Raman Spectroscopic Detection in Continuous Microflow Using a

Article Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Raman Spectroscopic Detection in Continuous Microflow Using a Chip-Integrated Silver Electrode as an Electrically Regenerable Surface-Enhanced Raman Spectroscopy Substrate Eva-Maria Höhn, Rajapandiyan Panneerselvam, Anish Das, and Detlev Belder* Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, Leipzig 04103, Germany

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S Supporting Information *

ABSTRACT: An electrochemical approach to enable surface-enhanced Raman spectroscopy (SERS) detection in continuous microflow is presented. This is achieved by the integration of a silver electrode as SERS substrate in a microfluidic chip device. By the application of actuation pulses of about 4 V, otherwise irreversibly adsorbed analytes are stripped off, which enables quasi-real-time SERS detection in a continuous microflow. The approach opens up a way for in situ SERS monitoring of compounds in microflow with high application potential in microseparation techniques like HPLC and lab-on-a-chip devices.

gold films,58 nanoporous gold discs,59 silver nanodot arrays,60 metal-elastomer nanostructures,61 anodic aluminum oxide,62 silver covered quartz substrates,33 sharpened silver wire,63 plasmonic nanopillar arrays,64 plasmonic nanodomes,65 and silver coated TiO2 nanotubes.66 Such stationary targets are in widespread use and commercially available as microscope slides for respective SERS applications. While stationary targets can provide improved reproducibility67 compared to colloidal solutions, a drawback is that they are intended for one-time use only. This is due to the so-called memory effect68,69 caused by the often irreversible adsorption of analytes to the SERS-active surface. Several approaches have been attempted to recycle SERS substrates driven by two main motivations. One goal is to improve the reproducibility in a series of SERS measurements as enhancement factors can vary from one substrate to another. Another motivation for multiple-use of precious SERS substrates is cost-effectiveness. Therefore, several target regeneration methods have been developed to enable multiple measurements with a single SERS substrate. Approaches to remove adsorbed analyte molecules from the target SERS active surface include treatment with chemical solutions such as acids, bases, or organic solvents,70−79 irradiation with light,80−85 or combinations of both including photocatalytic degradation.86 However, most of the methods mentioned above are rather time-consuming and laborious, and none of them would allow a quasi-instantaneous and reagent-free regeneration of an embedded SERS target. While the memory effect can be disadvantageous for typical SERS measurements, it especially circumvents the use of SERS targets in continuous flow cells.

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urface-enhanced Raman spectroscopy (SERS) is a powerful vibrational microspectroscopic technique which can provide chemical and structural information about a variety of chemicals and biomolecules without any labels in a noninvasive manner.1−6 Due to these advantages, SERS is widely used in the fields of analytical chemistry,7−10 biology,11−14 material sciences,15−19 electrochemistry,20,21 microfluidics,22,23 and food analysis.24,25 Compared to common Raman spectroscopy,1 SERS can achieve much higher detection sensitivities due to enhancement effects which rely on the interaction of laser illuminated analytes and the properties of SERS-active substrates.2,26−29 These are mainly noble metal substrates with rough surface morphology such as silver and gold nanoparticles (NPs) and respective nanostructured surfaces.30−32 A straightforward approach to realize SERS in analyte solution is the direct addition of suspended nanoparticles such as silver or gold NPs which act as SERS substrates. This approach has also been applied earlier to enable SERS detection in microfluidic devices, by adding NPs to the process fluid in both continuous33−40 and segmented41−53 microflow systems. The use of such particle suspensions as mobile SERS substrates in microfluidics, however, can be troublesome due to contamination and clogging issues. Furthermore, they can interfere with the (bio)chemical processes to be monitored or with further downstream procedures. Moreover, SERS analysis using such mobile substrates often suffer from poor reproducibility due to batch to batch variances of nanoparticle synthesis as well as aging of the colloidal suspensions, which impedes quantitation.33,54 Interesting alternatives are so-called stationary targets where SERS active nanostructures with defined morphology are permanently attached to substrates such as microscope slides. These are mainly immobile metal nanostructures with different morphology such as immobilized NPs,55,56 nanopillar forests,57 © XXXX American Chemical Society

