometry (ICPMS), which has a detection limit for Ni(II) ions of around 50 ppt.20,21 More recently, the application of much simpler optical detection methods that promote rapid in situ detection have started to emerge for a series of different heavy metal ions. The vast majority of these studies have focused on developing optical sensors for metal ions such as Hg(II), Pb(II), Cu(II), and Cd(II), with very few reports in the literature on Ni(II) ion © 2012 American Chemical Society

Received: September 6, 2012 Accepted: October 15, 2012 Published: October 15, 2012 10091

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Figure 1. Schematic showing the attachment of NTA onto a monolayer of a 1,6-hexanedithiol-modified Au SPR chip along with the polyhistidine functionalization of quasispherical nanoparticles (shown in boxed area) via an initial monolayer of 11-mercaptoundecanoic acid (MUA) and EDC/ NHSS linking chemistry. The surface sandwich assay for NP-enhanced SPR detection of Ni(II) ions is also shown.

achieve comparative detection limits of ∼5 ppm and 0.5 ppm respectively for each modality. This was performed with dual sets of nanoparticles functionalized with nitrilotriacetic acid (NTA) or L-carnosine, which is a dipeptide of histidine and βalanine. This approach is a variation of the hexahistidine-tag chemistry used in the affinity purification of proteins,31,32 where the Ni(II)−NTA complex has a much higher affinity toward (His)6 than NTA alone. This approach also has been applied to the fabrication of protein arrays on thin gold films.33 In this paper, we demonstrate the use of nanoparticleenhanced surface plasmon resonance (SPR) for the in situ detection of Ni(II) ions at concentrations as low as 50 ppt (211 pM), resulting in one of the most sensitive optical-based Ni(II) detection measurements achieved to date. The technique of SPR involves measuring changes in refractive index at the interface between a thin gold film and an aqueous dielectric, and its application has typically focused on molecular interactions involving molecules such as nucleic acids, peptides, and proteins rather than metal ions. Direct detection of the adsorption of both Hg(II)34 and Pt(II)35 ions onto a 1,6hexanedithiol monolayer at concentrations in the low ppm range has been reported. Also, modified SPR approaches such as flow injection SPR36 and differential SPR37,38 have been described for different heavy metal ions, with detection limits in the low parts per billion (ppb) range. The use of functionalized nanoparticles as part of a surface-sandwich assay to greatly enhance the sensitivity of SPR has now been described by a number of groups for a range of nucleic acid39−48 and protein44,49−52 targets. Recently, Wang et al.53 developed an assay using DNA−nanoparticle conjugates to displace Hg(II) ions bound to a mercury-specific oligonucleotide, which improved the detection limit to 5 nM (1.6 ppb). Here, we demonstrate a sandwich assay for the detection of Ni(II) ions where custom gold quasispherical nanoparticles functionalized

with polyhistidine (polyHis; molecular mass ∼9500 Da) are specifically adsorbed onto a NTA-functionalized SPR chip surface in the presence of Ni(II) ions. In addition to systematically comparing the detection signal for Ni(II) ions alone with that for both polyHis and also polyHis−nanoparticle (polyHis−NP) amplification, the improved specificity possible through a dual ligand binding approach was also emphasized by performing measurements in the presence of an excess of various divalent ionic species such as Cu(II), Pb(II), and Zn(II) ions.

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EXPERIMENTAL SECTION Due to space considerations, the chemicals and protocols used are described extensively in the Supporting Information. Functionalization of Quasispherical Au Nanoparticles with Polyhistidine. An overview of the functionalized nanoparticle preparation is shown in Figure 1, and a detailed protocol can be found in the Supporting Information. Prior to performing SPR sensing measurements, the formation of the polyHis−nanoparticle conjugates via 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHSS) cross-linking chemistry was verified by UV−vis spectroscopy and transmission electron microscopy (TEM) analysis. Creation of NTA-Modified Biochips for SPR Measurements. An overview of the surface attachment chemistry for immobilizing NTA onto a planar bare gold surface is shown in Figure 1. The Au chip was first soaked in 1 mM 1,6hexanedithiol in ethanol for 6 h at room temperature and then rinsed with ethanol and deionized (DI) water. The gold chip was then exposed to 15 mM maleimido-NTA in 100 mM triethanolamine (TEA) buffer (pH 8) solution for a minimum of 12 h. The NTA chip was then sequentially rinsed with DI water and dried under a gentle nitrogen stream. A Biacore 3000 10092

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was employed for all real-time measurements of surface-based interactions at a constant flow rate of 5 μL/min, and 100 mM Tris buffer (pH 7.4) containing 100 mM NaCl was used throughout the investigation. Two out of the available four sample injection channels were utilized to monitor nickel ion interactions with the NTA chip, and the remaining two channels were used for various control measurements. A minimum 1 h reaction time under a continuous flow was applied for all measurements at ppb nickel ion concentrations [prepared by dissolving NiCl2·6H2O salt in 100 mM Tris buffer containing 100 mM NaCl (pH 7.4)] to reach a steady-state Ni(II) ion surface coverage on the NTA chip surface. A relatively slow Ni(II) ion desorption rate [kd = (3.6 ± 0.5) × 10−5 s−1]54 from the NTA surface helped minimize target desorption during a buffer rinse step before injection of polyHis-coated Au nanoparticles for 1 h. More detailed procedures can be found in the Supporting Information, along with the preparation of polyHis-coated SPR chips.

