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Feb 8, 2017 - the interfacial SERS platform, for detecting trace epinephrine. (EP) in the serum. To realize sensitive and selective detection of EP in...
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Highly Selective and Repeatable Surface-Enhanced Resonance Raman Scattering Detection for Epinephrine in Serum Based on Interface Self-Assembled 2D Nanoparticles Arrays Binbin Zhou,†,‡ Xiaoyun Li,§ Xianghu Tang,†,‡ Pan Li,†,‡ Liangbao Yang,*,†,‡ and Jinhuai Liu†,‡ †

Institute of Intelligent Machines, Chinese Academy of Sciences, Anhui, Hefei 230031, China Department of Chemistry, University of Science & Technology of China, Anhui, Hefei 230026, China § Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201204, China ‡

S Supporting Information *

ABSTRACT: Target analyte detection in complex systems with high selectivity and repeatability is crucial to analytical technology and science. Here we present a two-dimensional (2D) surface-enhanced resonance Raman scattering (SERRS) platform, which takes advantages of the high selectivity of the SERRS sensor as well as the sensitivity and reproducibility of the interfacial SERS platform, for detecting trace epinephrine (EP) in the serum. To realize sensitive and selective detection of EP in a complex system, Au NPs are modified with α,βnitriloacetic acid and Fe(NO3)3 to form the Au NP-(Fe-NTA) sensor, and as a consequence, EP can be rapidly captured by the sensor on the surface of Au NPs and then delivered at the cyclohexane/water interface. More importantly, we synthesized the extremely stable Au NPs (PVP-stabilized Au NPs), where the presence of PVP prevents aggregation of Au NPs during the self-assembly process and then makes a more uniform distribution of Au NPs with analytes at the cyclohexane/water interface, approximately 2 nm interparticle distance between the Au NPs, which has been proved by synchrotron radiation grazing incidence small-angle X-ray scattering (SR-GISAXS) experiments. The selfassembly method not only effectively avoids the aggregation of Au NPs and decreases the influence of the background signal but also can capture and enrich EP molecules in the cyclohexane/water interface, realizing the sensitive and selective detection of EP in complex serum sample. This strategy overcomes the difficulty of bringing nanostructures together to form efficient interparticle distance with simple fabrication and maximum uniformity and also provides a powerful nanosensor for tracing amounts of analyte molecules in a complex system with the advantages of capturing and enriching of target molecules in the liquid/liquid interface during the self-assembly process. Our SERRS platform opens vast possibilities for repeatability, sensitivity, and selectivity detection of targets in various complex fields. KEYWORDS: interface, self-assemble, SERRS, epinephrine, serum

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INTRODUCTION Real target analytes, such as neurotransmitters,1 cancer biomarkers,2 and pollutants,3 are often in complex systems (for example, serum, tissue fluid and contaminated water), making detection of a target a real challenge. The problem is amplified when the signal of the analyte is swamped by the background signal in the complex system. Fortunately, surfaceenhanced Raman scattering (SERS) analytical strategy holds promise in this issue for fingerprint features from molecular vibrations and performing ultrasensitive biological and chemical detection.4−7 Recently, there has been much interest in interfacial SERS platform, such as multiphase detection,8 multiplex detection,9 and interfacial liquid-state detection.10 However, interfacial SERS platform cannot selectively recognize targeted molecules, especially for the molecules with lower Raman cross sections. © XXXX American Chemical Society

Surface-enhanced resonance Raman scattering (SERRS) analytical strategy holds great promise in this issue for modifying the surface of the nanoparticles with specific species that could trap the analyte of interest with sensitivity and keep it proximal to the nanoparticles surface.11 Recently, magnetically assisted SERRS detection of dopamine with selectivity in an artificial cerebrospinal fluid has been reported.12 However, noble metal nanoparticles are difficult to uniformly disperse on the surface of the magnetic material, leading to wide distribution of interparticles distance, and then resulting in irreproducible and incredible SERS signal.13 Compared with traditional approaches to fabricate highly uniform SERS Received: November 28, 2016 Accepted: February 8, 2017 Published: February 8, 2017 A