Received: March 26, 2019 Accepted: July 1, 2019 Published: July 1, 2019 A

DOI: 10.1021/acs.analchem.9b01514 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry An interesting approach toward SERS analysis in flowing streams has been published by Pothier and Forcé.87,88 They designed plexiglass cells incorporating three electrodes for potentiostatic control of a silver electrode working as a SERS substrate. With a potentiostat, they applied precisely controlled stepping potentials in the range from −0.6 to −1.3 V vs an incorporated Ag/AgCl or saturated calomel electrode (SCE) as a reference electrode. This enabled to control the adsorption and desorption process in a rather rapid manner. They observed that in less than 10 s of applying the desorption potential the signal for adenine returned to baseline.88 We have recently reported an approach for fast electrically assisted regeneration of SERS substrates inside microfluidic chips.89 The flow cell design was straightforward and facilitated microfabrication in the form of PDMS-glass hybrid chips. Chip-integrated inkjet-printed silver nanoparticles were used as SERS targets. Those were electrically connected via an ITO layer in a two-electrode setup omitting a reference electrode. In these experiments, we had to apply a comparatively high potential differences of about 100 V, which is far beyond any relevant electrochemical potential. The operation principle of this regeneration procedure was in part attributed to the electrolytic decomposition of the solvent and the corresponding generation of reactive and convection promoting gas. One of the motivations for the current study was to shed light on the unclear desorbing mechanism and to investigate this originally more coincidentally found effect in more detail. The present study is also aimed to explore the feasibility to enable SERS-detection in a continuous microflow. Such a detection technique which would allow the spectroscopic identification of compounds in microflow is highly demanded in various areas from micro separation techniques like HPLC to lab-on-a-chip technology.

was adapted from the literature.91 In detail, glass slides (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) were used as the bottom and top slides with powder blasted inlet and outlet holes. PET foils (Melinex ST506: König GmbH Kunststoffprodukte, Wuppertal, Germany) and adhesive transfer foils (467MP adhesive, 60 μm thickness: 3M, Neuss, Germany) were layered to assemble two different building blocks of foil (see Figure S1). Building block A consists of two layers of adhesive foil, and building block B consists of two layers of adhesive foil with one layer of PET foil in between them. The chip is assembled top to bottom beginning with the top plate on which one piece of laser cut (Lasercutter VLS 2.30: Universal Laser Systems, Vienna, Austria) building block A is placed for adhesion and to even out the surface. A chemically modified silver wire SERS substrate, and the counter electrode were placed in the measurement chamber. Subsequently, another piece of building block A was placed to compensate height differences and to prevent glass breakage and two pieces of building block B were placed to create the channel with sufficient height. After closing the chip with the bottom plate, the device was pressed at about 10 kN for 1 h. Altogether, the chip consists of seven layers: Two compensation layers, a SERS substrate, a counter electrode, two channel layers, and a top and bottom glass slide. The constant flow of electrolyte solution was provided via a syringe pump (PHD 2000 Infusion: Harvard Apparatus, Holliston, MA), which was equipped with a syringe (Omnifix Luer Lock: B. Braun Melsungen AG, Melsungen, Germany). The fluid flow was transferred via TFE Teflon tubing (0.5 mm ID, 1.58 mm OD) (Supelco Analytical Sigma-Aldrich Chemie GmbH, Steinheim, Germany) which was connected to the syringe with the P-835 Female Luer FTG system-1/16 (Postnova Analytics GmbH, Landsberg, Germany) and to the microchip via one-piece finger tight fittings (F-130: IDEX Cop., Lake Forest, IL). A three-port valve with a “T” flow path (Hamilton Bonaduz A.G., Bonaduz, Switzerland) was used to introduce analyte and electrolyte solutions into the chip. SERS Measurements. For SERS measurements, the microfluidic device was positioned on an inverted IX71 epifluorescence microscope (Olympus Corporation, Tokyo Japan), which was equipped with a LUCPlanFI 40-fold objective (NA 0.6) (Olympus Corporation, Tokyo Japan) and a part of a confocal modular Raman measurement setup (S&I Spectroscopy & Imaging GmbH, Warstein, Germany). The excitation light source was a Cobolt Blues 50 mW laser with 473 nm (Cobolt AB, Solna, Sweden) that was used with an excitation intensity of 2.5 mW for all measurements if not otherwise stated. The scattered light passed an Acton SP2750 monochromator (Princeton Instruments, Acton, NJ and Trenton, NJ) with an entrance slit of 150 μm and a grating of 600 lines/mm and was detected via a Peltier cooled ProEM 1600 × 200 CCD camera (Princeton Instruments, Acton, NJ and Trenton, NJ). For all experiments, an acquisition time of 1 s was used. As interface and recording software, VistaControl V4.2 Build 12596 (S&I Spectroscopy & Imaging GmbH, Warstein, Germany) was used. The SERS performance of the SERS substrates was evaluated by estimating the magnitude of enhancement factor (EF) in the detection of CV as shown in eq 1:92

