Organic Solvent as Organic Solvent as Internal Standard Internal

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Organic Solvent as Internal Standards for Quantitative and HighThroughput Liquid Interfacial SERS Analysis in Complex Media Fanfan Yu, Mengke Su, Li Tian, Hongyan Wang, and Honglin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00008 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

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Analytical Chemistry

Organic Solvent as Internal Standards Standards for Quantitative and HighHighThroughput Throughput Liquid Interfacial SERS Analysis in Complex Media Media Fanfan Yu,† Mengke Su,† Li Tian,† Hongyan Wang,§ and Honglin Liu*,†,‡ †

College of Food Science and Engineering, Hefei University of Technology, Hefei, Anhui 230009, China College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082, China § Department of Tumor Radiotherapy, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China ‡

ABSTRACT: Liquid-state interfacial nanoparticle arrays for surface-enhanced Raman scattering (SERS) promises a practical, substrate-free, and rapid analysis, but faces a great challenge to develop a batch and uniform fabrication strategy with stable internal standards (IS) due to the difficulties in precisely locating both the IS tags and analytes in the same local structure under the harsh conditions of biphasic liquid interface. Here, we develop a fast batch preparation of self-ordered dense Au nanoparticle (GNP) arrays on cyclohexane/water biphasic interface in 96-well plates with the assist of acetone as the phase-crossing inducer. The acetone can extract the pesticide molecules via a simple dipping sample peels, and can rapidly capture and locate the pesticide molecule into the plasmonic hotspots. Meanwhile, this phase-crossing solvent, acetone itself, generates stable SERS signal and is used as the IS tags to calibrate the signal fluctuation. This platform presents an excellent uniformity with a RSD of 5.9% compared to the RSD of 14.5% without the IS’s correction and a good sensitivity with a LOD of 1 nM thiram. This high-throughput strategy for analyzing pesticide residues at fruit peels reached detection levels of nanograms per square centimeter (ng/cm2). Combined with the 96-well plates, this platform greatly facilitates the self-assembly and multiplex sampling. The self-ordered arrays at two immiscible phases interface evidenced the detection of both the oil-soluble thiabendazole and the water-soluble thiram molecules and also realized the multiplex and two-phase detection of these two pesticides. This platform offers vast possibilities for on-site sensing of various analytes and paves a new way for the quantitative and high-throughput SERS analyzer just as convenient as the Microplate Reader.

Chemo-/bio-sensing of targeted molecules in complex media has a huge demand, e.g. pesticide residues in foods, pollutants in environments, biomarkers in body fluids, and etc. Usually, real samples contain mixture ingredients, interfering with each other and resulting in the difficulty of identification and quantitation. In addition, the analytes often have different solubility in various solvent phases (water, oil, or air), especially for the trace analytes, making the quantitative detection a real great challenge.1 Typically, GC,2 HPLC,3 and GC/MS4 are often used for analyzing of the complex media. Other alternative techniques are supercritical fluid chromatography,5 biosensors,6 immunoassay,7 and etc. Regardless of the sensitivity problem, most of these analyzers are complicated, time-consuming, labor-intensive, and not suitable for on-site and high-throughput measurement. Hence, the demand for developing novel methods for sensitive and time-efficient determination in real samples has urgently risen to date. Given the advantages of high sensitivity, unique molecular fingerprint and nondestructive detection capability, surfaceenhanced Raman scattering (SERS) is considered to be a promising label-free detection method for widespread applications.8-10 However, the bottleneck of SERS as a practical rapid analyzer is not about the Raman devices but about the total solution from SAMPLING to SPECTRUM. Typical SERS analysis often relies on the fixed solid substrates or the nanoparticle sol system, and is always an

inherent near-field enhancement phenomenon which is sensitive to excitation frequency, the detailed local nanostructure, surface roughness, field coupling, and even molecular structures, etc. Quantitative SERS analysis still faces many challenges which are originated not only from the uniformity of nanostructures11 but also from inconsistent molecule adsorption on metal surface and the steric hindrance in nanogaps.12 The use of an internal standard (IS) is considered to be an effective strategy for calibrating signal fluctuation originated from the interference of the samples and measuring conditions, i.e. IS tags can improve the irreconcilable properties problems on sensitivity and reproducibility.11,13 But discrete IS tags on metal surface are just like the targeted molecules facing similar reproducible problems. Promisingly, the uniformity of liquid-state interfacial GNP arrays is much superior to the random aggregates in sols or the fixed arrays on solid surface.1,14 The liquid-state SERS on interfacial arrays is emerging to a prospective sensing platform.15,16 The interfacial highly ordered nanoparticle array is more convenient and less costly, meanwhile owning variability and versatility, which ensures the analytes concentrating into the nanogaps and the stably controlling of the nanogap size. The stability of self-assembled array attributes to the reduced interfacial tension between bi-phase during the adsorption of nanoparticles process.17 Interfacial self-assembly not only can effectively avoid the aggregation of