DOI: 10.1021/acsami.6b15205 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces structures, such as electrochemical deposition,14 break junctions,15 and lithographic,16 the self-assembly techniques are more convenient and less costly to generate a 2D highly ordered nanoparticle array.17,18 Herein, we developed a 2D SERRS platform based on the large-scale self-assembly of Au NPs arrays at the cyclohexane/water interface. In this study, EP was chosen as a model compound for 2D SERRS platform detection, which was an important derivative of a neurotransmitter in the mammalian central nervous system, which played significant physiological roles in the central nervous system. Ultrasensitive and accurate detection of EP levels in biological fluids is beneficial to not only improve the quality of a patient’s life but also reduce the cost of treatment.19 However, many analytical methods suffer from low sensitivity and selectivity in the EP detection.20 For example, highperformance liquid chromatography (HPLC)21 and spectrophotometry22 need derivatization or combination with various detection approaches, so these methods are costly and complicated. Electrochemical methods cannot efficiently detect EP in the serum, because the oxidation potential of ascorbic acid is close to that of EP, resulting in an overlapping voltammetric response.23 Consequently, sensitive and selective detection of EP molecules in serum is still a challenge. Here, the Au NPs are modified with the iron-nitrilotriacetic acid (Fe-NTA), which can be used as SERRS sensor to capture EP efficiently because of the formation of the coordination compound NTA-Fe-EP. This approach offers multiple advantages for detection EP in a complex system, such as high selectivity and ultralow detection limits. Two critical factors can account for such high selectivity. First, EP in the serum is trapped by the SERRS sensor, thus bringing it proximal to the Au NPs surface. Then, Au NPs with the EP molecule form an orderly array on the liquid/liquid interface due to gradual reduction of the interfacial tension,24 providing high sensitivity and selectivity for detection of EP in the serum. The self-assemble nanoparticles arrays functionalized with SERRS sensor (Fe-NTA) demonstrated rapid, sensitive, and selective detection of EP in the serum within 3 min. What is more, compared to previous reports of magnetically assisted SERRS detection of dopamine, our method is simpler without any additional assisted materials. Most importantly, the ordered hotspots generating in a uniform array over a larger substrate is beneficial to obtain high repeatability of SERS signals. The performance of the approach with respect to selectivity, sensitivity, and repeatability will be discussed in detail.

