Ultrasensitive Plasmonic Sensor for Detecting Sub-PPB Levels of

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An Ultra-Sensitive Plasmonic Sensor for Detecting Sub-PPB Levels of Alachlor Elad Segal, Emir Haleva, and Adi Salomon ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b02164 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 4, 2019

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ACS Applied Nano Materials

An Ultra-Sensitive Plasmonic Sensor for Detecting Sub-PPB Levels of Alachlor Elad Segal¥, Emir Haleva¥, and Adi Salomon¥* ¥ Department

of Chemistry, Institute of Nanotechnology and Advanced Materials (BINA), Bar-

Ilan University, Ramat-Gan 5290002, Israel

ABSTRACT:

Alachlor is a globally used pesticide that has a significant impact on our worldwide ecological system owing to its penetration into soil and drinking water; thus, it can greatly affect human health. Owing to the increasing demand for sensor optimization, along with significant advances in nanotechnology, we present a one-micron scale label-free optical sensor, based on a plasmonic structure for detecting a minute amount of this highly toxic pesticide. The detection is achieved by surface-enhanced Raman scattering; our plasmonic device is designed systematically to overlap energetically with the Raman laser in order to maximize the signal enhancement. Furthermore, the studied device differs from conventional plasmonic systems because the electromagnetic field is confined to a flat surface between triangular nano-cavital features with a specific orientation, providing an area for deposition of molecules. An enhancement factor of ~106 allows for detection of trace amounts of alachlor, down to 0.4 partsper-billion and to differentiate it from a similar pesticide. Consequently, our device could potentially serve as a general, low-cost analyte sensing detector. The plasmonic sensors were characterized by linear transmission measurements, cathodo-luminescence, and surfaceenhanced Raman spectroscopy.

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KEYWORDS: SERS, pesticides, alachlor, plasmonic nano-structures, nano sensors, water contamination.

1. INTRODUCTION

Following significant advances in technology as well as changes in ecological regulations, there is an increasing need to detect small hazardous molecules such as ferbam, tiabendazole, chlorpyrifos, methyl parathion, picric acid, and others that have a deleterious impact on human health and/or ecological systems. Specifically, alachlor is an intensively used pesticide that significantly affects the human population and the ecological system worldwide. Alachlor is used in immense quantities as a herbicide for crops such as corn, soybeans, and potatoes.1 Surprisingly, only ~0.1% of the applied pesticides actually reach their pest target, meaning that most of these chemicals and their metabolites eventually leach into soil and ground water easily, making them even more spreadable and dangerous.2 Alachlor is not only considered an irritant for skin and eyes, it can also damage the liver, kidneys, and spleen, and it was found to be carcinogenic.3–5 In fact, it is so hazardous that the United States environmental protection agency (EPA) has set its goal to have a maximum contaminant level of zero in drinking water. This is significant, since the maximum level that is still considered not harmful for humans is only two parts-per-billion (ppb).6 The most common methodologies for detecting such substances is gas chromatography (GC) combined with mass spectrometry (MS), a relatively expensive and demanding method, with a limit of detection of ~0.2 ppb.1,3,7–10 Thus, there is no debate regarding the urgent need for accurate, yet rapid, simple, and cheap methodologies for detecting toxins and hazardous molecules in our food and water. 2,11–13


