Article pubs.acs.org/ac
SERS Assay for Copper(II) Ions Based on Dual Hot-Spot Model Coupling with MarR Protein: New Cu2+-Specific Biorecognition Element Yulong Wang,†,§ Zhenhe Su,†,§ Limin Wang,*,†,§ Jinbo Dong,† Juanjuan Xue,† Jiao Yu,† Yuan Wang,† Xiude Hua,† Minghua Wang,† Cunzheng Zhang,‡ and Fengquan Liu*,†,‡ †
College of Plant Protection (Key Laboratory of Integrated Management of Crop Diseases and Pests), Nanjing Agricultural University, Nanjing, 210095, P.R.China ‡ Institute of Plant Protection, Jiangsu Academy of Agricultural Science, Nanjing, 210014, P.R.China S Supporting Information *
ABSTRACT: We have developed a rapid and ultrasensitive surfaceenhanced Raman scattering (SERS) assay for Cu2+ detection using the multiple antibiotic resistance regulator (MarR) as specific bridging molecules in a SERS hot-spot model. In the assay, Cu2+ induces formation of MarR tetramers, which provide Au nanoparticle (NP)− AuNP bridges, resulting in the formation of SERS hot spots. 4Mercaptobenzoic acid (4-MBA) was used as a Raman reporter. The addition of Cu2+ increased the Raman intensity of 4-MBA. Use of a dual hot-spot signal-amplification strategy based on AuNP−AgNP heterodimers combined through antigen−antibody reactions increased the sensitivity of the sensing platform by 50-fold. The proposed method gave a linear response for Cu2+ detection in the range of 0.5−1000 nM, with a detection limit of 0.18 nM, which is 5 orders of magnitude lower than the U.S. Environmental Protection Agency limit for Cu2+ in drinking water (20 μM). In addition, all analyses can be completed in less than 15 min. The high sensitivity, high specificity, and rapid detection capacity of the SERS assay therefore provide a combined advantage over current assays.
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A substantial body of research has focused on these specificelement-based assays for Cu2+ biosensing. Our group has successfully developed an enzyme-linked immunosorbent assay (ELISA) for Cu2+ monitoring using an anti-Cu2+-chelate monoclonal antibody (McAb).14 The method has high specificity and satisfactory sensitivity. However, Cu2+ must be first reacted with a chelating agent to form a complex that is recognizable by the McAb. Prereaction of the sample with the chelating agent greatly extends the detection time and increases the complexity. Guo et al.18 have developed a colorimetric method for Cu2+ detection using papain-functionalized gold nanoparticles. The method is simple, cost-effective and rapid but lacks specificity for Cu2+. In addition, various chemistry-based recognition methods, such as Cu (I)-catalyzed click chemistry,19,20 and the use of other specific coordination reagents7,21,22 have been used in Cu2+ assays. Shen et al.19 and Zhou et al.20 have reported highly selective and accurate colorimetric methods for the detection of Cu2+ based on Cu(I)-catalyzed click chemistry reactions. Conventional click chemistry reactions have long reaction
he fast and accurate detection of metal ions at ultralow concentrations is a critical issue. Copper ions (Cu2+) are an essential trace element and have important roles in various physiological processes.1 In humans, the lack of Cu2+ may affect enzymatic activities and cell metabolism,2 whereas high levels of Cu2+ can lead to serious gastrointestinal disturbance,3 liver or kidney damage,4 and various neurological diseases.5,6 The U.S. Environmental Protection Agency has set a maximum contaminant level of 20 μM for Cu2+ in drinking water.7 Methods for highly sensitive, selective and quantitative determination of Cu2+ in environmental samples are urgently needed to enable the effective assessment of risks to human health. Direct measurements using Cu-signal-based instrumental techniques, namely, inductively coupled plasma mass spectroscopy (ICP-MS),8,9 atomic absorption spectroscopy (AAS),10,11 and electrochemistry,12,13 allow highly sensitive and specific detection of Cu2+. Furthermore, the use of Cu2+-specific recognition elements combined with various signal-transducing techniques is another major strategy used for Cu2+ sensing. Interest in highly specific Cu2+-regulated architectures or biorecognition elements for Cu2+ biosensing, including antiCu2+ antibodies,14,15 Cu2+-dependent DNAzymes,2,16 Cu2+specific single-strand DNA,17 and papain protein18 is growing. © XXXX American Chemical Society
Received: December 24, 2016 Accepted: May 31, 2017
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DOI: 10.1021/acs.analchem.6b05106 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Scheme 1. Schematic Diagram of the SERS Hot-Spot Model, Using MarR As Bridging Molecules, for Cu2+ Detection: (a) Traditional Model Using MarR-Coated AuNPs as Detection Probes and (b) Dual Hot-Spot Model Using Antibody-Modified AgNPs as Signal-Amplification Probes