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of sodium citrate solution (1% w/v), and 20 mL of NH2OH.HCl solution (0.02% w/v) were injected into the seed solution separately at room temperature under vigorous stirring. A total of 20 mL of HAuCl4 (0.1% w/v) was slowly added through Teflon tubes via a peristaltic pump under a rate of 1 mL/min. The resulting particles (∼50 nm, ∼5.68 × 1012 NPs/mL) are coated with PVP and hence are well suspended in water. Preparation of SERRS Sensor (Au−Fe−NTA). Fe-NTA is modified onto the surface of the Au NPs according to a strategy described by Kayat.11 Briefly, Fe-NTA is obtainable by dissolving NTA (0.28% w/v) and Fe(III) (0.24% w/v) in water to achieve a molar ratio of 1:1, then 0.1 M NaOH was used to adjust the pH of the solution to 7.0. A total of 1 mL of PVP-stabilized Au NPs was centrifuged and dispersed in the 1 mL of water again. Then, 0.6 mL of freshly prepared Fe-NTA was added to the 0.4 mL of redispersion PVP-stabilized Au NPs and then ultrasonication and centrifugation. After the supernatant fluid was discarded, the nanoparticles were washed by Milli-Q water to remove the uncoordinated Fe-NTA. Forced 2D SERRS Platform at Cyclohexane/Water Interface. A total of 1 mL of aqueous analyte solution, which maybe the serum or other distractions, poured into watch glass with the volume about 6 mL. Then 2 mL of an aqueous SERRS sensor (Au−Fe−NTA) was added into the analyte with intensive mixing. After that, 200 μL of cyclohexane was slowly added to the mixed solution to form a cyclohexane/water interface. Finally, 2 mL of ethanol was rapidly injected into the mixed solution to entrap the SERRS sensor and analyte of interest at the cyclohexane/water interface. Ultimately, the trapped Au−Fe−NTA-EP were self-assembled into highly close packed monolayer 2D SERRS platform over a large area at the cyclohexane/water interface. Surface Treatment of Silicon Wafer for Transferring the 2D SERRS Platform. The silicon wafer was cleaned by immersing in a piranha solution (30% H2O2 mixed with 98% H2SO4 with volume ratio of 1:3) for 2 h and rinsed with ultrapure water. Finally, the silicon wafer was dried by a nitrogen stream. The clean silicon wafer has been used to transfer the interfacial SERRS platform according to the method described by Li and co-workers.26 Briefly, the clean silicon wafer was slowly and carefully close to the interfacial SERRS platform with a small angle and then pulling it slowly from the interface. Measurements. Raman spectra were performed on a Lab-RAM HR800 spectrometer with a 633 nm laser excitation source and recorded with a 3 s accumulation time. The laser focal spot on the metal surface was about 0.9 μm in diameter with a measured power of 0.89 mW. Ultraviolet−visible (UV−vis) spectrum were performed on a Cary 5000 Varian UV−vis spectrometer and recorded at the range 200−800 nm, corrected against the background spectrum, and normalized to zero absorbance at 800 nm. Synchrotron radiation grazing incidence small-angle X-ray scattering (SR-GISAXS) data of the interfacial SERRS platform were obtained at Shanghai Synchrotron Radiation Facility (SSRF) by using the beamline BL16B1 with a focus beam size of 0.4 × 0.6 mm. Fourier transform infrared (FT-IR) spectra were collected on a Nexus-870 spectrophotometer with KBr pellets.

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EXPERIMENTAL SECTION

Reagents. Cyclohexane, anhydrous ethanol, iron(III) nitrate nonahydrate (Fe(NO3)3•9H2O), sodium citrate, and hydrogen tetrachloroaurate (HAuCl4·4H2O) were bought from Shanghai Chemicals Company. α,β-Nitriloacetic acid (NTA) was supplied from Sigma-Aldrich, and epinephrine bitartrate was obtained from Aladdin Company. Millipore water purification was used to produce ultrapure water (18.2 MΩ cm). Preparation of Au Seed. Seed particle solutions were synthesized by citrate reduction of HAuCl4.25 A total of 2 mL of HAuCl4 (1% w/v) in 198 mL of deionized water (198 mL) was heated with a heating mantle in a 250 mL three-necked round-bottomed flask. At the beginning of boiling, 10 mL of aqueous sodium citrate solution (1% w/v) was added quickly under stirring and kept boiling for 20 min. Preparation of PVP-Stabilized Au NPs. A procedure for the prepared PVP-stabilized Au NPs in water is described as follows. A total of 25 mL of seed solution was added into a three-necked roundbottomed flask. Next, 1 mL of aqueous PVP solution (1% w/v), 1 mL

RESULTS AND DISCUSSION Mechanism of the EP Trapped by the SERRS Sensor. Fe(III)-catechol complexes were famous for their exceptional stability and broad ligand-to-metal charge transfer complex in the visible region.27 However, Fe(III)-catechol complexes have shown electronegativity, which is electrostatic repulsion with Au NPs, Fe(EP)2 hold poor SERS performance (Figure S1). Therefore, it is urgently needed to keep Fe (III) proximal to the plasmonic surface. The NTA, citric acid, EDTA are the most widely used and studied organic chelating agents, especially for Fe(III).28 Hence, we studied the effect of these organic chelating agents to Fe (III)-EP. When the different organic chelating agents in aqueous solution were added into the EP solution in turn, they can obtain different ultraviolet absorption bands (Figure S2a). Moreover, the ultraviolet absorption bands B