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nanoparticles and reduce the influence of background signal, but also can quickly capture and enrich the analyte molecules from complex samples on the oil/water interfaces. Nevertheless, the bottleneck for the interfacial SERS analysis is the on-site and fast fabrication of liquid-state highly-ordered arrays. Furthermore, it is a great challenge to precisely locate both the IS and target molecules in the same local structure under the harsh conditions of biphasic liquid interface. Even if one can uniformly manipulate both the IS and target molecules, another problem is that these molecules may compete the adsorption sites on metal surface. Or the IS will be influenced by surrounding factors, again resulting in the fluctuation of SERS intensity.18,19 It has been speculated for a long time how to develop a batch fabrication strategy for liquid-state interfacial arrays with a stable IS tags. Herein, we developed a quantitative and high-throughput liquid interfacial SERS platform for analyzing pesticides in complex media by the use of a simple dip sampling. The organic solvent, acetone, acts as an excellent structure inducer for the fast batch preparation of dense GNP arrays on cyclohexane (CYH)/water biphasic interface in 96-well plates. Both acetone and CYH act as the extraction solvent concentrating the pesticide molecules into nanogaps and producing excellent detection sensitivity. Meanwhile, the acetone itself generating stable SERS signal is used as the IS tags to calibrate the fluctuation of the sample and microenvironment. The advantage of this kind of IS tags is that this phase-crossing solvent is homogenous filled in the nanogaps of GNP arrays and can well tolerate the coexisting of the targeted molecules. Combined with the 96-well plate, this platform greatly facilitates the self-assembly and multiplex sampling, shortens the preparation time, improves the detection efficiency, and promises the high throughput analysis of interfacial SERS. And the self-ordered arrays at two immiscible phases interface also evidenced the detection of both the oil-soluble and the water-soluble analytes. This platform paves a new way for quantitative and highthroughput SERS analysis just as convenient as a Microplate Reader.

EXPERIMENTAL EXPERIMENTAL SECTION SECTION Chemicals and Reagents. Chloroauric acid (HAuCl4·4H2O, 99.9%) was supplied from Nanjing Chemical Reagent Co., Ltd. Hydroxylamine hydrochloride (NH2OH·HCl, 98.5%) was purchased from Shanghai yuanye Biotechnology Co., Ltd. Trisodium citrate (C6H5Na3O7·2H2O, 99%), acetone (C3H6O, 99.5%), cyclohexane (C6H12, CYH, 99.7%), tetrahydrofuran (C4H8O, THF, 99%), ethanol (C2H6O, 99.7%), isopropanol (C3H8O, IPA, 99.7%), tween 20 and thiram (C6H12N2S4, 97%) were bought from Sinopharm Chemical Reagent Co., Ltd. Thiabendazole (C10H7N3S, TBZ, 98%) was purchased from Adamas Reagent Co., Ltd. Among of them, Tween 20 was chemically pure, other chemicals were analytical reagent and used without further purification. Ultrapure water (18.2 MΩ·cm) was produced using Millipore water purification system. The glassware in the experiments was cleaned with aqua regia before use. The fruits used were directly purchased from local supermarket. Characterizations. The morphology, structure and properties of as-prepared GNP sols were characterized by Shimadzu UV-2600 spectrometer, Sirion 200 field-emission SEM and JEOL 2010 high-resolution TEM, respectively.