structures,31,32 have been used for sample identification. Recently, AuNP−AgNP heterodimers have emerged as one of the most efficient and widely used configurations in SERSrelated studies because of their unique combination of properties such as long-term stability, controllable size distribution, good biocompatibility, and excellent SERS enhancement effects.33,34 On this basis, we developed a signal-amplification SERS assay based on a dual hot-spot model (Scheme. 1) using a MarR protein as a Cu2+-specific biorecognition element for rapid and ultrasensitive detection of Cu 2+ . The MarR-based Cu 2+ recognition mechanism was shown to occur through the oxidation of the cysteine residue to generate disulfide bonds between two MarR dimers, which was induced by Cu2+. Compared with the traditional model, the dual hot-spot model involving a signal-amplification strategy based on AuNP−AgNP heterodimers resulted in a high sensitivity. Moreover, the high catalytic efficiency of Cu2+ toward MarR accounted for the fast response of the assay. The SERS assay could be finished within 15 min by the dual hot-spot model or even 5 min by the traditional model if necessary. The proposed dual hot-spot model SERS assay has potential applications in environmental monitoring and clinical diagnosis. Most importantly, MarR is a potentially universal Cu2+-specific biorecognition element and can be conveniently designed to provide various analytical procedures for Cu2+ monitoring.
time (∼2 h) and the reduction of Cu2+ to Cu+ increases the complexity of the assay. Yin et al.7 and Li et al.21 developed cysteine-based ultrasensitive sensors for Cu2+. However, they generally involve long reaction times (∼1 h) or need masks to eliminate interference by other metal ions. In summary, although these Cu2+-regulated architecture-based techniques have all been used for Cu2+ analysis with excellent performance, they face certain challenges, as they can be time-consuming, require complicated sample-pretreatment, or have high cost, all of which limit their wide use. Therefore, it is important to explore new specific Cu2+-regulated architectures or elements with high selectivities and quick responses to enable the development of practical Cu2+ biosensors. The multiple antibiotic resistance regulator (MarR) family of transcription factors, exist as dimers and bind to specific DNA sequences, regulate diverse genes involved in multiple antibiotic resistance, synthesize of virulence determinants, and are involved in many other important biological processes.23,24 During one of our group’s recent studies of the function of MarR in Lysobacter enzymogenes OH11, we found that MarR (one member of the MarR family from Lysobacter enzymogenes OH11, including one cysteine residue) showed unexpected Cu2+ selectivity. Hao et al.25 reported that Cu(II) potentiated MarR (another member of the MarR family from Escherichia coli, including three cysteine residues) derepression in Escherichia coli and induced the dissociation of MarR from its cognate promoter DNA. Therefore, we thought that the MarR protein could be a potential biorecognition element of Cu2+. To the best of our knowledge, no research has been reported on the use of MarR in the field of Cu2+ detection. Surface-enhanced Raman scattering (SERS), which involves significant enhancement of the Raman intensities of molecules adsorbed on rough precious metal surfaces, is a powerful and extremely sensitive analytical technique in chemical and biological analyses.26,27 It is generally thought that a higher SERS efficiency is obtained within two adjacent nanoparticles, which is often referred to as “hot spot”.27 The SERS substrate is important and various substrates, such as shaped particles,28,29 bimetallic nanostructures,30 and functionalized metal nano-
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MATERIALS AND METHODS Materials and Reagents. HAuCl4, trisodium citrate, 4mercaptobenzoic acid (4-MBA), and copper sulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA). AgNPs (10 nm, 1 mg/mL) coated with PVP were purchased from XFNANO Materials Tech Co., Ltd. (Nanjing, China). AntiHis-tag monoclonal antibodies were purchased from Abmart Inc. (Shanghai, China). Other metal salts were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). The MarR protein (existing as a dimer) was prepared in our laboratory (see Supporting Information for details). All reagents were analytical grade and used without further purification. Ultrapure B
DOI: 10.1021/acs.analchem.6b05106 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 1. (A) Colorimetric response of WT MarR (MarRcys)- and mutant (MarRser)-functionalized AuNPs versus Cu2+ concentration and (B) SERS spectra of MarRcys- and MarRser-functionalized 4MBA−AuNPs probe at 5 μM Cu2+.