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Since its discovery in the mid-1970s,14,15 surface-enhanced Raman spectroscopy (SERS) has been increasingly used in many fields, including medicine, food/water quality control, and homeland security. 16–22 SERS is sensitive to ultra-low concentrations of various analytes, it can rapidly determine the presence of toxic residues, and it can be easily integrated into industrial tools. Being a nonlinear optical technique, SERS enhancement is often achieved by coupling to surface plasmnons (SPs), which give rise to enhancement of the electromagnetic (EM) field at proximity to the surface. By alignment of the SPs modes to the excitation laser frequency, one can maximize the enhancement, because both the incident beam, Einc, and the scattered one have a similar frequency and a quadratic enhancement can be achieved.23–28A common strategy to enhance the EM field is to fabricate metallic nanostructures (cavities or particles) with sharp edges, in proximity to each other, to achieve a deep wavelength confinement of the EM field.19,29–33 However, such a fabrication or synthesis, which involves using very sharp edges with a low radius of curvature is highly demanding and usually results in a truncated shape. In addition, a nano-sized gap between two or more structures, which is used to form a 'hot spot', is sometimes problematic in itself because molecules in a liquid phase cannot easily penetrate/diffuse into the effective “enhancing” gap region due to the high surface tension of the solution. Pronounced Raman enhancements have been reported in the literature; where many of these reports pertain to big molecules with high absorption cross-sections, such as rhodamine and crystal violet

21,22,34,35.

Detection of toxins molecules with SERS was reported

by several groups too.20,36–41 To the best of our knowledge, the lowest reported limit of (alachlor) detection by SERS is 5×10-9 M, using silver nanoparticles in a specific arrangement such as flowers/flakes, demanding state-of-the-art nano-synthesis.7


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Herein, we improve the detection by an order of magnitude using a sub-micron plasmonic structure for high-resolution SERS detection. This plasmonic structure is based on triangular cavities milled in a thin silver film, arranged in such way that the EM field is confined between the triangular bases, onto the flat surface, rather than at the triangular tips (bow-tie) structure.42 The energy of the spatially confined mode is determined by the distance between the cavities and it can be tuned throughout the optical regime.42,43 We show that this plasmonic device concept can serve as a potential SERS sensor. Specifically, it can be used to sense alachlor and reach a detection threshold of about 0.4 ppb. Such a detection limit does not diminish the sensitivity of our plasmonic sensor, and we further generalize the plasmonic detector concept by maximizing the number of 'hot spots' per given area and by covering the device with a thin layer of silica for robustness and future functionalization. In the last part of our manuscript we also show that we can distinguish between very similar molecules, at the sub-ppb level.


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2. MATERIALS AND METHODS

2.1. Materials. All chemicals and reagents were of analytical grade and were used as received. “Hellmanex” cleaning solvent, the pesticide (herbicide) alachlor, 2-chloro-N-(2,6diethylphenyl)-N-(methoxymethyl) acetamide (1000 µg/ml, in methanol), and the pesticide metolachlor,

2-Chloro-N-(2-ethyl-6-methylphenyl)-N-(-2-methoxy-1-methylethyl)

acetamide

(neat, further solvated in methanol) were purchased from Sigma-Aldrich (Israel). Pure methanol (ACS grade) was purchased from J. T. Baker Company (the Netherlands), and pure ethanol (99.9%) was purchased from Romical, Ltd. (Israel).

2.2. Preparation of a plasmonic device for SERS 2.2.1. Cleaning of glass substrates. To achieve high smoothness and quality, the substrates were cleaned by dipping them into a diluted aqueous solution of “Hellmanex III” cleaning solvent (1:100), followed by mild bath-sonication at 30° C for 20 minutes. Afterwards, the substrates were washed thoroughly with doubly distilled water (18.2 MΩ) and ethanol, and dried by a continuous stream of nitrogen. 2.2.2. Deposition of smooth and opaque silver film. The samples were covered by a smooth and thin (280 nm) silver layer using a direct current (DC) Magnetron sputtering tool (“PENTA Sputter") with a load lock. Before sputtering, the samples were pre-cleaned in mixed Ar/O2 plasma to remove organic contaminations and to promote better adhesion of the sputtered films. During sputter deposition, the sample holder was rotated to improve the sputtering uniformity. The working pressure was 3×10-3 torr, and the deposition rate was ~1 Å/sec.