Raman measurements were conducted with BCA151B Video Microscope equipped with a charge coupled device (CCD) detector in backscattered configuration using a 20×objective. The SERS spectra were acquired under a laser line of 785 nm with a power of 6 mW with 8 s exposure and one time accumulation. Synthesis of Au Nanoparticle (GNP). GNP sols were prepared by a modified sodium citrate reduction method.20 Briefly, 98.9 mL of water and 1 mL of trisodium citrate (30 mg/mL) were added into a conical flask on a hotplate set to 300 ℃ and heated to boil with gentle stirring. Then, 100 µL of 98.5 mg/mL HAuCl4·4H2O was rapidly added and kept boiling under vigorous stirring. After ~1 min the solution started to turn purple. After ~4 min the solution exhibited red color. Cooling the flask on the ice in 7 minutes and the GNP sols with the size of 15 nm was stored in a refrigerator at 4 ℃ for further use. The bigger diameter GNP sols were synthesized via hydroxylamine hydrochloride reduction method.21 First, 1 mL of above GNP sols and 37.4 mL of water and 0.4 mL of 100 mM NH2OH·HCl and 0.4 mL of 10 mg/mL trisodium citrate were mixed in a flask under magnetic stirring during 5 minutes. Then 0.8 mL of 1% (w/v) HAuCl4·4H2O was added into the above mixture for 1 hour. Finally, 64 nm of GNP solution was obtained with carmine color. The resultant solution was centrifuged to collect the products for further experiment. Self-Assembly of Liquid Interfacial Arrays. The array was prepared following the literature.22,23 A volume of 5 mL of an aqueous GNP sols with the addition of 250 µL 0.01% Tween 20 were centrifuged at 5000 rpm for 10 min to collect the bottom pellet. The pellet was diluted into a volume of 100 µL with ultrapure water and then transferred into a pore in 96well plate. 50 µL of CYH and acetone containing targeted analytes was slowly added into that pore in 96-well plate to induce nanoparticle self-assembly from the aqueous phase to the liquid-liquid interface. After approximate 60 s, the nanoparticles were simultaneously self-assembled into a large scale closely-packed mirror film. SERS measurements were performed on the resulting closed-packed mirror film at liquid interface. The acetone dispersed in water phase acted as the IS tags to calibrate the signal fluctuations. Detection of Pesticide Residues at Various Fruit Peels. 1 mM thiram stock solution was prepared and dissolved with acetone as original concentration. Following the gradient dilution into 5×10-4 M, 10-4 M, 5×10-5 M and 10-5 M, fruits in this experiment are thoroughly washed with ultrapure water before use. Then the thin peels were taken from fruits using a knife and formed a uniform piece with a metal punch of ~0.45 cm radius. Then 50 µL of as-prepared pesticide solution was spiked onto fruit peels and naturally dried at room temperature. Subsequently, the peel was immersed into acetone-CYH (v:v=1:1) prior to using for 20 min. Afterward, 50 µL extracted solution was used for the self-assembly GNP array and the direct SERS detection.

RESULTS AND DISCUSSION DISCUSSION Rapid and High Throughput Preparation of Liquid Interfacial Arrays on Two-Phase Interface. The successful synthesis of GNP sols was evidenced by UV-Vis absorbance spectra, SEM and TEM observations, respectively (Figure S1). The UV-Vis spectrum of GNP sols showed an absorbance maximum at 538 nm, implying a diameter of 64 nm,21 which is


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Analytical Chemistry consistent with the SEM and TEM observations, of which the statistical analysis gives a deviation of particle diameter of ±4 nm, indicating a relatively uniform and regular GNP sols. Scheme 1 presents the preparation process of the interfacial liquid-state GNP arrays. The array was transferred onto solid substrate by immersing the silicon wafer in the solution (tilt angle is 5-10 degree) and pulling slowly until it left the interface for further characterization directly (Figure S2). SEM observations evidenced that the dried GNP arrays were more disordered with some large voids because of the solvent evaporation during the drying process, which should make large fluctuations in SERS sensing.22

Scheme 1A and Figure S4). Interestingly, this phase-crossing solvent acetone not only can bring the GNP onto the phase interface, but also can concentrate the molecules both in organic and aqueous phase onto this interface. We speculated that the acetone can be chosen as the inherent IS tags in platform. This kind of IS tags might provide an effective and stable feedback to calibrate the fluctuation of analytes and surrounding environment.27 This point will be detailed discussed in the following text.

Figure 1. Inducer effects on of GNP interfacial self-assembling dynamics. (A) UV-Vis absorbance spectra of the lower sols before (black line) and after employing acetone (blue line) and ethanol (red line) as inducer, respectively, for the interfacial selfassembly of 0.06 nM GNP sols (inset: the scheme of dynamic test). (B) The corresponding self-assembling dynamics by monitoring the absorbance maximum at 538 nm with GNP concentration of 0.06 nM.

Scheme 1. (A) Illustration of the liquid-state interfacial GNP array for detecting targets with acetone as IS tags and the Raman fingerprints of targeted molecules and IS tags. (B) Detailed schematic of GNPs with adjacent molecules. (C) Microscope images of self-assembly process of the large-scale interfacial GNP arrays. (D) Optical observations a on concentration dependence of triplicate interfacial GNP arrays in perforated plate (from up to down: 0.06 nM, 0.3 nM, 0.6 nM, 0.9 nM, 1.5 nM and 3 nM, respectively). (a Each row of two holes were separately photographed for better focus and the whole image was supplied in Figure S5.)

