New Electrochemical Flow-Cell Configuration Integrated into a Three

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New electrochemical flow-cell configuration integrated into a three-dimensional microfluidic platform: Improving analytical application in presence of air bubbles Magno Aparecido Gonçalves Trindade, Cauê A. Martins, Lucio Angnes, Thomas Herl, Timo Raith, and Frank Michael Matysik Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02438 • Publication Date (Web): 20 Aug 2018 Downloaded from http://pubs.acs.org on August 21, 2018

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Analytical Chemistry

New electrochemical flow-cell configuration integrated into a threedimensional microfluidic platform: Improving analytical application in presence of air bubbles Magno Aparecido Gonçalves Trindade 4* Frank-Michael Matysik

1,2,4

1

3

4

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*, Cauê Alves Martins , Lucio Angnes , Thomas Herl , Timo Raith and

1

Faculdade de Ciências Exatas e Tecnologia, Universidade Federal da Grande Dourados, Rodovia Dourados-Itahum, km 12. Dourados-MS, 79804-970, Brazil. 2

Unesp, National Institute for Alternative Technologies of Detection, Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM), Institute of Chemistry, P.O. Box 355, 14800-900 Araraquara (SP), Brazil.

3

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, CEP 05508-000 São Paulo, SP, Brazil. 4

University of Regensburg, Institute of Analytical Chemistry, Chemo- and Biosensors, Universitätsstraße 31, DE93053 Regensburg, Germany.

ABSTRACT: A newly configured electrochemical flow cell to be used for (end-channel) amperometric detection in a microfluidic device is presented. The design was assembled to place the reference electrode in a separated compartment, isolated from the flowing microchannel, while the working and counter electrodes remain in direct contact with both compartments. Moreover, a three-dimensional coil-shaped microfluidic device was also fabricated using a nonconventional protocol. Both devices working in association enable to solve the drawback caused by the discrete injection when the automatic micropipette is used. The high performance of the proposed electrochemical flow cell was also demonstrated after in situ modifying the surface of the platinum working electrode with surfactant (e.g., using tween 20 at 0.10%). As the reference electrode remained out of contact with the flowing stream solution, there was also no trouble by air bubble formation (generated by accidental insertion or by presence of surfactants) throughout the measurements. This device was also checked regarding its analytical performance by evaluating the amperometric detection of acetaminophen, enabling determination from 6.60 µmol L-1 to 66.0 µmol L-1. This issue is an important finding, since at high concentration (e.g., as assessed in clinical analysis) the acetaminophen is known to passivate the working electrode surfaces by electrogenerated products, impairing the accuracy of the electrochemical measurements.

KEYWORDS: Microfluidics, discrete injection, three-dimensional coil-shaped microchannel, electroanalysis.

INTRODUCTION Undoubtedly, microfluidic devices have become an important tool in lab-on-a-chip analysis, which enables applications in biomedical, pharmaceutical and environmental fields.1–4 The advantages highlighted by the users arise from the possibility of manipulation very low volumes resulting in low consumption either as samples as reagents.5,6 Likewise, significant reduction in waste generation, quick analysis, manipulation of small particles, easy droplet formation for drug encapsulation, highthroughput drug testing and point-of-care diagnostics, among others applications, are emphasized.2,3,7 Because of the miniaturization requirements, the most critical part of the microfluidic chip platforms is the detection system and its integration into the microchannel.8 Admittedly, electrochemical detectors are the most suitable tool for

such integration, since they are well suited for miniaturization without losing their sensitivity. Additionally, their easy portability and notable cost-effectiveness are also characteristics that should be considered.8–10 From analytical viewpoint, the integration of electrochemical detection and microfluidic devices is also a suitable tool to attend the demand of flowing streams baseddrugs analysis either in environmental or bioanalytical context. Basically, a conventional electrochemical detector can be assembled into the microfluidic chip platforms in two main forms: placed inside the channel (onchannel) or nearby the end of the channel (end-channel).9 Despite the aforesaid versatility of the electrochemical arrangement, the microscopic electrochemical devices might not have all the requirements that can be found in the conventional macroscopic three-electrode arrange-

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ment.11 The main requirements involve: space to geometrically achieve the adjustment of the designed electrodes into the device system and stable electrodes able to maintain its surface-activity throughout the series of measurements.8,11,12 Once the electrochemical detector system is based on the interfacial electron-transfer processes, maintaining the surface-activity of working electrodes might be the most critical issue. In this sense, both the electroactive analytes and other electroactive substances (called contaminants) arising from complex samples (e.g., organic matrices) can be part of such electrochemical processes. Thus, both either the working electrode or the reference electrode unavoidably are susceptible to the surface coverage by electrochemical/chemical processes, which lead to fouling of their surfaces.9,11 A clean working electrode surface is a first requirement for accurate results in flowing stream analysis.13 The cleanliness of the reference electrode also significantly affects the efficiency and reproducibility of electroanalysis by shifts of the reference potential, which can cause variations in the magnitude of the peak current, in measurements with a fixed potential.8,12,13 In order to avoid undesirable results, in extreme cases, it is indispensable to perform a cleaning procedure between each measurement to remove the electrogenerated products and/or any adsorbed electroactive substances (contaminants) deposited on the working electrode surface.13 Literature survey indicates that the most recommended protocols to re-establish the electrode-surfaceactivity is the electrochemical activation (commonly, cathodic reduction) to remove the contaminants found to be passivating working electrode surfaces.11,12,14 For example, Tormin and coworkers used the multiple-pulse amperometry coupled to a flow-injection system to clean the glassy-carbon electrode, enabling the simultaneous determination of butylated hydroxyanisole and tertbutylhydroquinone in biodiesel samples.15 Unless the electrode assemblies are removable, another alternative would be adaptation on the conventional polishing of the working electrode surface between each measurement. In this sense, for the integration of electrochemical detection into microfluidic devices both protocols imply some disadvantages. This includes: time consuming procedures, possibility of removing the thin-film (solid electrodes) or destruction of the substrate (e.g., screen-printed electrodes) and/or, the most important issue, the challenges in coupling the miniaturized removable electrodes systems with the complex conventional microfluidic platforms. Alternatives such as in situ surfactant-modified electrodes have been proposed as anti-fouling agent by covering the metallic working electrodes surface.16–20 However, this process cannot be working appropriately at flowing streams where air bubble formation (or eventually, accidental insertion) can easily arise and becomes a critical obstacle in microfluidic applications.8,12,21–23 Indeed, surfactants and flowing stream analysis may not be an interesting combination, especially, when the interaction happens inside of the nano/microchannels and these are integrated into the electrochemical detec-