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EXPERIMENTAL SECTION Reagents. Nitric acid (69%) and ammonium hydroxide (35%) were purchased from Fisher Scientific GmbH, Schwerte, Germany. Potassium dihydrogen phosphate, disodium hydrogen phosphate, and hydrochloric acid 37% were obtained from Merck KGaA, Darmstadt, Germany. Crystal violet (CV), silver wire (diameter 0.25 mm), para-mercaptobenzoic acid (pMBA), and sodium hydroxide were acquired from SigmaAldrich Chemie GmbH, Steinheim, Germany. Malachite green (MG) was acquired from Kallies Feinchemie AG, Sebnitz, Germany. Copper foil was obtained from Conrad Electronic SE, Hirschau, Germany. All chemicals were used as received. All solutions were prepared using ultrapure water. SERS Substrate Preparation. Silver wire SERS substrates were prepared using an adaptation of a literature method.90 Briefly, commercially obtained silver wire was flattened using a PO10H (Paul Otto Weber, Remshalden, Germany) mechanical press for 120 min at 30 kN. Later, the wire was ultrasonicated in methanol for 3 min and immersed in water. The flattened silver wire (2 cm long) was then treated with 35% NH4OH for 30 s; then 10 s in 6 M HNO3 and rinsed with water and methanol subsequently. A maximum of three SERS substrates were fabricated in each batch. In most of the cases, more than one batch of substrates was needed to complete one set of experiments. The as-prepared chemically roughened silver wire was incorporated into the microfluidic chip for SERS measurements. Chip Fabrication. The chip building process was performed via a laser cutting and lamination method which

EF = B

ISERS Nvol IRaman Nsurf

(1) DOI: 10.1021/acs.analchem.9b01514 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure 1. Illustration of the microfluidic setup for SERS measurements.

ISERS is the SERS intensity of the CV indicator band, IRaman is the Raman intensity for the same band in the Raman spectrum, Nsurf is the estimated number of CV molecules on the surface of the substrate, and Nvol is the estimated number of CV molecules in the excited volume that contribute to the Raman spectrum. here, 1 μL of 1 μM CV was deposited onto the substrate, air-dried and 1% aqueous CV solution was used for Raman measurements. The EF value of the silver wire SERS substrate was approximately 2.75 × 104. Additionally, the SERS signal stability with this SERS substrate was examined for some time at stagnant conditions as well as under flow (Figure S3). Electrical Setup for SERS Substrate Regeneration. For electrical regeneration, an AFG-2005 function generator and GDS-3354 oscilloscope were acquired from Good Will Instrument Co. Ltd., New Taipei City 236, Taiwan. A McVoice NG1620-BL DC power supply was obtained from ETT Marketing GmbH, Braunschweig, Germany. As shown in Figure 1, copper foil was predominantly used as the anode and the SERS substrate is in most cases used as the cathode. Phosphate buffer (66 mM, pH 7) was used as a stock solution to ensure electrical conductivity and SERS substrate cleaning. The applied voltage refers to the potential difference between the SERS electrode and the copper counter electrode. A potential of 4 V, resulting in a current of about 150 μA, was applied for SERS substrate regeneration. To obtain comparable data, we normalized the SERS intensity of the analyte and considered the point at which the voltage was applied to be 100. For time controlled voltage application, an electronically DC switching circuit was designed using an Arduino Uno (Arduino AG, Italy) and IRFBG30 N-Channel MOSFET (Vishay Intertechnology Inc., Malvern, PA) which was coupled to the voltage source in order to periodically switch on and off the voltage supplied to the chip electrodes. Scanning Electron Microscopy (SEM) Measurements. SEM images of the samples were obtained using a NanoLab 200 (FEI, Hillsboro, OR) scanning electron microscope. An acceleration voltage of 15 kV was used for the SEM images.