The signal-amplification probe (McAb−AgNP, 4 μL) was added and the mixture was vibrated for 10 min. The sensing solutions were characterized using an in Via laser Raman spectrometer (Renishaw). Disposable 300 μL single wells were used. SERS spectral collection (500−2000 cm −1) was conducted using a diode laser operated at λ = 532 nm (10 mW incident laser power) coupled with a 50× long objective. All Raman spectra were obtained at an exposure time of 20 s. SERS spectra were collected from three different spots on each sample under identical experimental conditions. A standard curve was established based on the logarithmic relationship between the Cu2+ concentration and the SERS signal intensity. Analysis of Water Samples. Tap water samples were collected from our laboratory and commercial bottled water samples (Nongfu Spring brand) were purchased from a local supermarket (Nanjing, China). River water samples (Xuanwu Lake, Nanjing, China) were filtered with 0.45 μm cellulose acetate filters and the pH was adjusted to 7.0. Briefly, water samples (195 μL) with 40 μL of the detection probe were spiked with standard Cu2+ solutions (1 μL) of concentration 200 and 500 nM. The signal-amplification probe (4 μL) was mixed with the solution. The solutions were analyzed using Raman spectroscopy.
water with an electrical resistance greater than 18.2 MΩ was used for all experiments. Instrumentation. UV−vis spectra were measured on a multifunctional microplate reader SpectraMax M5 (Molecular Devices, USA). SERS measurements were performed with an inVia laser Raman spectrometer (Renishaw, UK). Solution vibrations were imparted by a microplate shaker (Thermo, USA). Transmission electron microscopy (TEM) images were obtained with a JEM-1200EX microscope (JEOL, Japan). Inductively coupled plasma optical emission spectrometry (ICP-OES) was performed with a PerkinElmer Optima 8000 series instrument (Waltham, MA, USA). Preparation of the SERS Detection Probe (MarR− 4MBA−AuNP). The MarR proteins exist as dimers35 and can be used as a whole to label the AuNPs through electrostatic interactions between the protein and the surface of the AuNPs.36 AuNPs with an average diameter of 20 nm were prepared using a slightly modified version of a reported citratemediated reduction of HAuCl4.37 Briefly, 0.01% HAuCl4 solution (100 mL) in a 250 mL conical flask was brought to boil with vigorous stirring, and then 1% trisodium citrate solution (1.8 mL) was added under constant stirring. After the solution color changed to red (about 45 s), it was boiled for another 5 min, the heating source was removed, and the colloidal Au solution was stirred for 10 min. The solution was stored in a dark bottle at 4 °C. Next, 8 μL of 1 mM Raman reporter molecule (4-MBA) was added to 2 mL of the prepared AuNP solution, and the mixture was shaken vigorously for 4 h. The pH of the MBA−AuNP was adjusted to 8.5 with 0.2 mol·L−1 K2CO3, and 15 μL of MarR (4.75 mg mL−1) was added under vigorous stirring. The mixture was incubated at room temperature for 2 h and then centrifuged at 12 000 rpm for 15 min. After removal of the supernatant, the sediment was resuspended in 0.01 M phosphate buffer (pH 7.0, 200 μL) and stored at 4 °C. Preparation of the SERS Signal-Amplification Probe (McAb−AgNP). Anti-His-tag monoclonal antibodies (1 mg mL−1, 15 μL) were added to an AgNP solution (pH 8.5, 2 mL) with rapid stirring, followed by incubation for 1 h. The mixture was stabilized by the addition of bovine serum albumin (1% w/ v) and stirred for 5 min. After incubation for 1 h, the mixture was centrifuged twice at 12 000 rpm for 15 min. After removal of the supernatant, the sediment was resuspended in 0.01 M phosphate buffer (pH 7.0, 200 μL) and stored at 4 °C for further use. SERS Determination of Cu2+. In a typical experiment, Cu2+ solution (1 μL, various concentrations) was added to 0.01 M phosphate buffer (pH 7.0, 195 μL) with 40 μL of the detection probe (MarR−4MBA−AuNP), and the mixture was vibrated by a shaker at 200 rpm for 2 min at room temperature.