In the two-phase self-assembly process, the organic phase CYH together with the phase-crossing solvent acetone were used for extracting pesticide in complex media. The GNPs array was prepared by the Langmuir–Blodgett film technology.24 The GNPs would gradually self-ordered on the incompatible two-phase interface, interestingly, we found the participation of acetone greatly speeded up this self-ordered process. A large scale nanoarray could be prepared in nearly 60 s at the liquid interface, owning to the total interface free energy minimum (the bottom of Scheme 1A).23,25 The microscope images in Scheme 1C evidenced the rapid assembly of the GNP arrays from micrometer to centimeter scales, and an obvious golden film was shaped by the container with increasing the concentration of GNP sols (Scheme 1D). The whole container used for detecting targets was shown in Figure S3. The prepared GNP arrays in 96-well plate produced reflective effect similar to a mirror when the GNP concentration is larger than 1.5 nM, indicating a collective plasmonic resonance mode.26 This whole assembly procedure is easy to handle and no sophisticated modification between targeted molecules and the substrate was structured here. Meanwhile, acetone itself has distinct fingerprint peaks in interfacial liquid-state SERS measurements (the top of

To evidence the superiority of selected phase-crossing inducer acetone, another common inducer ethanol was examined for contrast (Figure 1). The UV-Vis absorbance of lower sols after the array self-assembling was in-situ monitoring to examine the assembling dynamics about acetone and ethanol. It should be pointed out that the self-assembly system is 500 µL GNP sols and 250 µL organic solvent (the inset of Figure 1A). After the assembling equilibrium, the absorbance of the residual sol system by the use of ethanol as inducer is 0.917, but that with acetone is 0.75, nearly one times lower than that of ethanol (Figure 1A). The as-prepared GNP sols (1.0-fold) made an absorbance maximum of 1.05 at 538 nm, and that after adding acetone is 0.92 at zero time (Figure 1B). We must consider a dead time of ca. 3 s in instrument measurement, indicating a really fast assembly effect induced by acetone. In contrast, the addition of ethanol made an increase of the absorbance at zero time, maybe attributing to the aggregating phenomenon. For 0.1-fold GNP sols, the induced assembly could be finished in 100 s. When the concentration of GNP sols increased to 1.0-fold, this process could be completed in 300 s. Unexpectedly, the bottom part of GNP sols with higher concentrations, such as 5fold, 10-fold and 15-fold sols, showed a gradual increase in absorbance (Figure S6), although a mirror film could be formed in 60 s. The interfacial tension forces regulate and stabilize the self-assembly of GNP arrays. The residual GNPs in bottom sols have great tendency to reach the interface, however, the closely-packed interfacial arrays hinders more nanoparticles entering the interface. But the acetone as inducers and promoters has greatly altered the dielectric properties of GNP surface. In highly-concentrated GNP sols as used in Figure S6, interparticle coupling might contribute to the increase in absorbance of bottom residual GNP sols. Other organic solvents, such as THF and IPA, also showed lower efficiency of induced assembly of GNP sols (Figure S7). The



results indicated a much higher inducible efficiency and a much faster assembly speed. Particle Density Dependence of SPR and SERS Effects. To optimize the sensing performance, a series of amounts of particles were used to produce the self-assembled GNP arrays. The Microplate Reader was used to perform the absorbance mapping images of the self-assembled system in 96-well plate by the use of SPR absorbance at 538 nm (the inset of Figure 2A-D). And the SERS spectra were collected on each corresponding pore in 96-well plate (Figure 2A-D). The non-assembling liquid-state interface showed a complete green imaging (the inset of Figure 2A), and a rather weak SERS intensity of ca. 2270 cnts at 1386 cm-1 (Figure 2A), which should be originated from the ensemble contributions of individual particles in GNP sols. The interfacial self-assembly of the above GNP sols system produced the emerging of an obvious red image (the inset of Figure 2B), indicating strong interparticle plasmonic coupling. Furthermore, along with the GNP sols increased to 0.2 nM (Figure 2C) and 0.6 nM (Figure 2D), GNPs array is more orderly and the green color of center region indicates the precedence distribution of GNP arrays close to the wall of container because of the interfacial tension. Correspondingly, the SERS intensity at 1386 cm-1 increased to 19564 cnts, nearly 9-fold enhancement.

produced the best enhancement capability of nearly 16 times stronger than the non-assembled sols (Figure S8). In our platform, the liquid volume, pore size, interface area and other factors could be all fixed, the above results evidenced that the sensing performance of this dense GNP arrays could be quantitatively regulated just by controlling the number of particles and the target molecule,28,29 suggesting a good reproducibility of this interfacial liquid-state SERS platform. Liquid IS Tags for Reliable Raman Enhancement. To explore the liquid IS tags-advanced reliability of interfacial SERS sensing, we first examined the detection sensitivity by collecting the SERS spectra of thiram molecules with different concentrations (Figure 3A). The Raman bands at 1386 cm-1 and 1430 cm-1 could be assigned to the fingerprint vibrations of thiram and acetone molecules, respectively. The spectral intensities of thiram gradually increased along with the increasing of its concentration from 1 nM to 1 µM, while the fingerprint bands of acetone are almost stable. 1 nM of thiram produced an intensity of ca. 1017 cnts at 1386 cm-1, which could be clearly distinguished (Figure S9), while the band at 1430 cm-1 of acetone produced an intensity of ca. 531 cnts, i.e. the relative peak strength ratio (r1386/1430) is 1.91. In contrast, the blank sample also produced an intensity of ca. 235 cnts at 1386 cm-1 and its r1386/1430 value is 0.44. Naturally, we obtained a rather weak linear relationship with a correlation coefficient R2 of 0.552 between the intensity of thiram and the concentration in the range of 1 nM to 1 µM (Figure 3B, red solid line).