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2.2.3. Design and milling of plasmonic nano-cavities by using focused ion beam (FIB). The glass/silver samples were milled by a focused ion beam (FIB, Helios NanoLab DualBeam 600, FEI). The sub-units that comprise the nano-structures are isosceles triangular nano-cavities with a typical side-length of 215 nm and a base length of 200 nm. We will refer to a “cross” sensor system as one that comprises four triangular nano-cavity sub-units, where both triangular pairs face each other to form an overall cross-like structure. In addition, we will refer to a “hex-tri” sensor system as a hexagonal array of triangular nano-cavities with hexagonal periodicity. For the cross sensors, the internal distance between the triangular bases varies from 400 to 900 nm (base to base). For the hexagonal-triangular arrays, the periodicity also varies from 400 to 900 nm (center to center). Prior to the milling procedure, parameters such as the beam current and the number of passes were calibrated in order to obtain nano-cavities with high quality. The currents and voltages used for milling range from 10 to 50 pA and ~30 kV, respectively. Furthermore, in order to create a functional sensor and to maximize the number of sensor-analyte contact events, the solitary cross/hexagonal array were duplicated to form midsized arrays (6×4 matrices). We arranged these arrays to cover an overall area of ~250×250 µm2.

2.2.4. Deposition of a thin SiO2 layer to prevent oxidation/aging. In order to extend the shelf life of our plasmonic device and its durability, and to prevent oxidation of the silver layer, we deposited a thin 3 nm layer of silica on top of the silver layer. This process was done by electron-beam evaporation (besTec, Germany) at 2.4×10-5 mbar and a current of 1 mA.

2.3. Raman measurements, spectral acquisition, and sensor analysis. Prior to analysis, solutions of analytes in methanol (100 µL) with different concentrations (4×10-3 to 4×10-10 M) were drop-casted on the substrates and left to dry under ambient conditions. Spectra


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were obtained using a Renishaw InVia Raman microscope, integrated with a Leica DM2500 M microscope (Leica Microsystems) having an objective lens with ×50 magnification (N.A. = 0.75). All samples were excited by a λ = 514 nm laser with 0.16 mW power. For the linear concentrations range (0.4-2 nM), the output laser power was set to 1.80 mW, due to the minor changes between concentrations, which are more delicate to probe. We targeted our micron-sized sensors on the substrates, ensuring that the regions of interest were acquired; “coffee-ring” regions or bulk bulges were avoided. Each spectrum represents an average of ten different spots, each of which was repeated twice, with an acquisition time of 10 seconds. Two reference spectra were also acquired from clean glass and a thin silver film without plasmonic structures, both taken under the exact same parameters with the same concentration of alachlor.

2.3.1 Limit of detection calculation (LOD). The limit of detection (LOD) was calculated using the following formula: LOD=F•SD/b, where F is the confidence level factor (with a value of 3, when the confidence level is 90%), SD is the residual standard deviation of the linear regression, and b is the slope of the regression line. 2.3.2 Estimation of the number of probed molecules under Raman/SERS configurations. The maximum number of alachlor molecules (see SI section 1.2) which can contribute to the SERS signal has been estimated to be Nmax = 1.02x109 taking into account an active SERS area of ~0.33 μm3 (SI section 1.2). Considering a concentration of 4×10-9 M alachlor (100 μl drop), the total number of molecules over the whole sensor is ~2.22x1011, two orders of magnitude bigger than Nmax. It means that the sensor sites might be extensively overloaded. However, when the total number of molecules approaches ~Nmax (e.g., ~2.22x1010 molecules for a concentration of 4×10-10 M), it is unlikely to find a site which is overpopulated.


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2.4. Characterization 2.4.1. High-resolution scanning electron microscopy (HRSEM). Images were obtained by HRSEM as part of the integrated FIB system (Helios NanoLab DualBeam 600) to ensure the quality and morphology of our device. All images were taken at high resolution with a fieldemission gun (FEG). The emission current was set to 0.69 nA in all measurements. The secondary electrons were detected by a through-the-lens detector.