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RESULTS AND DISCUSSION Principle of the SERS Assay. Scheme. 1 illustrates the concept of the SERS assay for the detection of Cu2+. In our proposed approach, MarR and a Raman reporter (4-MBA) were simultaneously used to functionalize the AuNPs. The MarR was used as a bridging molecule and Cu2+ induced the formation of MarR tetramers by oxidizing the cysteine residue to generate disulfide bonds between two MarR dimers, resulting in aggregation of the MarR-coated AuNPs and generation of electromagnetic hot spots to enhance the SERS signal of the Raman reporters (4-MBA). A signal-amplification probe (AgNPs coated with anti-His-tag antibodies) was introduced; it combined with MarR (C-terminal His tag) to form dual hot spots and network of AuNP−AgNP heterodimers (Scheme 1b).The Raman signal was greatly strengthened as a result of high electromagnetic enhancement in the narrow gaps between the metal nanoparticles of the formed network of AuNP−AgNP heterodimers. The relationship between the concentration of Cu2+ and the SERS intensity can be used for sensitive, specific, and quantitative determination of Cu2+ at low concentrations. We mutated the cysteine on MarR to serine for colorimetric and SERS assays to further investigate the possible mechanism of Cu2+ recognition by MarR. In a typical colorimetric assay, we modified the AuNP with wild-type MarR (MarRcys) and the mutant (MarRser), respectively. Figure 1A shows that MarRcys C
DOI: 10.1021/acs.analchem.6b05106 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 2. (A) UV−vis spectra of AuNP−4MBA and AuNP−4MBA−MarR at 5 μM Cu2+. (Inset: photograph of the corresponding color changes.) (B) SERS spectra of AuNP−4MBA and AuNP−4MBA−MarR at 1 μM Cu2+ with 4 μL of the signal-amplification probe. TEM images of the SERS assay probe in the (C) absence and (D) presence of Cu2+.