Figure 2. Microplate reader mapping of the SPR peak at 538 nm after the interfacial self-assembly of GNP sols with concentration of 0.06 (B), 0.2 (C) and 0.6 (D), respectively. 0.06 nM of GNP sols without self-assembly was used as a reference (A). The corresponding SERS spectra collected on the interfacial self-assembly arrays in the presence of 1 µM thiram.

It should be noted that the GNP array film tend to be close to the wall of the container, i.e. the mapping images presented a thinner center area, which could be attributed to the liquid surface tension. Nevertheless, larger concentrations of GNP sols can produce perfect, mirror-like assembling arrays, our experiment showed that 3 nM of GNP sols can realize a closely-packed golden film just as shown in Scheme 1D. Although UV-Vis absorbance and mapping image could not be collected because of the strong absorbance, SERS signal

Figure 3. (A) SERS spectra of thiram with concentrations of 1 µM, 200 nM, 100 nM, 10 nM, 5 nM, 1 nM and the blank, respectively. (B) The linear relationship between Raman intensity at 1386 cm-1 and the thiram concentration before and after the IS calibration. (C) Raw spectra of 40 runs in triplicate experiments on interfacial liquid-state SERS analysis. (D) The corresponding IS-calibrated spectra by the use of acetone Raman band at 1430 cm-1 as reference. (E and F) The statistical deviation of Raman intensities at 1386 cm-1 before (E) and after (F) the IS calibration, respectively.


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Analytical Chemistry It should be noted that IS could not correct for non-linear adsorption behavior related to the Langmuir isotherm. Although a relatively higher signal strength at low concentrations of analytes might indicate a submonolayer or monolayer adsorption of analytes on the nanoparticle nanogaps,30 i.e. nonlinear enhancing effect is more significant at low concentrations. It should be clarified that the data in Figure 3B did not obey Langmuir isotherm fitting. Nevertheless, the intensity ratio of the thiram to acetone generated a much better R2 value of 0.932 (Figure 3B, cyan solid line), demonstrating a strong calibration capability of our IS tags. Moreover, the vibration at 564 cm-1 of thiram present a relative decrease in peak intensity, comparing with the normal Raman spectrum of thiram powder (Figure S10), which could be attributed to the S-S bond cleavage of thiram molecules after its adsorbing on gold surface.31,32 In fact, the stronger interaction with the gold surface makes the higher Raman shifts for this vibration. To evaluate the reproducibility, we collected a series of SERS spectra of thiram at 40 random spots on interfacial GNP arrays from triplicate experiments. The raw spectra is presented in Figure 3C, a statistical analysis of the peak strength at 1386 cm-1 showed a relative standard deviation (RSD) of 14.5% (Figure 3E). After peak strength calibrating by the peak at 1430 cm-1, 40 spectra were depicted in Figure 3D and their corresponding RSD value reduced to 5.9% (Figure 3F), implying a good reproducibility of this platform. The above results firstly demonstrated that the as-prepared interfacial liquid-state SERS platform has high sensitivity and excellent sensing performance. Secondly, the sensitivity and reproducibility are usually two irreconcilable parameters. But just by using acetone as reference to calibrate the relative band strength, one can easily distinguish the limit of detection and obtain the reliable peak intensity. The usage of ratiometric peak intensities can help to prevent the influence of complex environmental factors,33 and effectively reduce the variation of samples and fluctuation of the excitation. Multiplex and Multiphase Analysis. In the previous section, interfacial liquid-state SERS platform was successfully realized the detection of single analyte thiram. However, the targeted analytes often disperse in various phases and exist in a multicomponent form, making their distinguishing a great challenge, especially for the trace detection. We speculate that our platform holds great promise in this regard. Owing to the molecularly narrow band spectra, SERS is capable of multi-component detection without separation, including down to single molecule level.34,35 Herein, thiram and TBZ (Figure S11) as two commonly pesticides were examined in both dual-analyte detection and multiphase detection. Briefly, the experiment of dual-analyte detection was performed by dispersing TBZ and thiram in the organic phase (Figure 4A). The total mole number of two molecules is 0.25 nmol, the molar ratio of thiram in total molecules varies as follows: 0, 0.2, 0.4, 0.6, 0.8, and 1.0. The collected SERS spectra of sole thiram, sole TBZ, and the mixtures were present in Figure 4B for comparation. The major SERS peaks were listed in Table S1. The bands at 787, 1010, 1273, and 1562 cm-1 should be assigned to TBZ molecules, and the bands at 564, 1150, 1386, and 1517 cm-1 should be assigned to thiram molecules, indicating a good capability of this interfacial platform for distinguishing dual-analyte