unsatisfactory. The same was true for the necessary voltage level for surface regeneration which varied from device to device. This could be attributed to batch to batch variations of the silver ink, variances of the sintering process and also the quality of the ITO sublayer. As these resulting chip to chip variances impeded the intended systematic studies in this work, we developed a novel integrated microfluidic flow cell (see Figure 1) and a correspondingly alternative chip manufacturing process. As inhomogeneity and respective changes in electrical conductivity of the sintered silver nanoparticle layer were regarded as the primary source for the observed variances, a silver wire was chosen as a more robust SERS-active electrode. A flattened silver wire (width after pressing: 0.35 mm) was chemically roughened to enhance the SERS-effect in an adaption of a literature protocol90 by etching with an NH4OH− and subsequently by an HNO3-solution. In initial off-chip experiments, such etched silver wire SERS substrates showed good SERS enhancement effects and low target to target variances. Furthermore, electrical contacting was straightforward, and we observed better electrical regeneration effects as with our inkjet-printed electrodes. The morphology of the etched silver wire substrates was characterized using SEM images as shown in Figure S2. Additionally, Raman spectra and SERS spectra of CV were obtained to evaluate the SERS enhancement properties of our SERS substrates. As shown in an SEM image (Figure S2), the silver wire surface exhibited a roughened structure and promoted SERS enhancement as expected. To expedite the chip prototyping process and give access to a large number of different chips with varying layouts, we chose a straightforward lamination technique91 using laser cut adhesive foils layered between glass slides; see Figure S1 in the Supporting Information. This combination of simple SERS substrate preparation and respective straightforward chip building procedure leads to short preparation times for a fully functional device of about 1.5 h from scratch. A typical Raman spectrum of 10 μM crystal violet solution, obtained in a ca. 95 μL sized flow cell with an incorporated silver wire as a SERS substrate, is shown in Figure S2B,I in comparison to the Raman spectrum of solid crystal violet. The Raman background signals of an empty cell are negligible as evident from Figure S2B,III. The microfluidic chips for the electrical regeneration studies include a flow cell containing a silver wire as a SERS active electrode and a copper foil as a counter electrode as

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RESULTS AND DISCUSSION In initial experiments, we realized that our previous approach using inkjet-printed silver ink on an electrically connected ITO layer has some shortcomings. While this approach is appealing from the point of chip-microfabrication, it turned out that the chip to chip reproducibility of the signal enhancement was C


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Analytical Chemistry schematically shown in Figure 1. Such chips were placed on top of an inverted Raman microscope with electrodes attached to a voltage source to study electrical regeneration phenomena in a series of stagnant and flow experiments. The effect of several experimental parameters on the regeneration process has been studied systematically to gain a deeper insight into the phenomenon. For the evaluation of each experimental condition, a newly fabricated chip was used with a freshly prepared SERS substrate. Figure 2 shows the test

Figure 3. (A) Plot of time versus SERS intensity of 10 μM CV at 1180 cm−1 from silver wire SERS substrate as the cathode in the microfluidic device with different voltages. (B) SERS spectra extracted from (A) of 10 μM CV on the SERS substrate surface at different times during the electrical regeneration process. 4 V was applied at around 20 s to remove the adsorbed CV molecules from the SERS substrate.