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tors. The surfactants will cause undesirable troubles by air bubble formation blocking the reference electrode, which is losing the contact with the working electrode. The working electrode, in turn, can modify its electrochemically active surface area (ECSA). Thus, the potential difference — which is established in the absence of troubles onto the electrode/solution interface — is drastically affected, inducing an abrupt potential drop across to the electrodes arranged in a conventional electrochemical cell. 21–23 Considering all the highlighted shortcomings, the integration of the conventional three-electrode configuration into the microfluidic devices still offers great challenges to be overcome.12 Furthermore, there is still a need for a simple protocol to overcome the challenge regarding the adsorption of electrogenerated products from working electrode surface as well as the uses of surfactants in microfluidic devices. Thus, exploring this field of application, a newly designed electrochemical flow-cell (called NFC) was developed alongside with a three-dimensional microfluidic platform in order to overcome the challenge of minimizing the previously mentioned shortcomings. The configuration allows assembling the target NFC into the microfluidic chip platforms at the end of the microchannel and the most notable find is its two compartments. Thus, the reference electrode is isolated from the flowing streams phase in a separated compartment, while the working and counter electrodes are directly in contact with both compartments. For the previous check of its viability, we present a device made out of poly(methyl methacrylate), however, this device can easily be made by direct integration in polydimethylsiloxane (PDMS) platform mold or printed in the 3D-printer to design it as onchannel detector or as the preference of users. To check the analytical performance of the NFC, acetaminophen (named as APAP) was chosen as analyte due to its importance in the classes of pharmaceutical compounds widely used as analgesics. Thus, its quality control analysis in pharmaceutical formulations as well as its detection in biologic fluids as urine, blood or plasma for medical control is a permanent goal. Besides that, the APAP is frequently addressed in the list of the contaminants of emerging concern, which in turn, makes it an important analyte to be studied for analytical purposes to establish new methodologies able to detect at trace-level in microfluidics devices. However, as demonstrated in previous studies,24 the challenge in electro-oxidation of the APAP, especially at concentration in the range of µmol L-1 level (i.e., as assessed in clinical analysis), is to prevent the surface-electrode from fouling. On the one hand, the strong adsorption of APAP on the electrode surface could be suitable for uses on the renewable surface modified electrode which can results in a detection limit down to nmol L-1.25 On the other hand, in miniaturized analysis systems, where not just working electrode but also reference electrode requires considerable attention,11 the adsorption of electrogenerated products becomes a serious drawback to be overcome, and this issue will be studied aiming at analytical applications.


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EXPERIMENTAL SECTION Chemicals, solutions, and samples. The APAP, Figure S1 (Supporting Information, SI), was purchased from Sigma Aldrich and the standard stock solution was prepared weekly with a concentration of 0.01 mol L-1 by dissolving the powder in ultrapure water. The working solutions were prepared weekly by diluting (at the required concentration) the stock standard solution in the carrier. The sodium dihydrogen phosphate (0.10 mol L-1) and phosphate (0.10 mol L-1) buffer (PB) solutions were prepared using phosphoric acid (Sigma-Aldrich) by adjustment of the required pH with 1.0 mol L-1 of ammonium hydroxide (Sigma-Aldrich). Unless otherwise specified, the carrier serving as supporting electrolyte (at 0.10 mol L-1) was the sodium dihydrogen phosphate solution. The surfactants sodium dodecyl sulfate (ultrapure, Applichem, Germany), Triton™ X-100 (Sigma Aldrich) and Tween 20 (Sigma Aldrich) and other reagents/solvents used in this study were of analytical grade and used as received. The Polydimethylsiloxane (PDMS) elastomer used in this study was the cell guard polymer and the curing agent (crosslinking agent), both purchased from MLSolar (USA). Lake water sample was collected from the GeorgHegenauer Park (Regensburg, Bavaria, Germany) and spiked with APAP at three concentration levels (6.62, 33.1 and 66.2 µmol L-1). The target-spiked lake water samples were prepared by adding the required amount of the APAP working solution together with the phosphoric acid and further adjustment to the required pH value. The spiked tap water samples were stored in glass bottles, and were filtered through a 0.45 μm nylon filter disc (Millipore) prior to analysis. A Paracetamol solution having an orange taste (Ratiopharm) with 40 mg mL-1 of labeled APAP was also used as real sample. The 1.00 mmol L-1 stock solution was made by dissolving the Paracetamol oral solution (40 mg mL-1) in ultrapure water. Furthermore, the required concentrations of APAP (6.62, 33.1 and 66.2 µmol L-1) were diluted from the stock solution in sodium dihydrogen phosphate (0.10 mol L-1) solution (pH 5.0). Instrumentation, experimental setup and NFC design and fabrication. Electrochemical studies were performed using a µAUTOLAB TYPE III potentiostat/galvanostat connected to a microcomputer controlled by 2.1 NOVA software for data acquisition and experimental control. The measurements were performed using the newly designed electrochemical flow-cell (NFC) and a commercial flow-cell (CFC) from Micrux (Oviedo, Asturias, Spain) was used to compare the results. Figure 1 (I) shows a schematic diagram of experimental setup for the assembled microfluidic device, consisting of three main parts: (b) A portable automatic electronic micropipette to perform a hydrodynamic pressure for a discrete injections (in a T-junction way connection(c)) of the APAP standard and sample into the carrier stream (a), (d) an microdevice consisting of a three-dimensional microfluidic array, on which the main channel used in this study was 400 µm and (e) the newly designed electrochemical flow-cell (NFC) for chronoamperometric meas-