fingerprints. As expected, the decrease in TBZ mole number was accompanied with its weakening of spectral intensity (Figure 4C), triplicate experiments demonstrated a linear relationship of intensity vs mole number for TBZ with a R2 value of 0.8274. As a contrast, the increase in thiram mole number produced the significant enhancing of its spectral intensity (Figure 4C), triplicate experiments also demonstrated a good linear relationship of intensity vs mole number for thiram with a R2 value of 0.9835 (Figure 4D).

Figure 4. (A) Multiplex SERS analysis of both thiram and TBZ dissolved in the organic phase. (B) Interfacial SERS spectra of sole thiram, sole TBZ, and the dual-analyte, respectively. (C) Dual-analyte spectra with varied molar ratios of thiram in total 0.25 nmol molecules of TBZ and thiram, denoted as 0, 0.2, 0.4, 0.6, 0.8, and 1.0 from blue to red lines, respectively. The inset figured out the structural formulas of thiram and TBZ. (D) The relationship between the molar ratios and the peak intensities of thiram (red) and TBZ (cyan), respectively.

Multiphase detection as illustrated in Figure 5A was also carried out to evidence the capability of the interfacial SERS platform. The TBZ molecules were dispersed in organic phase while the thiram molecules was pre-dispersed in aqueous phase. Similar to the dual-analyte experiment, the total mole number of two molecules is 0.5 nmol, the molar ratio of thiram in total molecules varies as follows: 0, 0.2, 0.4, 0.6, 0.8, and 1.0. The interfacial SERS platform could be quickly prepared after mixing the aqueous solution of thiram, the GNP sols, and the TBZ solution in CYH/acetone. All the characteristic bands from each component can be clearly distinguished (Figure 5B). Similarly, the 787, 1010, 1273, and 1562 cm-1 bands assigned to TBZ were easily distinguished from the bands assigned to thiram at 564, 1150, 1386, and 1517 cm-1, and the band intensity of two analytes were proportional to their concentrations. The decrease in TBZ mole number was accompanied with its weakening of spectral intensity (Figure 5C), triplicate experiments demonstrated a good linear relationship of intensity vs mole number for TBZ with a R2 value of 0.9361 (Figure 5D). As a contrast, the increase in thiram mole number produced the significant



enhancing of its spectral intensity (Figure 5C), triplicate experiments also demonstrated a good linear relationship of intensity vs mole number for thiram with a R2 value of 0.9917 (Figure 5D). In brief, all of the above experiments demonstrated the excellent performance of the IS-assisted reliable interfacial SERS sensing and evidenced the powerful analytical capability.

Figure 5. (A) Multiphase SERS analysis of TBZ dissolved in organic phase and thiram dissolved in aqueous phase. (B) Interfacial SERS spectra of sole 0.5 nmol thiram in aqueous phase, sole 0.5 nmol TBZ in organic phase, and the dual-analyte with 0.2 nmol thiram in aqueous phase and 0.3 nmol TBZ in organic phase. (C) Dual-analyte spectra with varied molar ratios of thiram in total 0.5 nmol molecules of TBZ and thiram, denoted as 0, 0.2, 0.4, 0.6, 0.8, and 1.0 from blue to red lines, respectively. (D) The relationship between the molar ratios and the peak intensities of thiram (red) and TBZ (cyan), respectively.

Interfacial SERS Analysis of Pesticide in Complex Matrix. Based on the high Raman activity of the liquid interface platform, we confirm that it has the potential for detecting the analytes on the real sample. In general, the pesticides either adsorb at the surface of the peel or penetrate into the flesh.36 Here, a simple dipping sampling method was used to analyze the pesticide residues on fruit peels (Figure 6A). Briefly, the thin peels were taken from fruits using a knife and formed a uniform piece with a metal punch of ~0.45 cm radius. 50 µL different concentrations of thiram solution was spiked onto fruit peels, after drying at room temperature, the thiram-exposed fruit surface was dipping in CYH/acetone solution for 20 min. Afterwards the solution containing thiram was directly detected by the interfacial SERS platform in a 96well plate. Unique SERS spectra of thiram were collected from the peels of apple and pear, respectively (Figure 6B and D). As increasing the concentration of thiram at fruit peels range from 19 to 1900 ng/cm2, the SERS intensities gradually increased. The plot of the Raman intensity of thiram versus its concentration showed a linear relationship, and the correlation coefficient, R2, was showed as 0.8788. After normalized the