DOI: 10.1021/acsami.6b15205 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Schematic representation of Au-NTA-Fe used as a SERRS sensor for highly selective and sensitive detection of EP. (a) The chemical equation of Au-NTA-Fe and the EP to form coordination compound. (b1, b2, and b3) UV/vis spectra of Fe-NTA, EP, and Fe-NTA-EP aqueous solution, and the inset image shows the color of the solution, respectively. The dotted line indicates 633 nm, corresponding to the excitation wavelength used for SERRS spectra. (c1, c2, and c3) The corresponding SERS spectra of Fe-NTA (100 mM), EP (100 mM), and Fe-NTA-EP (1 mM) with Au NPs as SERS substrate, respectively.

than the only EP molecule. Third, the Fe-NTA and EP act as bidentate and tetradentate ligands respectively to yield a distorted octahedral geometry having catechol hydroxides at the axial and equatorial positions (Figure 1a), and the captured EP is proximal to the plasmonic surface. Furthermore, we studied the complexation time of the 1 μM EP and SERRS sensor (Au−Fe−NTA). The SERS signals were collected immediately, 5, 10, and 20 min after the addition of 1 μM EP aqueous solution on to the SERRS sensor substrate (Figure S6). The SERS banks at 1335 and 1564 cm−l are corresponding to benzene modes υ3 and υ8a, respectively.30 The primary band at 1482 cm−1, is corresponding to the ring stretching vibration (υ19b) mainly contributed from the stretching of the carbon− carbon bond to which the oxygen is attached.30 The SERS spectrum of the Fe-NTA-EP is similar to its intrinsic Raman signal (Figure S7). The SERS signal is almost the same form as the different reaction time. In other word, the whole SERRS process for SERRS sensor trapping the EP molecule proximal to the Au NPs surface lasted less than 30 s. The fast complexation time between target molecules and SERRS sensor confirmed that our approach has great potential for EP in the complex by using liquid/liquid interfacial self-assembled strategy. Self-Assembled Nanopaticles Array for 2D SERRS Platform. The SERS enhancement is not only dependent on the interaction between analyte and structure but also dependent on the interparticle distance.31 The subnanometer control of the gap size is the important issue to maximize the electric field amplifying Raman signals of the target molecules.32 Therefore, we seek a strategy for detection of the EP molecule that was ingenious combination of SERRS method and advanced self-assembled nanoparticle arrays to

of the Fe-NTA-EP is quite close to the energy of the incoming laser (633 nm) and then Fe-NTA-EP shows the most excellent SERS performance (Figure S2b), owing to the amines holding higher affinities for binding Au NPs, and keeping EP molecule closer to the NPs surface. Furthermore, we have studied the influence of the pH to the reaction (Figure S3). Ultraviolet absorption bands cannot be obtained, when the pH value of the solution is less than 7. Fe-NTA and EP cannot form the target compounds. Moreover, ultraviolet absorption bands blue shift when the pH is higher than 7. In alkaline conditions, the solution might able be to form the iron hydroxide precipitation, and the color of the solution shallow increasing pH value. Therefore, Fe-NTA was modified on the Au NPs to improve the selectivity and stability of the captured EP proximal to the NPs surface under the pH of the solution adjustment to 7. The FT-IR is provided to analyze to the PVP-capped Au and FeNTA-capped Au, which can prove the successful functionalization (Figure S4). What is more, FT-IR is also used to represent the combination of Fe-NTA and EP (Figure S5). In Figure 1, the Au-NTA-Fe was used as a SERRS sensor for highly selective and sensitive detection of Epinephrine. It is worth noting that the intensity of EP SERS signal has been greatly enhanced when the Fe-NTA was used (Figure 1c2− c3).Three essentially factors of the SERS activity can account for this issue.29 First, a new band arose at 600 nm when the FeNTA aqueous solution was added to the EP solution respectively (Figure 1b1−b3), with the color of the aqueous solution immediately changed from light yellow to blue (the inset digital photos in Figure 1b1−b3). This strategy obtains more favorable superposition of the electronic transition of the Fe-NTA-EP with the energy of the incoming laser (633 nm). Second, the cross-section of the Fe-NTA-EP molecule is larger C