2.4.2. Linear optical measurements and spectral acquisition. The transmission spectra of the nano-structures were acquired using a bright field inverted microscope (Olympus IX83). The samples were illuminated by non-polarized collimated light. The transmitted light was directed to a spectrograph (IsoPlane SCT-320, Princeton Instruments) and then detected by a high quantum efficiency CCD camera (PIXS1024b) having an objective used with ×20 and ×40 magnification (N.A. = 0.25 and 0.6, respectively). The grating used for acquiring the spectra was 50 grooves/mm (density), and the blaze wavelength was 600 nm (Princeton Instruments). The spectra were always collected under the same conditions (illumination intensity, objective, and input power). All acquired spectra were normalized to the reference glass spectrum taken using the same parameters.

2.4.3. Cathodo-luminescence (CL) measurements. CL measurements were performed on an Attolight Rosa 4634CL microscope, which tightly integrates an achromatic reflective lens within the objective lens of a field-emission gun scanning electron microscope (FEG-SEM). The focal plane of the light lens matches the FEG-SEM optimum working distance. CL was spectrally resolved with a Czerny-Turner spectrometer and measured with a UV-Vis CCD


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camera. The acceleration voltage and emission current of the electron beam were 7 kV and 20 nA, respectively. The integration time per spectrum was 100 milliseconds.

3. RESULTS AND DISCUSSION Our basic plasmonic device comprises four triangular nano-cavities milled in a thin silver film, as shown in Figure 1a. Within such a device, the hot-spot is confined to the flat region between the cavities, and its resonant modes are determined by the distance between the cavities.44 In such a configuration, the area between the triangular cavities allows for deposition of small and large molecules. The molecular structure of the chosen hazardous herbicide, alachlor, is shown in Figure 1b.

Figure 1. (a) A schematic illustration of the plasmonic sensing device - a glass substrate covered by a smooth silver layer. Triangular cavities with a side length of 200 nm have been fabricated by FIB to align with the Raman excitation laser. “d” is the distance between the bases of the cavities. The upper-left images were taken by HRSEM, whereas the two on the bottom-left are illustrations. The red region in the bottom left image illustrates the confined hot spot onto the flat surface. (b) The 3D chemical structure of the chosen analyte, alachlor.


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Figure 2 depicts the SERS fingerprint region of alachlor sensed on our cross sensor, with a concentration of 4 ppb (4×10-9 M). Alachlor`s vibrational modes shown in Figure 2, consists of: C-Cl stretch (650 cm-1), C-N stretch (750 cm-1), benzene ring “breathing” (1000 cm-1), benzene ring vibrations (1038 cm-1), C-C stretches (1127 cm-1), C-O-C stretch (1283 cm-1), CH2-CH3 deformations (1446 cm-1), benzene ring stretch (1600 cm-1), and the amidic C=O stretch (1650 cm-1).45 The detailed peak assignments of alachlor`s vibrational modes (reference on glass) are presented in Figure S1 in the supporting information (SI). Of note is the vibration range of 1020– 1055 cm-1, belonging to specific positions of ortho substitutions on the benzene ring (positions 2, 6).45 Within this range, specific functional groups were detected; they can potentially distinguish alachlor from other herbicides such as metolachlor, and acetochlor, which have a similar chemical structure as alachlor.

Figure 2. SERS spectrum of 4×10-9 M (4 ppb) of alachlor detected by our cross plasmonic sensor(s). The cross sensor consists four triangular cavities, with an inter-triangular distance of 700 nm. The asterisk-marked vibrations are the designated vibrations of interest for alachlor`s study in our case. The peaks are the 1000 cm-1, 1038 cm-1, and 1600 cm-1 vibrations, which are associated with the benzene ring “breathing”, ortho substituted benzene ring vibration, and the benzene ring stretch, respectively.