Figure 2. Schematic showing the electrical regeneration process for the silver wire SERS substrate in the microfluidic device: (1) analyte injection into the chip, 10 μM CV, (2) SERS detection, with an excitation wavelength of 473 nm, an excitation intensity of 2.5 mW, an acquisition time of 1 s and a 40-fold objective, (3) electrolyte (phosphate buffer 66 mM pH 7) injection and rinsing with a rate of 50 μL/min for 10 min, and (4) application of DC voltage for regeneration.

channel, we concluded that 4 V is the optimal voltage for efficient and rapid removal of the analytes from the SERSsubstrate. Figure 3B shows the extracted SERS spectra documenting the regeneration process with a voltage of 4 V. The SERS spectrum (blue) was acquired as a starting point of SERS measurements (Figure 3B, 0 s). The second SERS spectrum (orange) was acquired at 19.5 s to document the status immediately before the voltage application. The third (green) and fourth (red) spectra were acquired at 20.6 and 21.7 s to monitor the progressing regeneration process. These set of spectra demonstrate the effectiveness of the approach as the SERS signal disappears within seconds. In control experiments with comparable rinsing steps but applying 0 V instead no effect on SERS-spectra was observed as expected; see Figure 3A. These results confirm our earlier findings that a voltage above the decomposition voltage of the solvent is required to remove the analyte molecules from the electrode surface efficiently.94 The significantly higher potential difference of about 100 V in our earlier observation can be explained by overvoltage due to the poorer conductivity of the sintered silver ink used as SERS substrates as well as unsteady contacting of the inner ITO-sublayer. In another set of experiments, the effect of reversing the electrical polarity was studied. For this purpose, the silver wire SERS substrate was used as the anode and the copper foil counter electrode as the cathode. The chip was filled with 10 μM CV, and similar measurements as described above were performed to study the effect of different potential levels on the SERS signals. The observed correlation between the voltage level and the Raman indicator band during the process is plotted in Figure

sequence to study the electrical regeneration process. Initially, the microfluidic channel was filled with 10 μM CV, and in the second step a SERS spectrum of the analyte was acquired. Third, after a short equilibration time, the flow cell was rinsed with a continuous electrolyte flow of 66 mM phosphate buffer (pH 7) at 50 μL/min for 10 min to flush out the dissolved analyte. In the fourth step, SERS spectra of the adsorbed analyte were monitored for a period of 50 s with 1 s acquisition. In order to get as comparable data as possible in a series of experiments, the electrical regeneration process was initiated after a specific equilibration time (≈20 s). During this period, 19 spectra were acquired. With a constant electrolyte flow, the SERS signal of the analyte molecules slightly decreases which can be explained due to a rinsing effect and also by photobleaching (see Figure S3). This is evident from Figure 3A, where the equilibration time region is highlighted in blue while the pink region indicates conditions under voltage. The first studied parameter was the effect of the voltage level and polarity applied to the SERS-silver electrode. Figure 3A shows the effect of different voltages on the recorded SERS indicator band at 1180 cm−1. The obtained results indicate that 4 V with the SERS active electrode as the cathode is the voltage level of choice for removing analyte molecules from the SERS substrate. Below this voltage, the regeneration was not rapid and complete. However, the application of higher potential difference such as 6, 8, and 10 V also had the expected effect, but gas bubbles were observed due to water electrolysis.93 As the formation of gas bubbles can disturb the SERS measurements and also flow properties inside the D


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concentration) to make them as comparable as possible. This however induced the chip to variances that were noticeable in varying absolute SERS signal values as well in different courses of signal reduction during rinsing and equilibration. In order to enable a meaningful comparison of the data sets in one chart, the SERS intensities were normalized in respective studies by setting the moment of voltage application to a value of 100. To examine the influence of pH (Table 2), respective solutions were adjusted by addition of 1 M NaOH or 1 M