peak by IS tags, a much better linear relationship was found and the R2 was 0.9587 (Figure 6C). Similar results were presented with regard to pear (Figure 6E). The R2 values increased from 0.9663 to 0.9980 after the IS tags calculations. It should be noted that the strength of Raman signals varies at different fruit peels of the same pesticide dosage, indicating the differences in diverse contents of fruit peels and the interaction strength between the pesticide and the peels. These results suggested that the interfacial SERS platform is robust for analyzing the real complex sample, the continuous phase of IS tags could effectively calculated the signal fluctuation originated from surrounding environments.

Figure 6. (A) Schematic drawing for the detection of pesticide residues at fruit peels by liquid interfacial array platform. And the SERS spectra of apple (B) and pear (D) peels spiked with different concentrations of thiram (1900, 950, 190, 95, 19 ng/cm2 and the blank, respectively). (C, E) The plot of the ratio of peak height of thiram (1386 cm-1) and acetone (1430 cm-1) vs. thiram concentration (cyan); the plot of peak height of thiram vs. thiram concentration (red).

CONCLUSIONS The liquid interfacial SERS with optimized nanoparticle array exhibits a good sensitivity, repeatability and rapidity. Multiplex and multiphase analysis evidenced the excellent sensing of both the oil-soluble and the water-soluble analytes at ultratrace levels. Organic solvents as a phase-crossing inducer could extract targeted analytes from complex matrix, which could directly separate and enrich the analyte molecule into nanoparticles gaps for SERS enhancement. Furthermore, as an inherent IS tags, acetone disperses homogeneously in the nanoparticle sols which could effectively calculate the signal fluctuation and realize reliable quantitative detection. The rapid self-assembly promises the high throughput analysis with the smart assist of the 96-well plate, the array is easy to operate and no professional training is required. The simple, convenient, and quick operations provides the possibility of practical application for on-site determination and quantitation


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Analytical Chemistry of various chemicals in complex media. We believe that the platform provides vast possibility for quantitative and highthroughput SERS analyzer just as convenient as the Microplate Reader.

ASSOCIATED CONTENT Supporting Information Characterization of GNP sols; the SEM observation of GNP arrays on Si wafer surface; The picture of self-assembly nanoparticle arrays in the 96-well plate for the detecting targets directly; the normal Raman spectrum of acetone and the SERS spectrum of the blank; the whole image of different concentrations of arrays in the 96-well plate; inducer effects on of GNP interfacial selfassembling dynamics; the comparison of self-assembling efficiency of different inducers; SERS spectrum of thiram using liquid interface platform with the 3 nM GNP sols; the SERS spectra with AuNP arrays display the observable Raman signal of thiram at 1386 cm-1; the normal Raman spectrum of thiram and TBZ powders.

AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (U1632116), the Fundamental Research Funds for the Central Universities, Key Research and Development Project of Anhui Province (1704a07020067), Key Projects of Applied Basic Research of Hunan Province (2016JC2065), the China Postdoctoral Science Foundation (2015M582322 and 2016T90748), and Key Project of University Natural Science Research of Anhui Province (KJ2016A341). Special thanks to the Open Project of State Key Laboratory of Chemo/Biosensing and Chemometrics at Hunan University.

REFERENCES (1) Cecchini, M. P.; Turek, V. A.; Paget, J.; Kornyshev, A. A.; Edel, J. B. Nat. Mater. 2013, 12, 165. (2) Patel, K.; Fussell, R. J.; Macarthur, R.; Goodall, D. M.; Keely, B. J. J. Chromatogr. A 2004, 1046, 225-234. (3) Huang, G.; Ouyang, J.; Baeyens, W. R.; Yang, Y.; Tao, C. Anal. Chim. Acta 2002, 474, 21-29. (4) Nguyen, T. D.; Han, E. M.; Seo, M. S.; Kim, S. R.; Yun, M. Y.; Lee, D. M.; Lee, G. H. Anal. Chim. Acta 2008, 619, 67-74. (5) Norman, K. N.; Panton, S. H. J. Chromatogr. A 2001, 907, 247255. (6) Rekha, K.; Gouda, M.; Thakur, M.; Karanth, N. Biosens. Bioelectron. 2000, 15, 499-502. (7) Wigfield, Y. Y.; Grant, R. B. Environ. Contam. Tox. 1992, 49, 342-347.