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Figure 2. Schematic representation of the detection process of the 2D SERRS platform. Schematic illustrations and optical images of the (a) sample, (b) capture, (c) interfacial self-assemble, and (d) transfer the SERRS platform to a silicon wafer.

control the gap size.17 SERRS detection needs to modify the surface of the Au NPs with Fe-NTP that are capable of trapping the EP and keeping it proximal to the plasmonic surface. However, the Au NPs, which are variations of the classic Turkevich−Frens citrate reduction route,25 are vulnerable to aggregation when its surface modify Fe-NTP, due to the increasing solution ionic strength changes the particle surface charge and then undermines the balance between particles.33 Moreover, their reversible aggregation of Au NPs severely restricts their SERS detection, especially for the repeatability of the signal. Therefore, it is necessary to explore more stable Au NPs in the complex system for SERRS detection. Zhou has reported on an environmentally benign method for rapid synthesis of highly stable Au NPs with the PVP, which is used as a crystal growth inhibitor and solubilizing agent.34 Nonetheless, the size of the PVP-stabilized Au NPs, synthesized by the general procedure, is in the range of 6−17 nm. Recent studies have shown that the SERS intensity is greater for the larger particles while nanoparticles are self-assembled at a liquid−liquid interface.8 Hence, we modified the general seedgrowth procedure to synthesis of PVP-stabilized Au NPs with the average diameter of 48 nm. In comparison with PVPstabilized Au NPs and citrate-stabilized Au NPs, one can observe that the PVP-stabilized Au NPs modified with the FeNTA still exist extremely stable (Figure S8). This result reveals that the PVP plays an important role in balancing the electrostatic repulsion force and the van der Waals attractive force between Au NPs as well as preventing Au NPs from aggregating. Therefore, the surface tension was the most critical issue in the interface self-assemble of PVP-stabilized Au NPs, and EP can be rapidly captured by sensors on the surface of Au NPs, then delivered at liquid/liquid interface because of the decrease of the interfacial tension. Consider an Au NP (p) of radius r which is initially totally in water and is subsequently adsorbed to the water−cyclohexane (w/o) interface. Figures S9 presented the Au NP is ultimately at the cyclohexane/water interface with a contact angle (θ). The interfaces p/w, p/o, and w/o have interfacial tensions γ associated with them. The effect of gravity is negligible because the particle is small enough, and the particle removed from the cyclohexane/water interface requires the energy E (more details in Figure S8):

Calculation results also suggest that the larger PVP-stabilized Au NPs are propitious to interface self-assembly. Figure 2 presents the formation process of the 2D SERRS platform. First, EP in the sample was captured by the added Au−Fe− NTA (Figure 2b), to form the bidentate ligand compounds (Fe-NTA-EP). Then, Au NPs with the Fe-NTA-EP molecule form an orderly array on the cyclohexane/water interface because of gradual reduction of the interfacial tension (Figure 2c).The clean silicon wafer was slowly and carefully close to the interfacial SERRS platform with a small angle and then pulling it slowly from the interface (Figure 2d). The electronic transition of the Fe-NTA-EP is a favorable superposition with the energy of the incoming laser (633 nm); therefore, we can obtain the SERRS platform through the interface self-assembly. Moreover, the geometric morphology of the SERRS platform was clearly observed by SEM. Figure 3a shows that the selfassembled nanoparticles are packed closely in the 2D SERRS platform. The scale of the monolayer Au NPs array is more than 50 μm2 (Figure S10), and the laser focal spot on the

Figure 3. Characterization of the interfacial SERRS platform. (a) SEM image of the interfacial SERRS platform. Inset shows higher magnification image. (b) Typical 2D SR-GISAXS pattern and (c) the corresponding q−I curve obtained by integrating over a rectangle region as indicated by red lines in b.