Figure 2. Series of real-time SPR sensorgrams for adsorption of polyhistidine onto a surface Ni(II)−NTA complex. Ni(II) ion concentrations were 1 ppm (4.21 μM), 50 ppm (210.5 μM), 100 ppm (421 μM), 300 ppm (1263 μM), 750 ppm (3157.5 μM), and 1000 ppm (4210 μM). The concentration of polyhistidine subsequently introduced was fixed at 400 nM.

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RESULTS AND DISCUSSION Ni(II) Ion Detection Methodology. A simple two-step assay was used where Ni(II) ions were first exposed to a NTA ligand-modified SPR chip surface, followed by the introduction of gold nanoparticle−polyHis conjugates (see Figure 1). Since both the NTA and histidine moieties have been previously shown to simultaneously have good specificity and affinity toward Ni(II) ions,55 we adapted this model system to demonstrate that the limitations of SPR associated with the sensitive detection of small ionic species can be overcome. This builds on our recent efforts highlighting that relatively larger nanoparticles, 40−50 nm in size, can be used to enhance the SPR signal for the sensing of proteins by a factor of up to 106 compared to that for unamplified SPR detection.49 Prior to the use of NPs for signal enhancement, a series of real-time SPR measurements for the adsorption of Ni(II) ions onto either a NTA- or polyHis-coated chip surface were first performed to verify the binding affinities of both ligands for Ni(II). A plot of fractional surface coverage versus Ni(II) concentration is shown for both chip surfaces in the Supporting Information (Figure S1), from which Langmuir adsorption coefficient values of 1.3 (±0.2) × 109 M−1and 1.7 (±0.2) × 108 M−1 were obtained for NTA and polyHis, respectively. These values are similar to what has been previously reported for Ni(II)−NTA56 and Ni(II)−histidine57 interactions. Note here that the direct detection of Ni(II) ions with no secondary binding probes to further amplify the SPR signal is limited to target concentrations above ∼10 μM. A second set of measurements was then performed to verify the feasibility of the sandwich assay format involving both the NTA and polyHis probes and establish the resulting improvement in the detectable concentration range of Ni(II) ions. Figure 2 shows a series of representative SPR curves obtained for formation of the NTA−Ni(II)−polyHis surface complex. The NTA chip surface was first exposed to Ni(II) concentrations ranging from 1 to 1000 ppm, followed by the introduction of a fixed polyHis concentration of 400 nM; the latter is sufficient to ensure that a high percentage of the available NTA−Ni(II) surface complex sites are occupied. All the SPR curves are normalized with respect to the signal in the negative control channels where 100 ppb Cu(II) ions were exposed to the NTA surface, followed by the adsorption of polyHis. Cu(II) ion adsorption was chosen since Cu(II) ions also exhibit reasonable binding affinity toward either NTA58 or

His.37 The SPR response linearly increased as the Ni(II) ion concentrations increased from 4.2 to 1260 μM, and above 3000 μM no further changes in the SPR response were obtained. The quantitative analysis of the sandwich assay without NPs will be further discussed by comparing the difference in refractive unit (ΔRU) values with that for the NP-enhanced sandwich assays (Figure 5). It can be clearly seen that this sandwich assay format can be quantitative and a Ni(II) concentration of below 1 ppm (4.2 μM) could be easily detected without nanoparticle amplification. This value is similar to or better than nanoparticle colorimetric detection approaches30 but relatively poor compared with ICP-MS.20,21 To regenerate the NTA surface for repeat use, a series of washing steps were developed. This involved rinsing with 8 M urea for 20 min, followed by sequential rinsing with DI water for 30 min, 0.1 M HCl for 20 min, 100 mM Tris buffer containing 100 mM NaCl (pH 12) for 20 min, and DI water for a minimum of 30 min. Next, the chip was washed with 100 mM Tris buffer containing 100 mM NaCl (pH 7.4) prior to any injection of different concentrations of target ions. Finally, the background SPR signal was checked to show a similar level as that on the first use of the NTA chip, and further washing steps were continued until a similar background signal was achieved. The NTA chip could typically be reused a minimum of 5 times before poor recovery of the background signal resulted in the chip being discarded. Nanoparticle-Enhanced SPR Sensing. Next, we investigated the enhancement of the SPR detection signal through the use of polyHis−nanoparticle conjugates and then assessed the specificity of the assay toward Ni(II) ions in the presence of a variety of divalent metal cations. Recently, we showed that with robust control over the functionalization of both nanoparticle and chip surfaces combined with the use of larger quasispherical nanoparticles, ∼40−50 nm in size, large increases in sensitivity could be obtained49 compared to previous efforts with smaller nanoparticles.59 Representative TEM images of the nanoparticles used in this study with an average diameter of 50 (±5) nm are shown in the Supporting Information (Figure S2). As described earlier in the text, a carboxylic acid-terminated alkanethiol was formed on the nanoparticle surface to which polyHis was covalently attached. 10093