urements. In all studies, the performance of the NFC (Figure 1e1) was compared with a commercial flow-cell (CFC) by changing the detection system as showed in the schematic representation (Figure 1e2). The new flow-cell system (see Figure 1e1) was made in the workshop (University of Regensburg) and was composed of two compartments. The first one was a liquid reservoir including a platinum wire (420 µm diameter) which served as quasi-reference electrode (QRE) separate from the carrier stream. The second one was the channel for the flow stream (i.d.: 1000 µm) equipped with vertically aligned platinum wires (420 µm diameter) serving as working electrode (WE) and counter electrode (CE) in direct contact with both compartments (Figure 1e1). In this way, the potential applied on the WE (controlled in the upper compartment) will be the same in the flowing channel. The commercial flow cell consisted of a thin-film electrode integrated into a 2 mm diameter cell. The working, quasi-reference and auxiliary microelectrodes were made by a 150 nm thin-film platinum deposited on a glass substrate (Figure 1e2). The geometric area of the working electrode (WE) was 1.0 mm (0.8 mm2). Unless otherwise specified, all potentials are versus the platinum as QRE.

Figure 1. Schematic representation of the experimental setup (I) and the detection system (II). Transport of the carrier stream by a syringe pump (a), hydrodynamic analyte/sample injection by an automatic electronic micropipette (b), Tjunction (c), three-dimensional coil-shaped microfluidic platform (d) and detection system (e). A novel electrochemical flow cell (e1) as well as a commercial flow-cell (e2) were applied for detection. Real image is showed in Figure S2 (SI).

A portable automatic electronic micropipette (Eppendorf, model Multipipette Xstream) was used to perform a hydrodynamic pressure for discrete injection (T-junction array) of the APAP standard and the samples. The device was assembled as shown in Figure S2 (SI). Micropipette tips of 100, 500 and 1000 µL were used to inject discrete volumes of the APAP solution and samples. A syringe pump (Razel, model A-99) and (KDScientific, model 101). Common syringes of 10 mL were used to drive the carrier into the microfluidic device. All pH measurements were performed using a combined glass electrode (Schott) connected to a digital pH-meter (Schott, model CG 837). An ultrasonic cleaner (Elma, model Elmasonic S 30H) was used to dissolve the ABS filament inside the cured PDMS.



A vacuum pump controller (KNF, Newberger GmbH) was used to eliminate bubbles from PDMS before the curing process. The images were collected using a digital camera (20.1 megapixels) super HAD CCD sensor 63X optical zoom (Sony, DSCH400). Microfluidic device fabrication. The microfluidic device (Figure 2) was fabricated based on some adaptations of the previously published method by Saggiomo and Velders.26 Target protocol was preferred due to the simplicity in fabricate the PDMS platform without familiarity or experience in microfabrication. This protocol provides some advantages in our proposed flow system, such as no valve systems, high flexibility and modularity of setup. In the schematic illustration (Figure 2, I), firstly, it was necessary to extrude the acrylonitrile butadiene styrene (ABS) filament (1.75 mm) in a diameter of 400 μm, using a 3D-Printer Pen (Myriwell, model RP-100A) with nozzle diameter 400 µm. To obtain the target coil-shaped microchannel, a mold was created by manually wrapping the extruded ABS wire. In the next steps, the coil-shaped wire was stretched and placed in the platform model (consisting of a movable plastic case) to create the required length and design. (II) Afterwards, the cell guard PDMS and curing agent (cross-linking agent) were mixed in a ratio of 9:1 (w/w) for about 10 min., followed by elimination of bubbles under vacuum pumping control for about 30 min. The mixture was discharged into the platform model containing the spiral and left (overnight) for cure. (III) In this step, the cured PDMS, which showed the coilshaped ABS wire inside the microdevice was removed from the plastic mold. (IV) Finally, the ABS wire was removed by swelling in hot (45 °C) acetone/water (7:3, v/v) under sonication for approximately 75 min., leaving an empty cavity and the designed three-dimensional coilshaped microfluidic device was prepared.

Figure 2. Schematic representation for the fabrication process of the three-dimensional microfluidic array. (I) Extrusion and fabrication of the coil-shaped microchannel by manually wrapping ABS wire, stretching and fixing in the a movable plastic mold, (II) pouring of the PDMS polymer and curing agent (9:1, w/w) into the mold, (III) coil-shaped ABS wire inside of the PDMS after overnight curing, (IV) the designed three-dimensional coil-shaped microfluidic device after removal of the embedded ABS wire.