ΔE = −πr 2γow(cos θ − 1)2 D

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ACS Applied Materials & Interfaces SERRS platform was about 1 μm2. Therefore, the 2D SERRS platform provides enough enhanced area for SERS detection, which is important for reproducibility. An excellent analysis method is not only dependent on the repeatability but also dependent on sensitivity. The interparticles distances play an important role in the SERS enhancement. SR-GISAXS is the most suitable technology to obtain more information on the spatial configuration of the 2D SERRS platform directly on the silicon wafer surface. The X-ray beam holds a focus beam size of 0.4 × 0.6 mm, which was grazing the surface of the sample stage. A typical 2D SR-GISAXS pattern can be observed during the SR-GISAXS experiment (Figure 3b). Figure 3c shows the one-dimensional GISAXS intensity profiles using projection integration by Fit2D, and the integrated area is the rectangle region showing as Figure 3b, the average particles size and interparticle distance were obtained by size distribution P(r) which was fitted by Irena software packages.4 The theoretical basis is shown as follow:

laser spots on the 2D SERRS platform. The main characteristic peak of the EP molecule was at 1482 cm−1; therefore, the standard deviation (σ) of its intensity in each spectrum set was calculated as the indicator of signal repeatability. 10−5 M of EP in solution produced an σ value of 3.6%, and σ values of 10−6, 10−7, and 10−8 M are 6.9%, 9.4%, and 11.6%, respectively. The detection limit of our 2D SERRS platform can reach the single nmol/L (Figure S12). What is more, we taken over a larger area Raman maps of the substrate at all stages-before functionalization with Fe-NTA, signal of the raw AuNPs layer, after fucntionalization, signal of Fe-NTA and with EP, signal of EP (Figure S13). All of these results indicate that the repeatability shows a concentration dependence and a good repeatability of detection signals. Additionally, the other characteristic peak also has a good repeatability (Figure S14). The liquid−liquid interfacial self-assembly techniques drive the Au NPs into the 2D SERRS platform of closely packed spheres creating uniform hotspots and ensuring that the EP captured into liquid−liquid interface. More importantly, the results above show that the different concentration of EP molecules in the solution can be trapped into the 2D SERRS platform with high repeatability. Excess EP can cause high blood pressure or heart failure, so rapid real-time monitoring of EP in serum is of utmost significance. However, it is still a challenge to establish a proper analysis method for detection of EP in the serum. It is not only because of the ultralow detection limits but also a lot of interferences in serum, such as various amino acid, ascorbic acid, glucose, etc. NTA-Fe-EP exhibited the strong SERS signal at 1482 cm−1, while ascorbic acid, glucose, and various amino acids cannot. In this sense, the combination of NTA-Fe and EP is unique (Figure S15). Therefore, the SERS signal at 1482 cm−1 has been chosen to characterize EP in complex systems. In the classic SERS detection, 3 μL of mixed solution (10 μM interference and 10 μM EP) and the SERRS structures (Au -NTA-Fe) are added on the silicon wafer (Figure 5a). The signal of the EP is swamped by some amino acid molecules, which bring difficulties for tracing EP in the complex system just on the silicon wafer. As the liquid volatilization, all molecules (interference and EP) deposition on silicon wafers, so the interference molecules inevitably exist in the laser test range. While Figure 5c shows the self-assembly method, 2 mL of SERRS sensor (Au−Fe−NTA) was poured into a watch glass, where it contains 1 mL of sample (10 μM interference and 10 μM EP), and then 200 μL of cyclohexane was added to the colloidal to form cyclohexane/water interface. Then 2 mL of ethanol was rapidly injected into the mixed solution to entrap the SERRS sensor and analyte of interest at the cyclohexane/water interface and eventually form a 2D SERRS platform. 2D SERRS platform holds great promise in this issue for capture, transport, self-assembly of the EP molecules, greatly reducing the interference of the background signal. As a consequence, the SERRS signal of the EP can be clearly distinguished from glucose or various amino acid interferences (Figure 5b). Figure 5c presents the intensity of 1482 cm−1 (SERRS characteristic spectra of Fe-NTA-EP) through the different analytic strategies. It is noteworthy that more fingerprint peaks should be considered while similar catechol amines in the complex systems (Figure S16). It is easy to get SERRS signal of EP in a complex system by interfacial liquid− liquid nanoparticle self-assemble analytic strategy. In other words, compared with the classic SERS detection methods, 2D SERRS platform can effectively avoid the influence of the