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A ∼100-fold molar excess of polyHis compared to NPs was used to ensure a suitably high fractional surface coverage. UV− vis spectra obtained before and after polyhistidine conjugation (see Supporting Information, Figure S3) exhibited a red shift of 11 nm from an original λmax at 534 nm for the stock Au NPs. The prepared NP−polyHis conjugates were suspended in 100 mM Tris buffer solution and typically used within 2−3 days. Prior to quantifying the enhancement in the SPR signal for Ni(II), the concentration of polyHis-coated nanoparticles to be used in the assay was first optimized. This was achieved by performing a series of repeat measurements for a fixed Ni(II) concentration of 100 ppb while the concentration of the injected polyHis−NP conjugates was varied. The resulting SPR curves (Supporting Information, Figure S4) were obtained for NP solutions with optical density (OD) values at 535 nm of 0.8, 1.0, 1.2, 1.4, and 1.6. Above an OD of 1.4, no further improvement in the SPR response was obtained and this concentration (estimated at 0.18 nM on the basis of an extinction coefficient60 of 7.66 × 109 M−1·cm−1) was used for all subsequent NP-enhanced SPR measurements. Various control measurements were also performed to ensure the validity of Ni(II) ion measurements in the parts per trillion (ppt) concentration range. Figure 3 compares a

channel featured nonspecific adsorption of polyHis−NPs onto a NTA chip previously exposed to 1 ppb Cu(II) ions. Figure 4 displays a series of measurements at Ni(II) ion concentrations ranging from 50 ppt to 20 ppb followed by the

Figure 4. Representative SPR sensorgrams for NP-enhanced analysis of Ni(II) ions using a NTA-modified Au chip in conjunction with polyHis−NP conjugates for signal amplification. The Ni(II) ion concentration was (i) 0.05 ppb (0.21 nM), (ii) 0.1 ppb (0.42 nM), (iii) 0.4 ppb (1.68 nM), (iv) 1 ppb (4.2 nM), and (v) 20 ppb (84.2 nM), whereas the concentration of the NP conjugate solution was fixed at 0.18 nM as established in Figure S4 (Supporting Information). The SPR signal was normalized with respect to the reference channel signals (see text for details).

injection of polyHis-coated Au NPs fixed at the optimized concentration established earlier. Analysis of the normalized SPR curves in Figure 4 show that Ni(II) concentrations as low as 50 ppt could be detected, which is a remarkable discovery in comparison to a conventional Ni(II) analysis using various detection methodologies such as ICP-MS with a detection limit of 50 ppt.20,21 A relatively small dynamic range of ppt level (or lower nanomolar concentration) is obtained (see Figure S5 in Supporting Information). This can be improved by use of diluted NP−polyHis conjugates, which may result in loss of sensitivity.49 At concentrations above 1 ppb (4.21 nM) up to 1 ppm (4.21 μM), the SPR response becomes nonlinear but continues to increase as the Ni(II) ion concentration increases (see also Figure 5). It is also interesting to note that a fast adsorption kinetic curve shape taking a very short time to reach a steady-state response by the NPs was observed, which may come from strong binding of NP−histidine conjugates. In order to further highlight the differences in the dynamic range and sensitivity for each of the Ni(II) detection approaches described above, Figure 5 displays a side-by-side comparison of the measured changes in RU as a function of Ni(II) concentration for (a) amplified detection via polyHis− NP conjugates, (b) use of polyHis alone to form a NTA− Ni(II)−polyHis surface complex, and (c) direct adsorption of Ni(II) ions onto a NTA chip surface. Some of the data points were obtained from measurements shown previously in Figures 4 and 2 and in Figure S1b (Supporting Information), respectively. All RU values are normalized with respect to control SPR signals performed simultaneously in both reference channels (further details are given in Supporting Information). The plot in Figure 5 clearly shows the NP-enhanced detection results in a remarkable 106 enhancement in signal compared to Ni(II) ion detection only and ∼104 greater than when polyHis is used. The enhancement is mainly due to refractive index changes induced by the presence of highdensity gold NPs upon the formation of surface polyHis−NPs−