S4A. The recorded data show that the application of 2 V results in a significant decrease of the SERS signal. However, higher potentials affected the surface properties as evident from the altered SERS spectra of CV acquired in subsequent experiments. This is shown in Figure S4B, which also reveals that the substrate regeneration was not complete with the subsequently applied voltage of 4 and 6 V. This can be explained by oxidation of the silver95 which then leads to a decay of the SERS intensity. This is in agreement with the literature data.96 In the context of this study applying DC potentials of different polarity, an interesting question was how the application of AC would perform. This was studied in a set of experiments applying various AC frequencies and voltages. These experiments revealed the basic suitability of AC frequencies in the range of about 10 to 100 Hz (Figure S5A) and a voltage of about 3 to 4 V. Respective data are shown in the Supporting Information in (Figure S5B). Besides the applied voltage and polarity, other parameters potentially affecting the regeneration process such as the electrolyte concentration and pH were studied as well. This was explored in a set of experiments applying the proven 4 V DC voltage with the SERS active electrode as the cathode with phosphate buffer solutions of varying concentrations including pure water as shown in Table 1. From the

Table 2. Conductivity of 66.6 mM Phosphate Buffer at Different pH Values

conductivity (mS cm−1)

0.666 3.33 16.7 33.3 66.6

0.123 0.551 2.33 4.36 8.03

conductivity (mS cm−1)

3 5 7 9

10.17 8.70 8.05 9.64

HCl. As shown in Figure 4B, the regeneration speed and efficiency was not significantly affected by the pH of the electrolyte in a range from pH 3 to pH 9. These and further experiments (Figures S6 and S7) indicate that the regeneration approach works reliably providing that the solution exhibits a sufficient electrolyte concentration (above ≈3 mM) and a respective conductivity (above ≈0.5 mS/cm); see Figure 4. In further studies, we evaluated the effect of the electrode configuration. So far, one silver wire SERS substrate and one piece of copper foil were utilized in a two-electrode configuration. Three different electrode configurations (Figure S8) were evaluated applying the regeneration voltage of 4 V DC with a 66 mM phosphate buffer at pH 7. Analogous to previous experiments, the indicator band at 1180 cm−1 of CV was monitored for 50 s without and with applied voltage using either one, two or three electrodes configurations. The results are shown in the Supporting Information (Figure S8). In summary, it can be concluded that the electrical regeneration process only occurs when the SERS substrate is at lower potential and that a simple two-electrode configuration is sufficient. Such a fast regenerable SERS substrate inside a microfluidic chip is very appealing as an analytical tool, providing that a single SERS substrate is stable and reusable in repetitive experiments. In eight consecutive experiments with the same device, it could be shown (Figure S9A) that the indicator band at 1180 cm−1 was reproducible and reliable.

Table 1. Conductivity of Different Concentrations of Phosphate Buffer concentration (mM)

pH

data (Figure 4A), it is evident that the regeneration is not efficient in pure water, most probably due to limited conductivity. With a diluted phosphate buffer (0.66 mM) the decay in the SERS signal was slower than at higher electrolyte concentrations. A rapid and complete SERS substrate regeneration was achieved at buffer concentrations in the investigated range from 3.33 mM until 66.6 mM. A closer look at Figure 4 reveals that the individual curve progressions during the equilibration period differ in some cases. As described in the experimental part, a new chip was taken for each set of experiments (e.g., each buffer

Figure 4. (A) Plot of time versus SERS intensity of 10 μM CV at 1180 cm−1 from silver wire SERS substrate with different concentrations of phosphate buffer 0, 0.666, 3.33, 16.7, 33.3, and 66.6 mM. (B) Plot of time versus SERS intensity of 10 μM CV at 1180 cm−1 from the SERS substrate with different pH 3, 5, 7, and 9 of 66.6 mM phosphate buffer. E