Electrochemical measurements in stationary and flow configuration. In order to obtain reproducible measurements without compromising the performance of

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the electrodes/microelectrodes, the working electrodes were cleaned by recording successive cyclic voltammograms prior to use, following the protocols previously published by Martins and coworkers.27 For the commercial flow-cell having the microelectrode acquired from Micrux (Figure 1), the pre-cleaning procedure was done by cycling between -1.50 and +1.5 V (20 cycles) at a scan rate of 100 mV s-1 in KCl (0.10 mol L-1) as background electrolyte solution. For the new electrochemical flow-cell (Figure 1 and S2, SI), the same procedure was used followed by additional 20 cycles (at a scan rate of 100 mV s-1) between -0.70 and +0.95 V in H2SO4 (0.50 mol L-1) as background electrolyte solution. The NFC setups serving as electrochemical detectors for the microfluidic platform as shown in Figure 1 were tested by evaluation of some characteristics, such as: voltammetric measurements in stationary and flowing stream configurations (Figure S3 and S4, SI) to determine the electrochemically active surface area (ECSA) and to determine the oxidation behavior of APAP. The ECSA of Pt electrodes is usually obtained from the hydrogen under potential deposition (HUPD) region considering the charge density of a hydrogen monolayer desorption on the Pt active sites. However, such protocol is limited to O2-free solutions, which is unlikely to be used in real applications where the samples usually contain dissolved-air. This issue was previously addressed by Fonseca and coworkers.28 The authors suggested the use of the charge involved in the reduction of platinum surface oxides (QPtO). It is possible to find the QPtO by comparing it with the charge of the hydrogen desorption region (QH = 210 µC cm-2), easily obtained from a characteristic Pt plate profile in a H2SO4 (0.50 mol L-1) solution performed in a conventional O2-free three-electrode cell. We found QPtO = 400 µC cm-2 as the approximate reduction charge density involved in the reduction of a surface oxide monolayer. Therefore, although the profile of the developed system showed a characteristic Pt profile (Figure S3, SI), the presence of small amounts of dissolved O2 led us to the calculation of the current densities found here by normalizing the current through the surface area calculated as follows: ECSA = (experimental charge of PtO / QPtO). The designed three-dimensional coil-shaped microfluidic device hosting the new electrochemical flow-cell and the commercial flow-cell (Figure 1 and S2, SI), which was used for APAP detection, had a dimension of 40 × 10 × 12 mm (length × width × height), with the microchannel measuring 400 µm of inner diameter. The connections between the main parts of the devices (Figure 1 and S2, SI) were made by PTFE tubing segments of 500 µm (i.d.). The T-junction way for connecting the automatic electronic micropipette to perform a hydrodynamic sample injection was created by three-way PEEK Tee (1/16''). For the electrode characterization regarding the ECSA, the H2SO4 (0.50 mol L-1) was placed in a 10 mL syringe and pumped into the NFC system at 20 μL s−1. The absence of bubbles was checked carefully to not trouble the system. After filling the cell compartment containing the electrodes, the pumping was stopped and the cyclic voltammograms


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were recorded. The ECSA was obtained as presented in the Results and Figure S3 (SI). For amperometric measurements in presence of surfactant, after cleaning the Pt electrodes in the NFC and CFC using the aforesaid protocol, the carrier (NaH2PO4 at 0.10 mol L-1) containing the surfactant (0.10%) was driven through the microdevice at a flow rate of 0.35 µL s-1, cycling (at scan rate of 100 mV s-1) between -0.70 and +0.95 V (10 - 20 cycles). For the measurements operated with the CFC, the air bubbles (which are persistent in surfactants medium) troubled the electrochemical detection. So that, after filled the syringe with the carrier solution, the trapped air bubbles were eliminated by carefully tapping the syringe until the bubbles rise the needle exit. Then, part of the content was dispensed until bubbles are no longer visible in the syringe vessel. For the amperometric detection, unless otherwise specified, the appropriate carrier was continuously pumped into the microdevice at 1.77 μL s−1. The proposed microdevice was also tested in the practical application by determining the concentration of APAP within a real sample such as Paracetamol oral solution as well as the spiked lake water (Section 2.1). In that case, the measurement of the APAP concentration was performed by calibration curves generated using the peak current values from the chronoamperograms under optimized parameters.

RESULTS AND DISCUSSION Voltammetric measurements under stationary and flowing stream configurations. The setup shown in (Figure 1 and S2, SI) was chosen based on the influence of the flow cell configuration for the electrochemical profile of the Pt working electrode in sulfuric acid (0.50 mol L-1). Figure S3 (SI) shows a representative cyclic voltammogram of the Pt working electrode of the NFC with the QRE placed at the separated compartment (Figure S3 A1 and A2, SI) as well as placed in parallel with the flowing stream (Figure S3 B1 and B2, SI). The characteristic Pt electrochemical features are found for stationary conditions with the reference electrode placed at the independent upper side compartment, as shown in Figure S3 (A1, SI). Although there is a small amount of O2 dissolved in solution, we can clearly see the HUPD region between 0.70 and -0.30 V vs. Pt QRE, formation of Pt surface oxides starting at 0.06 V (vs. Pt QRE) and the reduction of such oxides through the cathodic current with a peak at 0.00 V. Moreover, the beginning of the oxygen evolution reaction can be seen at potentials greater than 0.70 V vs. Pt QRE (Figure S3 A1, SI). It is worth noticing that potentials close to the onset of oxygen evolution were reached in order to clean the surface by electrooxidizing remaining organic fragments of the previous use. In this sense, the previous usage of the working electrode does not influence the measurements, assuring reproducibility in further analysis. The injection of supporting electrolyte (continuous phase) at 0.35 µL s-1 through the microchannel into the NFC did not modify the characteristic electrochemical behavior of Pt when the QRE was kept at the upper side