Figure 3. Series of SPR sensorgrams investigating different nonspecific adsorption behaviors when NP-enhanced SPR is used for Ni(II) ion detection. NC 1 is the adsorption of MUA−NP conjugates onto the surface Ni(II)−NTA complexes; NC 2 is the adsorption of MUA− NPs onto the NTA surface in the absence of Ni(II) ions; and NC 3 is the adsorption of polyHis−NP conjugates onto a NTA surface in the absence of Ni(II) ions. For comparison, the SPR response for the specific adsorption of polyHis−NP conjugates after the NTA chip was exposed to 0.05 ppb (0.21 nM) Ni(II) ion is also shown.

representative SPR curve for Ni(II) detection at 0.05 ppb (50 ppt) with three different control variations to indicate the level of nonspecific adsorption onto the NTA-modified Au chip surface: NC 1 is the adsorption of MUA−NP conjugates onto a NTA-modified Au chip previously exposed to 100 ppb Ni(II) ions; NC 2 is the MUA−NP adsorption onto a NTA chip surface in the absence of Ni(II) ions; and NC 3 is the adsorption of polyHis−NP conjugates onto a NTA surface in the absence of Ni(II) ions. It can be clearly seen in Figure 3 that the SPR response for 50 ppt Ni(II) detection is at least 2 times greater than the largest of the three negative control responses. Note that, in a typical NP-enhanced Ni(II) assay measurement, the SPR signal from the two measurement channels was normalized by subtracting the average response from the two control channels. One channel featured the third example described above, where polyHis-NPs are nonspecifically adsorbed in the absence of target ions, and the other 10094

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considerably higher than each of the other divalent species tested, with the assay showing negligible affinity toward Zn(II) ions and a stronger interaction with Cu(II) ions than for Pb(II) ions. Further measurements in the presence of mixtures of different ions were also performed. In Figure 6b the SPR data for 0.05 ppb Ni(II) ion detection alone (plot i) is compared with measurements where two-component mixtures (plots ii−iv) are introduced to the chip surface, involving 0.05 ppb Ni(II) ions plus a second divalent ion species at 10 ppb. In the final plot (v) in Figure 6b, a mixture containing all four of the divalent species used in this study was utilized. Additional data are shown in the Supporting Information (see Figures S6− S8). It can be clearly seen that the SPR response increases in each case compared to the 0.05 ppb Ni(II)-only curve except for the Ni(II) + Zn(II) ion combination. At such low target concentrations there is no direct competition between Ni(II) and other species for NTA surface sites, resulting in an accumulating increase in the SPR response. It is also important to note that if the concentration of the Cu(II) species was reduced to 0.05 ppb rather than 200-fold excess compared to Ni(II) ions used in Figure 6, then no significant change in the SPR signal could be observed. The excellent selectivity for the detection of Ni(II) ions at ppt levels can be attributed to the dual use of specific NTA and polyHis ligands in a sandwich assay format.

Figure 5. Comparison of plots of refractive unit changes as a function of Ni(II) ion concentration for (a) NP-enhanced method, (b) NTA− Ni(II)−polyHis surface sandwich assay in the absence of NPs, and (c) only Ni(II) adsorption onto a NTA chip surface. All experimental conditions are the same as for (a) Figure 4, (b) Figure 2, and (c) Figure S1b in Supporting Information.

Ni(II)−NTA sandwich complexes.49 Also clearly evident is the differences in the dynamic concentration range over which measurements can be performed. For NP-enhanced detection, SPR measurements can be successfully performed at concentrations in the 0.2−1 nM range, compared to micromolar concentrations for the other two cases. One of the major concerns with this sandwich assay approach is the selectivity for Ni(II) ions in the presence of other divalent cations such as Zn(II), Pb(II), and Cu(II) ions, which potentially have reasonable binding affinities toward either the histidine or NTA moieties. Figure 6a shows a series of SPR curves for separate measurements where polyHis−NP conjugates were introduced to chips previously exposed to either a solution of 0.05 ppb Ni(II) or 10 ppb solutions of Cu(II), Pb(II), and Zn(II) ions. Despite the Ni(II) ion concentration being 200-fold lower, the SPR response was

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CONCLUSIONS In this paper, we have demonstrated that ppt concentrations of Ni(II) ions can be detected by SPR via a simple sandwich assay composed of polyhistidine-functionalized gold nanoparticles and a NTA-modified gold chip surface. Importantly, this was achieved in situ without any of the sample preconcentration steps that are often used by established techniques such as ICPMS to achieve comparable levels of sensitivity. This is also one of the first reports showing that NP-enhanced measurements can be successfully applied to the SPR detection of much smaller MW (