(8) Cialla, D.; März, A.; Böhme, R.; Theil, F.; Weber, K.; Schmitt, M.; Popp, J. Anal. Bioanal. Chem. 2012, 403, 27-54. (9) Schlücker, S. ChemPhysChem 2009, 10, 1344-1354. (10) Li, J.-F.; Huang, Y.-F.; Ding, Y.; Yang, Z.-L.; Li, S.-B.; Zhou, X.-S.; Fan, F.-R.; Zhang, W.; Zhou, Z.-Y.; Wu, D.-Y. Nature 2010, 464, 27-27. (11) Bell, S. E.; Sirimuthu, N. M. Chem. Soc. Rev. 2008, 37, 10121024. (12) Zhang, Y.; Huang, Y.; Zhai, F.; Du, R.; Liu, Y.; Lai, K. Food. Chem. 2012, 135, 845-850. (13) Chen, L.; Choo, J. Electrophoresis 2008, 29, 1815-1828. (14) Fang, P.-P.; Chen, S.; Deng, H.; Scanlon, M. D.; Gumy, F.; Lee, H. J.; Momotenko, D.; Amstutz, V.; Cortés-Salazar, F.; Pereira, C. M.; Yang, Z.; Girault, H. H. ACS Nano 2013, 7, 9241-9248. (15) Ma, Y.; Liu, H.; Mao, M.; Meng, J.; Yang, L.; Liu, J. Anal. Chem. 2016, 88, 8145-8151. (16) Böker, A.; He, J.; Emrick, T.; Russell, T. P. Soft. Matter. 2007, 3, 1231-1248. (17) Du, K.; Glogowski, E.; Emrick, T.; Russell, T. P.; Dinsmore, A. D. Langmuir 2010, 26, 12518-12522. (18) Ansar, S. M.; Li, X.; Zou, S.; Zhang, D. J. Phys. Chem. Lett. 2012, 3, 560. (19) Kasera, S.; Biedermann, F.; Baumberg, J. J.; Scherman, O. A.; Mahajan, S. Nano Lett. 2012, 12, 5924. (20) Lee, P.; Meisel, D. J. Phys. Chem. 1982, 86, 3391-3395. (21) Haiss, W.; Thanh, N. T.; Aveyard, J.; Fernig, D. G. Anal. Chem. 2007, 79, 4215-4221. (22) Shin, Y.; Song, J.; Kim, D.; Kang, T. Adv. Mater. 2015, 27, 4344-4350. (23) Xu, X.-W.; Zhang, X.-M.; Liu, C.; Yang, Y.-L.; Liu, J.-W.; Cong, H.-P.; Dong, C.-H.; Ren, X.-F.; Yu, S.-H. J. Am. Chem. Soc. 2013, 135, 12928-12931. (24) Tao, A.; Sinsermsuksakul, P.; Yang, P. Nat. Nanotechnol. 2007, 2, 435-440. (25) Bollhorst, T.; Shahabi, S.; Wörz, K.; Petters, C.; Dringen, R.; Maas, M.; Rezwan, K. Angew. Chem. Int. Edit. 2015, 127, 120-125. (26) Lin, M.-H.; Chen, H.-Y.; Gwo, S. J. Am. Chem. Soc. 2010, 132, 11259-11263. (27) Shen, W.; Lin, X.; Jiang, C.; Li, C.; Lin, H.; Huang, J.; Wang, S.; Liu, G.; Yan, X.; Zhong, Q. Angew. Chem. Int. Edit. 2015, 54, 7308-7312. (28) Constantino, C. J.; Lemma, T.; Antunes, P. A.; Aroca, R. Anal. Chem. 2001, 73, 3674. (29) Ru, E. C. L.; Etchegoin, P. G.; Meyer, M. J. Chem. Phys. 2006, 125, 1102. (30) Liu, H.; Yang, Z.; Meng, L.; Sun, Y.; Wang, J.; Yang, L.; Liu, J.; Tian, Z. J. Am. Chem. Soc. 2014, 136, 5332-5341. (31) Kang, J.-S.; Hwang, S.-Y.; Lee, C.-J.; Lee, M.-S. B. Korean. Chem. Soc. 2002, 23, 1604-1610. (32) Sanchez-Cortes, S.; Domingo, C.; Garcia-Ramos, J.; Aznarez, J. Langmuir 2001, 17, 1157-1162. (33) Guerrini, L.; Pazos, E.; Penas, C.; Vázquez, M. E.; Mascareñas, J. L.; Alvarez-Puebla, R. A. J. Am. Chem. Soc. 2013, 135, 10314-10317. (34) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I. Phys. Rev. Lett. 1997, 78, 1667. (35) Nie, S.; Emory, S. R. Science 1997, 275, 1102-1106. (36) Liu, B.; Han, G.; Zhang, Z.; Liu, R.; Jiang, C.; Wang, S.; Han, M. Y. Anal. Chem. 2012, 84, 255.



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