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The strong SERS signal of the cross sensor is attributed to two characteristics of the device: the ability to tune the plasmonic device to resonate with the incident excitation beam wavelength as well as its strong EM field confinement on the surface. The cross device has two main plasmonic modes: the triangular cavity modes, which are discussed elsewhere,43 and a confined mode between the triangles, which results from hybridization between the triangular cavities and the plasmonic modes of the surface.42,43 Figure 3a shows the linear transmission spectrum (red curve) as well as the cathodoluminescence (CL) spectrum (black curve) of a specific cross device with an inter-triangular distance of d=700 nm. Two plasmonic modes are observed in the transmission spectrum: one at ~650 nm for the glass-silver interface (not relevant for this study); the second is a higher energy mode at ~514 nm for the silver-air interface. The latter is in agreement with the CL measurements, which can detect only the silver-air interface. The CL measurements, which were taken from the center of the structure (denoted by X in the inset of Figure 3a) shows an intense mode at ~480 nm. The observed mode is blue-shifted due to a superposition with the bulk silver plasmonic mode.43 In general, both CL and linear transmission measurements for this device (with d=700 nm) indicate that the mode at the center of the flat surface is in resonance with the Raman laser wavelength at 514 nm, as indicated by the green line.


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Figure 3. (a) A comparison between the transmission spectrum of a d=700 nm cross sensor and its complementary CL spectrum, showing the strong plasmonic photon emission at λ~480 nm. The inset, denoted by an X, indicates the central region from which the CL spectrum was extracted. The green line denotes a wavelength of 514 nm, which corresponds to our laser excitation as well as to the overlapping plasmonic mode in the transmission spectrum. (b) Transmission spectra of the plasmonic cross sensors for different inter-triangular distances (400 nm-900 nm). The green line indicates the overlap of the 514 nm laser wavelength with the 700 nm cross plasmonic sensor. (c) The Raman signal of: 1000, 1038, and 1600 cm-1 peaks as a function of an inter triangular distance, d, for various cross plasmonic sensors, with a constant concentration of 4×10-7 M alachlor. The values were normalized with respect to the maximum enhancement at d=700 nm. All datasets collapse on a single curve. The strongest signal is obtained with the d=700 nm sensor, as can be seen in all three curves for the designated vibrations, due to the overlap of the plasmonic modes with the excitation laser (514 nm).

As mentioned before, the plasmonic mode that resides on the surface at the middle of the plasmonic structure can be energetically tuned by changing the distance, d, between the


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triangular cavities. Next, we show that an overlap of the plasmonic mode at the middle of the structure is crucial for the device performance. Figure 3b shows a set of transmission spectra of our plasmonic cross devices in which the distance between the cavities is varied between d=400 and 900 nm (base to base), while other geometrical parameters are kept identical, i.e., the size and orientation of the four triangular cavities. Again, we are interested in the high-energy mode for the air-silver interface; its wavelength spans from λ=400 to 600 nm, where the maximal overlap with the Raman laser wavelength (λ=514 nm) is obtained by an inter-triangular distance of d=700 nm (see the green vertical line in Figure 3b). The maximum overlap is manifested by the enhancement of the Raman signal. To show this, we measured the Raman spectra for a set of plasmonic devices at a fixed alachlor concentration (4×10-7 M). We then plotted the intensity of three representative vibrations: the 1000, 1038, and 1600 cm-1, as a function of inter-triangular distance, as shown in Figure 3c. These selected vibrational modes are associated with the benzene ring vibrations and its chemical group substitutions as was mentioned above. The three curves (Figure 3c) show a maximum SERS enhancement for an inter-triangular distance of d=700 nm, thus assuring the consistency that indeed the plasmon-laser overlap greatly contributes to the signal intensity. The SERS spectra for the plasmonic cross devices having different inter-triangular distances are shown in Figure S2. The optical active area, namely, the hot-spot, is located between the triangular cavities. Given a laser beam diameter of about two microns, the number of hot spots using a cross sensor is approximately one (see Figure 4a). In attempting to improve the performance of our plasmonic device, we extended our study to a hexagonal triangular array (“hex-tri”) where the number of hot spots is at least five times higher than the previous configuration, as illustrated in Figure 4b. Such a configuration leads to an increase in the overall enhanced area within each laser cross-


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section and consequently increases the resulting Raman signal intensity, enabling more sensitive detection per surface area unit. This is also manifested in the stronger signal obtained by a hex-tri sensor, as shown in Figure 4c.