has a more sensitive response to Cu2+ than does the mutant (MarRser). Previous reports mentioned that Cu2+ could oxidize the cysteine residue to generate disulfide bonds between two MarR dimers, inducing tetramer formation25 and leading to aggregation of MarRcys-coated AuNPs, as well as a color change. However, the MarRser-coated AuNP changed color at Cu2+ concentrations above 20 μM. This is mainly because the functional groups in the MarR protein (e.g., carboxyl, amino, and hydroxyl) can interact with Cu2+,18 and charged, aromatic, and hydroxyl-containing amino acids can also interact with metal ions through noncovalent interactions.38,39 However, the bonding strength with Cu2+ is not as high as that of a cysteine residue. In addition, we used our proposed SERS method to verify these results. Figure 1B shows that the Raman signal response of the MarRcys-functionalized probe to Cu2+ was stronger than that of the MarRser-functionalized probe. Treatment with Cu2+ had almost no effect on the Raman signal response of the MarRser probe. These results suggest that the Cu2+-induced aggregation may be caused by the strong catalytic efficiency of Cu2+ in the oxidation of the cysteine residue of MarR. Viability of the Design for Detection of Cu2+. UV−vis spectroscopy was used to investigate the Cu2+-induced aggregation of monodispersed AuNP−4MBA−MarR. Figure 2A shows that AuNP−4MBA−MarR aggregated immediately in the presence of Cu2+, resulting in a significant decrease in the absorbance at approximately 520 nm and significant broadening and red shifting of the absorption spectrum (curve a), along with a red-to-purple color change (photograph a). The color of the AuNP−4MBA solution was red before and after Cu2+ treatment (photographs c and d), and the absorbance peak at 520 nm (curves c and d) was stable, indicating that AuNP aggregation was mainly caused by interactions between MarR and Cu2+. The SERS spectrum of the monodispersed MarR-coated AuNPs after Cu2+-induced aggregation was also examined. Figure 2B shows that the 4MBA−AuNP probe gave a weak
SERS signal before and after Cu2+ addition, whereas the MarR−4MBA−AuNP probe had a strong SERS intensity, which increased after Cu2+ treatment. These results further indicate that interactions between MarR and Cu2+ play a decisive role in the formation of AuNP aggregates, which can be used in the hot-spot SERS model to greatly enhance Raman scattering. TEM images showed that the SERS probes were well dispersed in the absence of Cu2+ (Figure 2C); aggregation of the probe occurred upon addition of Cu2+ (Figure 2D). The change in SERS intensity corresponded to AuNP aggregation induced by Cu2+, therefore, Cu2+ detection using this strategy was feasible. Condition Optimization. The experimental parameters (the volume of the detection probe and signal-amplification probe, pH, and incubation time) that could affect the analytical performance and results were optimized. The recognition element (MarR) and signal molecule (4MBA) were coated on the AuNPs. Therefore, the volume of the detection probe (MarR−4MBA−AuNP) is one of the most important parameters that determines the assay sensitivity. The ratio of the Raman intensities recorded in the presence and absence of Cu2+, I/I0, was chosen as the evaluation criterion. The concentration of Cu2+ was set at 1 μM, and the volume of the detection probe was varied from 10 to 50 μL. Figure S1 shows that the highest signal ratio was obtained at a volume of 40 μL; this corresponded to the strongest target recognition and signal response. Therefore, the optimum volume of the detection probe was 40 μL, which was used in all subsequent experiments. The change in volume of the signal-amplification probe (McAb−AgNP) was also investigated. Figure S2 shows that the signal ratio increased gradually with increasing signalamplification probe volume up to 4 μL, and then decreased at higher volumes. Too much signal-amplification probe resulted in high background and a low volume did not contribute to signal amplification. Therefore, the optimum D
DOI: 10.1021/acs.analchem.6b05106 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 3. SERS spectra for Cu2+ sensing using (a) the traditional model and (b) the dual hot-spot model; (c) linear calibration curve for the dual hot-spot model for Cu2+ concentrations in the range 0.5−1000 nM; and (d) specificity of the dual hot-spot model SERS assay for Cu2+ over other competing metal ions. I/I0: The ratio of the Raman intensities recorded in the presence and absence of metal ions. Raman signal was measured based on the peak area at 1583 cm−1. Concentrations of Cu2+ and other metal ions were 0.1 and 0.2 μM, respectively. Error bar at n = 3.
Table 1. Various Sensing Strategies for Cu2+ Detection
a
probe element
analytical method
linear range
LODb
antibody papain protein Click chemistry pyoverdine E2Zn2SODc cysteines MarR protein
ELISA colorimetric assay colorimetric assay fluorescent assay fluorescent assay colorimetric assay SERS assay
4−127 μM N/Aa 0.5−10 μM 0.2−10 μM 0.1 μM−1 mM 30−90 μM 0.5−1000 nM
500 nM 200 nM 250 nM 50 nM 10 nM 2.23 μM 0.18 nM
time (min)
interference ions
ref
>90
none Hg2+, Pb2+ none none none none none
14 18 19 40 41 42 this work
N/Aa 20/60 30 >60 15