Article

Analytical Chemistry To monitor compounds in a continuous flowing system in quasi-real-time, it would be necessary to recover the SERS substrate in a nearly continuous manner. To achieve this, we connected the device to a programmable voltage source controlled by a microcontroller (Arduino Uno). The microcontroller was programmed to provide periodic voltage pulses to the gate terminal of the MOSFET to function as a switch. To evaluate this, a time period of over 700 s was recorded. Starting with an empty chip, a constant flow of a 10 μM CV solution in 66 mM phosphate buffer pH 7 was started after approximately 10 s. A waiting period of 3.5 min followed to ensure that the analyte reached the SERS substrate before the preprogrammed switching circuit was started. As can be seen in Figure 5, the analyte solution took ca. 170 s to reach the SERS

Figure 6. SERS spectra of MG and CV on a silver wire SERS substrate in alternating detection processes after and before regeneration.

As we used just two popular SERS analytes MG and CV in this proof of concept study, the wide applicability of the approach still has to be shown and will be subject of future studies. Since we succeeded in the first try also with strongly absorbing compounds such as para-mercaptobenzoic acid molecules as shown in Figure S11, we are quite confident.

Figure 5. Effect of voltage changes on the SERS signal response in the microfluidic flow cell using a preprogrammed switching circuit. Program: automatically switching the applied voltage of 4 V on (10 s) and off (20 s). “Constant analyte flow” stands for analyte dissolved in electrolyte with a flow of 50 μL/min (473 nm; 2.5 mW; 600 lines/ mm; objective: 40-fold; exposure time: 1 s; electrolyte (50 μL/min): 66.6 mM phosphate buffer pH 7; analyte: 10 μM CV; indicator band: 1180 cm−1).

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CONCLUSION A rapid and straightforward electrical regeneration method for SERS substrates was successfully established in a microfluidic flow cell. It was found that DC voltage of about 4 V with the SERS active substrate as the cathode is effective in conductive aqueous solutions. By applying pulsed voltages, it is possible to monitor different compounds in a continuous flow, which makes the technique very attractive for quasi-real-time analytics of effluent flows from chemical microreactors or separation devices.

substrate and exhibited a clear analyte signal. After 200 s, the preprogrammed switching circuit switched the applied voltage of 4 V DC on and off with off periods of 20 s and on periods of 10 s. Figure 5 clearly shows that the proposed strategy can rapidly clean the SERS substrate which makes it a promising strategy for quasi-real-time SERS detection in a continuous flow. In order to use an analytical unit as a flow-through cell, another essential criterion is the absence of carry-over effects. As a proof-of-concept, two different analytes were analyzed in the same device in an alternating manner. Beside CV, MG was chosen as an additional model analyte due to its strong SERS signals,97 which makes it ideal for probing carry-over effects. The two analytes can be distinguished with the bands at 1213 and 1391 cm−1, which appear in the spectrum of MG but not in the signal of CV (see Figure S10).98 After injection of each analyte, SERS spectra of analytes were recorded, and continuous electrical regeneration was carried out in a constant flow. These results shown in Figure 6 demonstrate that the electrical regeneration worked successfully in a continuous flow without carry-over effects. After each regeneration, the analyte signal raises approximately to the same level as before. With the same microfluidic device, it was possible to perform consecutive regeneration cycles with alternating analytes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b01514. Schematic of the lamination technique; SERS substrate characterization; comparison of different microfluidic conditions on signal stability/effect of photobleaching; evaluation of SERS active electrode as anode; effect of AC voltage in electrical regeneration; comparison of different channel widths on the regeneration strategy; effect of potential in electrical regeneration; effect of chip configuration; evaluation of the SERS substrate lifetime in a microfluidic flow cell; SERS spectra of the used model analytes; regeneration experiment with 4mercaptobenzoic acid (PDF) F


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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 341 97 36091. Fax: +49 341 97 36115. E-mail: [email protected]. ORCID

Detlev Belder: 0000-0001-6295-8706 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Deutsche Forschungsgemeinschaft (DFG) Project BE 1922/16. The authors thank Dipl.Phys. Jörg Lenzner (Felix-Bloch-Institut für Festkörperphysik, Universität Leipzig) for recording the SEM images.

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Raman Spectroscopic Detection in Continuous Microflow Using a

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