Figure 4. HRSEM sensor images of (a) a cross device, and (b) hexagonal triangular (hex-tri) array geometry. The images include illustrations of the excitation laser beam with a diameter of 2 μm (green) and the suggested plasmonic hot spots (red). Note that for the hex-tri configuration, this arrangement suggests an average of five hot spots within the laser beam, rather than one for the cross device. “p” denotes the distance between cavities (center to center). (c) Comparison of the SERS signal obtained by the most optimized sensors, the 700 nm cross (red), and the 700 nm hex-tri (black), both measured with equal concentrations of 4x10-9 M of alachlor (4 ppb), the cross spectrum is enlarged by x5 for clarity.

A large-scale hexagonal plasmonic structure (Figures S3a-b) and its spectral characterization are shown in Figures S3a-c. The transmission spectrum shows that indeed the plasmonic mode


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energetically overlaps with the Raman laser (Figure S3c). As shown above (Figure 4), for 4×10-9 M of alachlor with p=700 nm, the hex-tri sensor obtained a signal superior to that of a cross sensor. We shall mention that the higher sensitivity of the hex-tri unit can also be contributed by the inter-tip regions, which may show plasmonic “hot-spot” activity. This can be seen in Figure S4. Yet, the biggest contribution at λ=520 nm (near our Raman laser wavelength) is from the hotspots between the cavities, while the contribution from the other hot-spots (between the tips) for this configuration is relatively moderate as was supported by other studies.42,43 A comparison of both plasmonic sensors with a silver thin film reference with a concentration of 4×10-3 M is shown in Figure S5. We have repeated our results tens of times, measuring different samples and multiple devices. The results we obtained are very reproducible as is shown in figure S6. In S6a-b, we present the 3D cascades of multiple spots (alachlor`s SERS spectra) obtained from our optimized features: p=700 nm hex-tri, and d=700 nm cross sensors, respectively. In general, each of our devices was probed several times by thoroughly washing (removal) and re-drop casting different concentrations of alachlor (not before making sure our matrix of sensors is clean and clear by dark-field light microscopy and Raman acquisition. Because of the high number of hot-spots per area in case of the hexagonal array, we have measured its sensitivity down to 4×10-10 M. Figure 5a shows a SERS comparison (hex-tri sensors) of different alachlor concentrations ranging from 40 ppm to 0.4 ppb, with a calculated limit of detection (LOD) 46,47 of ~0.33 ppb. Figure 5b shows the signal intensity as a function of the concentration (log scale), for 1038 cm-1 vibration mode. There are two regimes in the graph (figure 5b), a linear regime at low concentrations (see inset), and a logarithmic regime in which


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the signal intensity is slightly changed with concentration. A very good linear fit is obtained for the low concentrations regime, with R-squared value of 0.9998. The red data points also show minor STD values, indicating the accuracy of our measurements. At the low concentration regime, the signal intensity is strongly dependent on the analyte concentration, yet, above some concentration threshold, this dependency is moderate as was already reported before by Hakonen et al. 48 and Fang et al 49 for other plasmonic systems. This behavior is attributed to the so-called “hot-spot saturation” effect, which suggests that there is a “finite” number of “hot-spots sites (volume)” which contribute to the signal enhancement. That is, when the number of the deposited molecules is higher than the volume of the hot-spot multiplied by their number, i.e., high concentration, then, a fraction of the deposited molecules is not affected (enhanced) by the enhanced EM field (hot-spots) (See SI section for estimation of number of molecules per hotspot 1.1 & 1.2). On the other hand, at lower concentrations (

Ultrasensitive Plasmonic Sensor for Detecting Sub-PPB Levels of

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