rmin

(q) = |Δp|2 ∑ |F(q , r )|2 V 2(r )NP(r )Δr rmax

where I(q) is one-dimensional SAXS intensity profiles from the spherical Au particles, Δp is the electron contrast density, F(q,r) is the form factor of the sample, and V(r) is the scattering volume. Eventually, the average size of the Au NPs base on the Irena software fitting is 47.7 nm, and the interparticle distance is 2 nm; these results were confirmed with statistical data observed in TEM images (Figure S11). The monolayer and subnanometer gap size of the SERRS platform presented an excellent repeatability and sensitivity in the SERS detection. Repeatability, Sensitivity and Selectivity of 2D SERRS Platform. To examine possible fluctuations in every location of self-assembled array in each interface, Figure 4 shows the SERS characteristic spectra of different concentrations EP analyte, each set of the SERS spectrum was collected from ten random

Figure 4. SERS spectra of trace EP at interfacial SERRS platform. The EP solution has different concentrations of 10−5, 10−6, 10−7, 10−8, and 0 M, respectively. The data set are offset for clarity, and each set contains ten spectra from different runs and illustrates the standard deviation (σ) of peak intensity at 1482 cm−1. The spectra were collected with an integration time of 3 s and an objective lens of LWD 50 × /0.5 NA. Inset: the linear relationship between the intensity of 1482 cm−1 band and the concentration logarithm. E

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nanostructures, which is beyond the scope of the current work. Figure 6 shows that the SERS peaks at 725 and 744 cm−1 were

Figure 6. Comparison of 2D SERRS platform and sol detection of 1 μM EP in the serum. SERS spectra of sole EP (orange line), serum (blue line), 2D SERRS platform detection (red line), and sol detection (black line).

Figure 5. (a) SERS spectra of Au-NTA-Fe in the presence of EP and other various relevant analytes were directly detected on a silicon wafer. (b) SERS spectra of Au-NTA-Fe in the presence of EP and other various relevant analytes in the form of 2D SERRS platform transferred onto the silicon wafer surface. (c) SERS intensity at 1482 cm−1 based on the spectra in (a) and (b). Yellow histogram: the complex system analytes were directly detected on silicon wafer. Red histogram: the complex system analytes were trapped in interfacial nanoparticle arrays and then transferred onto the silicon surface. Each data point represents the average value from three SERS spectra. Error bars equal to the standard deviations.

attributed to the C−H in-plane bending mode of coenzyme A and adenine, 1099 cm−1 due to the C−H plane stretching vibration band. The SERS bands at 1440 and 1582 cm−1 can be assigned to the CH2 bending mode of collagen and phospholipids and CC bending mode of phenylalanine, which were the characteristics of serum peaks (blue line in Figure 6).37 Compared with the Raman spectra of the pure FeNTA-EP sample in Figure 4, the characteristics of Fe-NTA-EP peaks (1275, 1335, and 1482 cm−1)30 can be clearly identified (red line in Figure 6). The details of the characteristic peak are listed in Table S1 (Supporting Information). What is more, more serum samples were used to support the claim (Figure S17). The classical sol method was also used to detect epinephrine in the serum. A total of 1 μL of the EP sensor (Au−Fe−NTA) was mixed with 1 μL of pretreatment serum (contain 1 μM EP) and then dipped on the silicon wafer to form thin solid films for SERS detection. The comparison of the cyclohexane/water interfaces 2D platform and the sol SERS spectrum shows that the main characteristic peaks of analytes and the background signal could be recognized and detected. Furthermore, 2D SERRS platform strategy indicated excellent selective and sensitive detection of EP in the serum in comparison with that of classical sol detection, which has confirmed that the EP sensor can efficiently capture EP in a complex system by self-assembly at the liquid/liquid interfaces, which is further substantiated by multivariate analysis methods (Figure S18). As a consequence, 2D SERRS platform strategy hold more promise in complex systems of detection than the classic sol methods for SERS detection.