compartment (having 300 µL of 0.50 mol L-1 H2SO4), as shown in Figure S3 (A2, SI). The Pt profile under flowing stream of supporting electrolyte matched the profile under stationary conditions. Moreover, both configurations displayed an ECSA of ~0.70 cm2 for the working electrodes, which guaranteed an efficient use of the whole surface area without trouble to further electroanalysis. The proper use of a cell in such configuration was not previously reported, however, the characteristic profiles (Figure S3 A1 and A2, SI) shown in this study indicate the appropriate connection between both compartments. The contact of the lower and upper side, which was enabled by the Pt wires (see inset image Figure S3, SI), generated electric conductivity without the contribution of the migrational mass transport to the current. Additionally, target results indicate that the potential applied on the WE is satisfactorily controlled in both compartments. A single channel containing the electrochemical cell in a three-electrode configuration would be preferable; however, here we prove that further electroanalysis may be compromised. Figures S3 (B1, SI) shows the profile of exactly the same Pt working electrode connected to the same counter electrode, but with the Pt QRE placed in the microchannel under stationary conditions. The thin layer configuration of the electrochemical cell limited the charge compensation between counter and working electrode, so that the QRE may be polarized, adding a migrational contribution to the current and possibly increasing cell resistance. As a result, the reduction potentials of Pt surface oxides were displaced towards lower values and the HUPD region was shifted to a region out of the studied scale (Figure S3, B1, SI). Similarly, this electrochemical behavior was seen under flowing stream conditions (Figure S3, B2 SI). This behavior demonstrated that the stream itself does not compromise the surface reactions taking place on the Pt working electrodes, but the position of the QRE. Most interestingly, the ECSA was ~0.40 cm2 when the QRE was placed inside the microchannel, which is ~40% smaller than the real area found with the QRE independently placed. This evidence led to the hypothesis that the real charge involved in the reduction of Pt surface oxides is underestimated, since the working electrode and scan rates were the same. In addition, the ECSA values calculated for both configuration are in agreement with those (Cottrell-type chronoamperometric experiments) obtained by using the ferricyanide as usual redox probe (Figure S4, SI). Therefore, the investigation of an analytical signal from an anodic transient in terms of charge or peak height may be misinterpreted when the QRE is placed in parallel to the flowing channel. Linear sweep voltammograms (Figure S5, SI) were recorded for the 100 µmol L-1 APAP and used (as electrochemical probe) to investigate the performance of the NFC compared to the CFC. The voltammograms of the Pt working electrode in the presence of APAP clearly showed an anodic current for both flow-cells with a peak potential at around 0.60 V (See Figure S5 A and B, SI), which is related to the two-electron process, as shown in Figure S5 (Inset). The anodic peak observed at the same potential



region when using the NFC (Figure S5 B, SI) ensured the reliability of our newly designed system compared to the commercial one. In this context, this work presents a NFC to be used as electrochemical detector, with the QRE isolated from flowing streams carrier, as detailed in the next sections. Amperometric measurements in presence of surfactants and pH dependence. It is well known that surfactants can adsorb at the surface of carbon based electrodes 16,29 and noble-metal based electrodes such as platinum and gold.16,17 Herein, these previous protocols were also used (after some adaptations) to obtain a surfactantmodified Pt in order to protect its surface from electrogenerated products of APAP. Subsequently, the NFC performance was evaluated alongside with the comparison of the data obtained by the CFC by measuring the amperometric response of APAP (50 µmol L-1) in the absence and presence of surfactants. The surfactants sodium dodecyl sulfate (SDS), triton X-100 and tween 20, all at a concentration of 0.10%, were added to the carrier (NaH2PO4, 0.10 mol L-1). Firstly, the experimental setup (Figure 1 and S2, SI) was mounted by connecting the potentiostat cables to the WE and CE (lower part of the NFC, Figure 1) and placing the QRE in the separate compartment (reservoir containing 300 µL of carrier, Figure 1). After using the protocol described in the experimental part (section 2.4), amperometric measurements were accomplished setting the applied potential at 0.55 V (NFC) and 0.60 V (CFC). The potentials were chosen according to the data presented in Figure S5 (SI). To enable the comparison of results, Figure 3 shows the performance of both flow-cells; tween 20 (at 0.10%) seemed more appropriate than the SDS and Triton X-100. Here, the tween 20 yielded more reproducible peak currents (Figure 3, amperograms A-C) throughout the six discrete injections. Despite the generation of lower peak currents when using the NFC in comparison to the CFC, there was no significant variability in the intensity observed within the studied conditions. To perform a more accurate check of the reproducibility, eight discrete injections (25 μL) with the selected tween 20 (at 0.10%) were carried out. Figure 3D shows that — even in presence of air bubbles introduced via the inlet flow (Figure 3D, inset image) —, the electrochemical response had no significant variance that affected the accuracy measurements of the peak current intensity (Ip = 64.8±2.0 nA). This result confirmed the high performance of the designed NFC, working properly without the common problems associated with the presence of air bubbles adhered onto the surface of working and references electrodes. Moreover, the use of the NFC ensured that the QRE (in a separated compartment) remained its clean surface throughout the process of covering the Pt-WE surface with surfactant. It is important to point out that this particularity avoids variability in the working potential and consequently the peak current intensity, leading to more accurate measurements. Based on the previous work,21–23 the surfactants will form air bubbles at the flowing streams analysis, which

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also gives rise to inaccuracies in the measurements. The presence of bubbles has particular effects in microdevices working under electrochemical detection with microscale electrodes, where they can easily block the electrical contact between the electrodes. Unquestionably, the hypothesis of air bubble formation and its effect in the modification of the active area of WE and QRE can be seen in Figure 3 (amperograms A-C). This reinforces that, for a conventional three-electrode configuration (such as assembled into the CFC), the QRE in direct contact with the flowing streams solution is more susceptible to blocking its surface by air bubbles. At this point, the presence of surfactants significantly affected the electrochemical detection for the CFC, having major effect on the electrodes activity, which, in turn, led to a poor reproducibility of the amperometric responses.

Figure 3. Amperograms registered for APAP (50 μmol L-1) to compare the performance of the NFC and CFC in the presence of the surfactant. (A) SDS (0.10%), (B) Triton X100 (0.10%), (C) tween 20 (0.10%). (D) Amperogram registered for eight injections (25 µL) after visualization of bubble into the microchannel adhered onto the Pt-WE surface. Applied potential of 0.55 V (NFC) and 0.60 V (CFC), injection volume of 25 µL and flow rate of 1.77 µL s1 . Carrier: NaH2PO4 (0.10 mol L-1) at pH 5.0. The pH dependence study and the evaluation of the best concentration of tween 20 are presented in Figure S6 (SI), testing the proposed NFC and the CFC as detection system. Then, comparing the performance of both flowcells, the amperometric data (Figure S6A, SI) shows that the peak current for APAP was affected by the influence of pH and the presence of tween 20 (0.10%) in the carrier. However, in absence of tween 20, there was an evidence of pH dependence, where at around pH 5.0 the peak current was largest. Indeed, the APAP has a strong acidic pKa (9.45, regarding the 4-hydroxyphenyl group) and its deprotonated form arises at pH higher than 11.5 (99% dissociated form). Accordingly, it would be expected that there is a small effect at pH lower than 9.0 and this is evidenced in the Figure S6A (SI). From this study, it would be expected that the APAP cannot electrostatically interact with tween 20 due to the unfavorable pH condition and the non-ionic nature of the target surfactant.


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Figure S6B (SI) shows the data generated to compare the performance of both flow-cells under the optimized pH and the presence of tween 20 at different concentrations. At a concentration of 0.10%, the maximum of the peak current may mean that the critical micellar concentration (CMC) has not yet been reached and under this condition, the surface coverage reaches its highest efficiency. Although it is widely reported that the surfactants play an important role in protecting the surface of electrodes against adsorption of electrogenerated products,16,30 the CMC should be controlled. This issue involves the micelle aggregates formation, which is sometimes undesired due to its effect on the electrochemical detection (in electroanalysis) as well as hindering the electrodeposition process (by surface coverage in surfactant-modified electrodes). In this sense, the tween 20 is recognized to have a low CMC value, which is highlighted as being around of 0.35%.31 Likewise, due to the non-ionic nature, the concentration of the electrolytes does not significantly change either the CMC or the surface tension at the CMC.32 Accordingly, the concentration of tween 20 at 0.10% is understood to be lower than its CMC and this condition was chosen to be used for further measurements in order to avoid losing the effectivity of Pt-surface coverage. Figure 4 shows a set of discrete injections (30 µL) of APAP (75 μmol L-1), air and the carrier itself carried out to effectively evaluated the response of the electrochemical device in the presence of bubbles. The current responses were approximately identical (Figure 4, amperograms a, c and e), demonstrating the highperformance of NFC as in absence as in presence of tween 20 (0.10%). In addition, the controlled generation of bubbles performed by the injection (30 µL) of air (Figure 4b) as well as the injection of the carrier itself confirms the high-performance of the NFC.

Figure 4. Discrete injections (30 µL) to compare the performance of the NFC under different conditions. (a) APAP (75 μmol L-1) diluted in tween 20 solution (0.10%) and injected into the carrier (in absence of tween), (b) injection of air into the carrier (having tween 20 at 0.10%), (c) injection of APAP (75 μmol L-1) in into the carrier (both in absence of tween 20), (d) injection of the blank solution (carrier in absence of tween 20) into the contin-

uous phase (carrier having tween 20 at 0.10%) and (e) carrier and the injected APAP (75 μmol L-1) both in presence of tween 20 (0.10%). Carrier: NaH2PO4 (0.10 mol L-1) at pH 5.0, applied potential of 0.55 V vs. Pt and flow rate of 1.77 µL s-1. According to the data and supported by previously published works,16,17 a schematic representation for electrodeposition of tween 20 on the Pt surface is illustrated in Figure 5. Briefly, the deposition was performed by using the experimental procedure described in section 2.4. Figure 5(I) shows the representation for the head and tail group present in the tween 20 and Figure 5(II) shows the schematic model for interaction of tween 20 with the Pt surface. In Figure 5 (IIA), the scheme shows the continuous phase with the carrier in presence of tween 20, while in Figure 5 (IIB) the discrete injection of APAP to the Tjunction way and its flowing stream into the designed three-dimensional coil-shaped microfluidic device (Figure 5, IIC) is shown. The Figure 5 (IID) shows the insights into how the tween 20 interacts (by its head group) with the Pt-surface and thus serves as antifouling agent to repel the electrogenerated products. Here, while the wellarranged films ensure the protection of the Pt-surface, the electron transfer to the APAP can still occur properly. This process avoids the fouling by repelling (to the downstream) the electrogenerated products from the surface. In addition, as tween 20 is a non-ionic surfactant, it is understood that under the cycling process (section 2.4), the head group (Figure 5I) interacts with the Pt and substantially changes its chemical properties by surfacecoverage, resulting in a well-arranged film as shown in Figure 5 (IID). In this case, due to the configuration of the NFC, the accurate analyses of the chemical composition of the surface regarding the interaction of tween with Pt is difficult to be performed by conventional techniques such as the XPS analyses. Thus, a series of amperometric measurements was used to demonstrate the analytical applicability and the results will be discussed in the following sections.

Figure 5. (I) Representation of the head and tail group present in the tween 20 surfactant and: (II) the schematic model for: (A) the continuous phase having the carrier and presence of tween 20, (B) the discrete injection (on



the T-junction way) of APAP and its flowing streams into the (C) designed three-dimensional coil-shaped microfluidic device and (D) the head group of the tween 20 attached to the Pt electrode. Evaluation of the injection volume and flow rate. It is well known that most of the microfluidic applications require stable flow rates, in general, performed by an external syringe pump.33 However, even when using both carrier and sample phases in a continuous pumping system, it is difficult to control some trouble because the flow rates inevitably tend to change over time. Here, the automatic micropipette is used to introduce a discrete volume of analyte/sample to the continuous phase (carrier), resembling a high-cost standard syringe pump commonly used in microfluidic devices. The automatic micropipette is a suitable tool to perform discrete injections into microfluidic devices, because it does not require complex automated workstations (e.g. automatized/ programmable syringe pump systems). Its drawback is the tendency to cause troubles (particularly in electrochemical detection) due to the pressure caused by the propulsion which influences the equilibrium between the carrier and the injected phases.34,35 Thus, this study also deals with the feasibility of using the automatic micropipette as discrete injector, on which working in association with the three-dimensional coil-shaped microfluidic device and the NFC, enable to solve the aforesaid drawback. The typical hydrodynamic voltammograms (Figure S7, SI) showed that the best amperometric detection is obtained at potential of 0.55 (NFC) and 0.60 (CFC) V vs. Pt QRE. In a set of experiments, firstly, the amperometric response was measured varying the volume of the electronic micropipette for discrete injections of APAP (50 μmol L-1). This variation increased the difference in the composition of the carrier and injected aliquot and this is known to cause perturbations in the equilibrium between both phases.36 Figure S8 (A1 and A2, SI) shows that this operation mode presents proper repeatability of peak heights for the NFC both in absence and in presence of tween 20 (0.10%). The amperometric response increased with the injected volume (ranged from 10 to 50 μL) and the accurate measurement of the peak current intensity could be performed without any trouble caused by the perturbation of the equilibrium between the continuous phase (carrier) and the discrete phase (analyte/sample). Contrarily, for the CFC (Figure S8 B1 and B2, SI) the variation of the injected volume (discrete phase) had a slight difference when comparing the results in the absence (Figure S8 B1) and presence (Figure S8 B2) of tween 20 (0.10%) in the carrier. Here, unlike observed for the NFC, the results for the CFC in presence of tween 20 and the increase in the injection volume (from 10 to 40 μL) clearly gave rise to troubles of signals at volumes higher than 30 µL, leading to variability in the measurement of the peak current intensity. This happened even when the experiments are handled carefully to avoid accidental insertion of air bubbles. Thus, the CFC configuration proved to be susceptible to the random errors associated with the vari-

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ations in the injection volume and accidental insertion of air bubbles into the carrier stream. In a subsequent set of experiments, Figure S9 (SI) shows typical amperograms registered for variation (from 0.58 to 4.53 µL s-1) of the flow rate. For this study, the electrochemical response (signals, a-j) registered for the NFC was characterized by a small variability in the peak current intensity. The average of 66±8 nA (absence of tween 20, Figure S9, A1) almost complies with the 65±2 nA obtained in the presence of tween 20 (Figure S9, A2). Otherwise, the CFC, although the electrochemical response was always greater than generated by NFC, showed a variability in the peak current intensity (especially in presence of tween 20) throughout the increase of the flow rate from 0.58 to 4.53 µL s-1. Here, the peak current intensity registered in absence of surfactant (Figure S9, B1) increased from 162±12 nA (Figure S9 B1, a-e) to 238±10 nA (Figure S9 B1, g-h) with ∆Ip = 98±4 nA (throughout the flow rate variation between a and j). Similar observations could be made, but with random changes in amplitude, when the tween 20 (0.10%) was present in the carrier (Figure S9, B2). The most concerning fact is the lack of reproducibility in the results — (Ip = 168±30 nA, Figure S9B2, a-e) and (Ip = 252±10 nA, Figure S9B2, f-j), with ∆Ip = 108±5 nA — generated by the CFC under the flow rate variation. This could be explained by the fact that, when the tween 20 was present in the carrier and the flow rate was decreased, the bubbles became more active in blocking the microelectrodes activity. When this happened, eventually, the electrical contact between the QRE and the WE was lost, resulting in more variability in the electrochemical signal. As one of the main effects, Figure S9 (B1 and B2, SI) shows that the automatic micropipette alongside with the presence of tween 20 in the carrier, created unsuitable conditions when using the CFC. This is more evident at flow rates lower than 2.55 μL s-1, which eventually, can trouble the amperometric response and increase the variability of peak current intensity. The insights in how the flow rate and presence of tween 20 affect the electrochemical response are shown in Figure S10 (SI). After performing three injections (at 53, 350 and 650 s), both flow-cell configurations were expressively affected by a disturbance of signals observed at the moment of each discrete injection into the continuous phase. The movement of the piston to execute the automatic injection/propulsion generated pressure that perturbed the equilibrium between both phases, resulting in highly noisy peaks, which superimposed the main peak. On the one hand, due to the conventional arrangement of the microelectrodes in the CFC, this perturbation was more pronounced (Figure S10A, SI). On the other hand, the NFC proved again to be useful when attempting to reduce the noise and the turbulence/troubles caused by the micropipette. For that reason, due to its design (with QRE separate from the flowing streams solution), it allowed maintenance of its performance even in the variations associated with the flow rate, injection volume and accidental insertion of air bubbles. It is worth noting that a


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higher flow rate is advantageous for decreasing the peak width at half-height as well as increasing the throughput time (Figure S9, f-j). Taking into account these outcomes, the injection volume of 30 µL and flow rate of 1.77 μL s-1 were chosen for the further experiments. Analytical performance: Analytical characteristics and sample analysis. When comparing the analytical performance of the NFC both in absence (Figure 6, A1) and presence (Figure 6, A2) of tween 20 in the carrier, it can be seen that it fulfilled the requirements over the studied concentration range. Repeatable results with high-performance linear relationship between the APAP concentration (from 1.00 to 100 µmol L-1) and the electrochemical response (Figure 6, A3) were achieved. Likewise, in absence of surfactant (Figure 6, B1), the CFC satisfied with good coefficient of determination (R2) for the regression of the linear range. However, in presence of the tween 20, the magnitude of the peak current reached a limiting value, which was evidenced at a concentration higher than 25 µmol L-1. Due to this issue, the linear range was limited between 1.00 and 25 µmol L-1 (Figure 6, B3). Expressions of the linearity are reported in Table S2 (SI), where, in particular, the high-performance of the NFC in generating more accurate responses is highlighted, fulfilling the requisites for the analytical application in a miniaturized electrochemical flow-cell. To access the reliability of the analytical response for both flow-cells, the performance regarding limits of detection and quantitation (LoD and LoQ) values were calculated as highlighted by Ruggieri and co-workers.36 At this point, the data produced by ten discrete injections of the lake water samples spiked at three concentration levels (low, middle and high) were evaluated to check the matrix influences (so called LoD and LoQ under matrixspecific). The equations reported in Table S2 were used as reference test for the establishment of acceptable LoD and LoQ values at each spiked concentration level. Figure 7 shows the results of eight discrete injections performed to demonstrate the applicability of the target NFC with respect to the repeatability after changing the medium between absence and presence of tween 20 (0.10%) in the carrier combined with the concentration of APAP. Again, the high-performance of the NFC, when

measuring the amount of APAP in spiked lake water sample and in an oral solution of Paracetamol, is evident. Figure 7 (blue curves) shows that, despite the attenuation in the magnitude of the peak current in the presence of tween (0.10%), it remained constant and repeatable throughout the discrete injections. A well-established electrochemical response was observed and the peak current intensity measured using the proposed NFC maintained more than 94% of its initial value after eight discrete injections both in presence and in absence of tween 20. Otherwise, for the CFC, the variability in the amperometric response was evident in both: absence (Figure S11A1, SI) and presence of matrix (Figure S11B1, SI). Similarly, the surfactant caused serious troubles (Figure S11 A1 and A2, SI) and, in some cases, no electrochemical response due to the presence of strong noise (Figure S11A2, SI). Again, it can be ascribed that the air bubble formation blocked the surfaces of the microelectrodes (especially QRE and WE), resulting in decreased activity. This behavior can be responsible for the non-linear response observed in Figure 6 (B3), and the ability of the method to quantify the analyte might vary with the sample matrix, frustrating the requirements of highperformance analytical applications. To demonstrate the functionality of this method, the quantitative application was confirmed using the APAP as model analyte to spike lake water samples and in a pharmaceutical formulation (oral solution of Paracetamol). Table S3 (SI) shows the quantification performed by diluting target samples at three spiked levels (low, middle and high). This procedure allows to reduce the uncertainty associated with the matrix effect and to validate the method regarding its ability to detect the target analyte from high to low level of concentration. As summarized in Table S3 (SI), the claimed concentrations versus the amount found were acceptable with values of recovery greater than 81% for the NFC and 78% for the CFC. There was a tendency of lower recovery values at high concentration of APAP, possibly due to the electrode fouling that takes place even after protecting the Pt-surface with surfactant coverage.



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Figure 6. Amperograms and the calibration plots for triplicate injections (30 µL) of APAP at different concentrations. Concen-1 tration range: (a) 1.00, (b) 5.00, (c) 10.0, (d) 25.0, (e) 50.0, (f) 75.0, (g) 100 µmol L . Applied potential: (NFC) 0.55 V and (CFC) 0.60 V. Other conditions as in Table S1 (SI).

Figure 7. Amperograms to compare the performance of the NFC under: (A1) injection of paracetamol solution having APAP (66.2 µmol L-1) in absence and presence of tween 20 (0.10%), (A2) injection of lake water spiked with APAP (66.2 µmol L-1) in the absence and presence of tween 20 (0.10%), (B1) standard of APAP (100 µmol L-1) injected in the absence and presence of tween 20 (0.10%), (B2) standard of APAP (50 µmol L-1) injected in the absence and presence of tween 20 (0.10%). Other conditions are in Figure 6 and Table S1 (SI).

CONCLUSION As conclusion, the successful use of a portable automatic electronic micropipette for discrete analyte/sample injections and a newly designed electrochemical flow-cell as detection system integrated into the three-dimensional microfluidic platform was demonstrated. This integration

ASSOCIATED CONTENT

allows to overcome the challenge in reducing the perturbation of the equilibrium in the continuous phase (carrier streams), a drawback caused by the discrete injection performed by micropipettes. Indeed, the most notable finding was the high performance of the flow-cell configuration regarding generation of accurate results even in the presence of surfactant, where the electrochemical response is known to be troubled. The benefit of using the NFC over a conventional electrochemical detector system is the ability to be miniaturized and integrated within a microfluidic platform without losing the sensitivity. These novelties have significant effect on system simplification and cost reduction avoiding complicated fabrication or expensive equipment to solve the problems of air bubble formation. To prove the viability and versatility of this finding, we also have successfully applied a developed method to enhance the determination of APAP even at high concentration. The electrochemical detection of APAP at high concentration has been reported as big challenge due to the formation of passive films of its products over electrode surfaces, which critically affected the accuracy and precision of the electrochemical measurements. We foresee that this system also will find broad applications in studying biological, pharmaceutical and environmental applications that involve complex matrices and the requirements of a miniaturized system such as an electrochemical detector with noblemetal electrodes. For the next step, our goal is to incorporate all main parts directly into one microfluidic platform to obtain microfluidic chip platforms in scalable manufacturing methods and explore its application in clinical analysis and/or the electrochemical detection of biomarkers from cell cultures on microfluidic reactors, in which the bubbles are recognized to have a damaging impact. Supporting Information contains: chemical structures, voltammograms under stationary and flowing stream configura-


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tions, amperograms and Tables summarizing the evaluation of the experimental conditions and sample analysis.

AUTHOR INFORMATION Corresponding Author *Magno Aparecido Gonçalves Trindade [email protected] and [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version. Note: The authors declare no competing financial interest.

ACKNOWLEDGMENT The financial support by: Brazilian funding agency CNPq/INCT (Grant: 465571/2014-0), CNPq (Grant: 306504/2011-1 and 402832/2016-7). Especially, CAPES Foundation, Brazilian Federal Agency for the scholarship (Grant: 88881.119436/2016-01).

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New Electrochemical Flow-Cell Configuration Integrated into a Three

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