background signal in complex systems. In a nutshell, the 2D SERRS platform can provide a closely packed monolayer of nanoparticle functionalized EP sensor, bringing sensitive, selective, and high reproducible detection of EP. Analysis of Epinephrine in the Serum by 2D SERRS Platform. We further examine the capability of the 2D SERRS platform for serum detection, not only confined to the interferences with single-analyte but also with multiple interference. The serum came from CAS Hefei Cancer Hospital, and it was pretreated according to the strategy described by Colantonio.35 Briefly, 3 mL of serum was mixed with 2 mL of cold ethanol and then incubated at 4 °C for 60 min, and then the mixture was centrifuged at 10000 r/min for 15 min. The precipitation was removed, 0.8 M C2H5COONa was used to adjust the pH of the supernatant to 5.7, and then the mixture was then centrifuged at 10000 r/min for 15 min. The supernatant was transferred to a centrifuge tube, and the pH was adjusted to 7 using cold 0.1 M NaOH. This method can effectively remove albumin from the serum. A total of 1 μM EP was added into 1 mL of pretreatment serum, and then 1 mL of the EP sensor (Au−Fe−NTA) and 200 μL of cyclohexane were sequentially added into the serum. Eventually, 1 mL of ethanol was used to induce Au NPs to form the interfacial 2D arrays for the following 2D SERRS platform detection. As previously described the EP sensor has fast capture of the EP molecule before being transported into the liquid/liquid interfaces during the self-assemble process. Moreover, selfassembly of nanoparticles arrays are self-healing, invertible, and easily renewable.36 More EP molecules can be enriched into the cyclohexane/water interfaces by shaking the serum bottle for multiple self-assembly. However, it does not mean that the 2D SERRS platform can be reused. It is worth noting that the key issue for reused substrate is to clean the SERRS sensor of metal

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CONCLUSIONS In summary, we have rationally designed and fabricated a novel 2D SERRS platform that allows highly repeatable, sensitive, and selective detection of EP molecules in serum through interface self-assembly. This SERRS platform can be rapid formation and then realize the efficient concentration of EP at the cyclohexane/water interface. Furthermore, the SERRS platform can easily transfer on the silicon wafer to avoid the interference of background signal in the complex system. The detailed spatial configuration information on the SERRS platform on the silicon wafer has been acquired by SR-SAXS. More importantly, the strategy for detection of EP molecules in serum is general F

DOI: 10.1021/acsami.6b15205 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

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and can be extended to other small-molecular targets by substitution of the EP sensor for other sensors. We believe that this SERRS platform holds great promise for repeatable, sensitive, and selective detection of targets in various complex fields, such as contaminated water, urine, and tissue fluid.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b15205. Optimization organic chelating agents and pH of the reaction; FT-IR spectra characterization; effect of the complexation time on the SERRS signal strength and intrinsic Raman signal; optimization the stability of the Au NPs in interface self-assembly; schematic representation the influence of surface tension to form interfacial SERRS platform; the repeatability of 2D SERRS platform; the selectivity of 2D SERRS platform; analysis of EP in the serum by 2D SERRS platform (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (+86)551-65592420. Tel: (+86)551-65592385. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (21571180 and 21505138), Special Financial Grant from the China Postdoctoral Science Foundation (2016T90590) and the China Postdoctoral Science Foundation (2015M571950), Anhui Natural Science Foundation (1508085MB26), and the Open Project of State Key Laboratory of Physical Chemistry (201405).

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REFERENCES

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DOI: 10.1021/acsami.6b15205 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsami.6b15205 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX