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Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems
C H A P T E R
16 Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems Mario Castaño-Álvarez, Diego F. Pozo-Ayuso, Ana Fernández-la-Villa MicruX Technologies, Gijón, Asturias, Spain
16.1 Background Nowadays, new analytical devices that fulfill features such us portability, automation, user-friendliness, low cost, low power requirements, etc., are required. Therefore, a current trend in Analytical Chemistry is focused on the development of micro total analysis systems (mTAS) or lab-on-a-chip (LOC) platforms [1]. Microfluidics is the engineering or use of devices that applies fluid flow to channels smaller than 1 mm in at least one dimension. Microfluidic devices can reduce reagent consumption, allow well-controlled mixing and particle manipulation, integrate and automate multiple assays, and facilitate imaging and tracking. Electrochemical transducers offer also multiple advantages such as low cost, portability, and low power requirements. Moreover, they can be easily integrated on microfluidic devices because of their compatibility with the microfabrication technologies. Thus, the possibility of integrating both systems, electrochemistry and microfluidics, contributes to the development of true LOC platforms [2,3]. Microfluidic devices and electrochemical sensors can be coupled into a flow injection analysis (FIA) system to improve the automation and high throughput of the platforms (see also Chapters 5, 9 and 28 with other electrochemical FIA systems). Flow cells facilitate analyte delivery to electrode surfaces for a range of applications such as clinical diagnostics or food and pharmaceutical analysis. The most common flow cells used in an FIA system are wall-jet and thin-layer arrangements (Fig. 16.1) [4].
Laboratory Methods in Dynamic Electroanalysis https://doi.org/10.1016/B978-0-12-815932-3.00016-4
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Copyright © 2020 Elsevier Inc. All rights reserved.
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16. Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems
(A)
(B) Inlet Outlet
Electrode surface
Top view
FIGURE 16.1
Inlet
Outlet
Electrode surface
Top view
Sketch of typical (A) wall-jet and (B) thin-layer flow cells.
In a wall-jet cell, the axis of the solution stream is normal to the electrode surface and the solution is drained away completely from the electrode vicinity after contacting the electrode. In a thin-layer cell, the solution flows through a thin flat channel parallel to the electrode surface, which is embedded in the channel wall. The use of microfluidics improves the design and performance of thin-layer flow cells. Thus, the solution layer thickness is easily controlled through the dimensions of the microfluidic channels. Microfluidics enable a perfect control of the dimensions of microchannels and the positioning of electrodes, decreasing the cell volume, the sample, and reagents requirements and enhancing the efficiency and sensitivity of the system. Therefore, integration of microfluidics and electrochemical detection on a single chip allow enhancing the control of fluids over the electrode surface. The use of a thin-layer-based flow cell in FIA systems is ideal for (bio)chemical sensors development. Acetaminophen, N-acetyl-p-aminophenol, or paracetamol (APAP) is a common analgesic and antipyretic drug formulated in a variety of dosage forms. APAP is used for the relief of fever, headaches, and other minor aches and pains. Their determination in pharmaceuticals is of paramount importance because an overdose of APAP can cause fulminating hepatic or renal necrosis and other toxic effects. Hepatic toxicity begins with plasma levels of APAP in the 120 mg/mL range 4 h after the ingestion and an acute damage is presented with plasmatic levels up to 200 mg/mL 4 h after the ingestion. p-aminophenol (pAP), the primary hydrolytic degradation product of APAP, can be present in pharmaceutical preparations of APAP as a synthetic intermediate or as a degradation product. pAP is limited to the low level of 50 mg/mL (0.005% w/w) in the drug raw material and 0.1% w/w in tablet formulations by the European [5], United States [6], and Chinese [7] pharmacopoeias. The low level ensures APAP drug safety because pAP has significant nephrotoxicity and teratogenic effects.
II. Electroanalysis and microfluidics
16.2 Flow injection analysis system setup
163
Therefore, establishment of simple, economical, and accurate analytical methods for determination of APAP and pAP would be useful to medical manufacturers, etc., for investigation of the stability of APAP, pharmaceutical analysis, and quality control. This lab experiment is focused on undergraduate and postgraduate students of different areas (Chemistry, Pharmacy, Biology, etc.) and can be performed in two/three sessions of 3 h. The students will be able to acquire practical skills in the use of microfluidic devices integrating electrochemical sensors in an FIA system.
16.2 Flow injection analysis system setup The FIA system setup for using the microfluidic electrochemical sensor and thin-film electrodes is shown in Fig. 16.2.
FIGURE 16.2 Flow injection analysis system setup for microfluidic sensors and thin-film electrodes. AIO, all-inone; PC, personal computer.
Microfluidic electrochemical sensors (Fig. 16.3A) consist of a three-electrode system fabricated on gold (150 nm) or platinum (150 nm) deposited on a glass substrate (10 6 mm). The working electrode has a geometrical area of 0.3 mm2. An SU-8 resin layer is used for building the microfluidic channel on the glass substrate with the electrodes. The channel is 40 mm height with a width of 250 mm and 1 mm for the electrochemical cell. The single channel
FIGURE 16.3 (A) Microfluidic electrochemical sensor and (B) thin-film single-electrode layouts. AE, auxiliary electrode; RE, reference electrode; WE, working electrode.
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16. Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems
(A)
(B)
Bo om view
Top view
FIGURE 16.4
Top view
Bo om view
(A) Thin-layer and (B) wall-jet flow cell add-ons.
has a length of 3 mm. Finally, an SU-8 film containing the inlet/outlet is used as cover for closing the microfluidic channel. The basic thin-film single electrodes (Fig. 16.3B) also consist of a three-electrode system fabricated on gold (150 nm) or platinum (150 nm) deposited on a glass substrate (10 6 mm). In this case, an SU-8 layer is simply used for delimiting the electrochemical cell (2 mm diameter). The working electrode has a diameter of 1 mm with a geometrical area of 0.8 mm2. Two different flow cell add-ons are used with the all-in-one (AIO) platform depending on the sensor. For the microfluidic sensors, the flow cell add-on (Fig. 16.4A) consists of two independent inlet and outlet isolated with two O-rings enabling a thin-layer approach. For the thin-film single electrodes, the flow cell add-on (Fig. 16.4B) consists of two inlets/outlets embedded in the same O-ring enabling a wall-jet approach. Both approaches integrate standard fluidic ports (1/4 00 -28 UNF) with inlet channel of 0.5 mm ID.
16.3 Chemicals and supplies Reagents: Analytes: APAP and pAP. Preparation of electrolytes: Sulfuric acid (H2SO4), phosphoric acid (H3PO4), hydrochloric acid (HCl), and sodium hydroxide (NaOH). Solutions: Background electrolyte: The carrier used in the FIA system is 0.1 M H2SO4 and 0.1 M phosphate buffer (PB) pH 7.4. Prepare 1000 mL of each solution. Stock and standard solutions: 10 mM stock solutions of APAP and pAP are prepared in 10 mM HCl. Prepare 100 mL of each stock solution. For the FIA system, standard solutions of APAP and pAP (1, 10, 50, 100, 250, and 500 mM) are prepared by diluting the stock solutions in 0.1 M H2SO4 or 0.1 M PB pH 7.4. Prepare 25 mL of each standard solution.
II. Electroanalysis and microfluidics
16.5 Experimental procedure
165
Instrumentation and materials:
Potentiostat AIO platform (drop-cell and thin-layer/wall-jet flow cell). Thin-layer microfluidic single electrodes (TL-SE1-Pt/Au). Thin-film single electrodes (ED-SE1-Pt/Au). Additional instrumentation for the FIA system:
Peristaltic pump or syringe pump. Six-port injection valve (20 mL sample loop). Tubing and fittings. Volumetric material (flasks, pipettes, vessels, micropipettes, etc.) and all the materials necessary for the preparation of solutions, which should be of analytical reagent grade. Syringes and syringe filters (0.1e0.45 mm), used for removing particles of working solutions and samples.
16.4 Hazards Acid and alkaline solution preparations should be carried out under a fume hood. Protective garment and gloves should be worn at all times.
16.5 Experimental procedure 16.5.1 Electrochemical procedures 16.5.1.1 Electrode precleaning The metal surface of thin-film electrodes integrated into microfluidic chips should be cleaned to get the best electrochemical signals. The electrode surface is cleaned by a simple electrochemical pretreatment. With this aim: 1. Using the AIO cell with thin-layer (for TL-SE1 sensors) or wall-jet (for ED-SE1 sensors) flow cell add-on, drive the carrier throughout the FIA system (be sure the solution covers all the electrode surfaces in the cell without any bubble). 2. Stop the flow and perform a cyclic voltammetry experiment between 1.5 and þ1.5 V with a scan rate of 0.1 V/s (at least 10 cycles) for platinum-based electrodes and between 1.0 and þ1.0 V with a scan rate of 0.1 V/s (at least 12 cycles) for gold-based electrodes. 16.5.1.2 Amperometric measurements A flow cell (thin-layer or wall-jet) is used in the FIA system for obtaining the amperometric measurements in dynamic conditions. To get the signals: 1. Connect the flow cell to the FIA system and to the potentiostat. 2. Perform the electrode precleaning, as commented in Section 16.5.1.
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16. Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems
3. Drive the carrier continually throughout the system using the peristaltic pump (be sure no bubbles are formed in the tubing or flow cell). 4. Apply a constant detection potential to the electrode and record the baseline on the corresponding software window. 5. When the current of the baseline is stable, inject the standard solution (or sample) by using the six-port injection valve of the FIA system. This valve enables the injection of a sample volume in a very fast and reproducible way without disturbing the flow of carrier. In all the experiments, a sample volume of 20 mL is used. Record the signals on the software window.
16.5.2 Influence of the electrode material, carrier solution, and detection potential The electrode material, carrier (composition, ionic strength, pH, etc.), and detection potential affect the analytical signals obtained in the FIA system. The optimal detection potential is determined by performing the hydrodynamic voltammogram (HDV, see Chapters 5, 9 and 28). Using the microfluidic sensors in the FIA system with the thin-layer flow cell, 1. Study the influence of the detection potential for APAP by varying it between 0.0 and þ1.0 V, for a 100-mM solution, with microfluidic gold- and platinum-based electrodes using different carrier solutions (0.1 M H2SO4 and 0.1 M PB pH 7.4). 2. Study the influence of the detection potential for pAP by varying it between 0.0 and þ1.0 V, for a 100-mM solution, with microfluidic gold- and platinum-based electrodes using different carrier solutions (0.1M H2SO4 and 0.1 M PB pH 7.4). For all the experiments, fix a flow rate of 1.0 mL/min. The flow rate in the system can also affect analytical signals (shape and height of the peaks). Then, a study of the flow rate can be also accomplished. After these studies, the most appropriate detection potential, carrier, and electrode material is selected for the detection of each analyte.
16.5.3 Analytical parameters Using the optimal detection potential, electrode material, and carrier: 1. Study the precision (repeatability) for successive injections (at least 15 measurements) of standard samples of 10 and 100 mM APAP solutions as well as 10 and 100 mM pAP solutions. Evaluate the intrachip (in the same chip) and interchip (in different chips) precision (Fig. 16.5). 2. Study the effect of the compound concentration on the analytical signals (calibration plot). Prepare solutions of different concentrations of APAP and pAP (values ranging from 1 mM to 1 mM) to be injected in the flow system. Perform the calibration plot, starting first by the lowest concentration to the highest. Then, repeat injections from the highest concentration to the lowest one. This study also allows checking the durability of the electrodes without spotting an electrode fouling effect (Fig. 16.6). II. Electroanalysis and microfluidics
16.5 Experimental procedure
FIGURE 16.5
167
Fiagrams for successive injections in (A) the same and (B) different microfluidic chips.
16.5.4 Comparison of flow injection analysis systems: wall-jet versus microfluidic thin-layer flow cells Microfluidic sensors enable the use of a thin-layer approach in an FIA system. Thus, they enable an excellent control of fluids through the electrode surface, decreasing the volume of sample and reagents as well as enhancing the sensitivity with very low dead volume. To check the high performance of the thin-layer approach, a comparison study between the microfluidic sensors with the thin-layer approach and a thin-film single electrode with a wall-jet configuration is accomplished. Perform successive injections of standard samples of 100 mM APAP and 100 mM pAP (separately) in the FIA system with the microfluidic thin-layer sensors using the optimal
FIGURE 16.6
Amperometric response for increasing/decreasing concentrations of acetaminophen.
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FIGURE 16.7
16. Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems
Comparison of analytical signals for (A) wall-jet and (B) microfluidic thin-layer approaches.
conditions. Repeat the same experiments but using the thin-film single electrodes with the wall-jet approach. In this case, as the electrode area is different, for a better comparison, the analytical signals are normalized (current density, J) considering the area of the electrodes (Fig. 16.7).
16.5.5 Paracetamol determination 16.5.5.1 Sample preparation 1. Pulverize finely a tablet of paracetamol in a mortar. 2. Weigh accurately three samples. Powder can be directly weighed. 3. Solve each weighed sample directly in deionized water and transfer quantitatively to a 100-mL volumetric flask, making up with deionized water. 4. Filter the solutions using a syringe filter (0.1e0.45 mm). 5. Dilute the sample to the desired concentration with the background electrolyte. Prepare 25 mL of each diluted sample. 6. Inject directly into the FIA system. 16.5.5.2 Sample measurement The analysis of the paracetamol samples is performed using the microfluidic thin-layer sensors under optimal conditions. APAP determination in drug samples is carried out using two different methodologies: 1. Calibration with standard solutions: The calibration plot accomplished in Section 16.7 is used for the determination of the paracetamol concentration in the sample. Thus, the sample (paracetamol tablet/powder dissolved in 100 mL of deionized water) diluted in the carrier is directly injected into the FIA system using the six-port valve. Perform at least three replicates of the sample. The mean current intensity of the peaks is used to calculate the paracetamol using the calibration curve.
II. Electroanalysis and microfluidics
16.7 Additional notes
169
2. Standard addition method: It is used in instrumental analysis to determine the concentration of a substance (analyte) in an unknown sample by comparison to a set of standards of known concentration included in the sample matrix. Standard addition method can be applied to most analytical techniques and is used instead of a calibration curve to solve matrix effect problems and get a better precision. Prepare four drug samples (paracetamol tablet/powder dissolved in 100 mL of deionized water) in separated flasks. One of the samples is directly diluted with the carrier to the desired concentration (as in the previous methodology). In the other samples, increased volumes of an APAP standard solution (solution of known concentration of analyte) are added. For obtaining the standard addition curve, the intensity of current is plotted against the concentration of standard added. The intercept in the x-axis corresponds to the concentration of APAP in the sample.
16.6 Lab report At the end of the experiment, write a lab report including an introduction, experimental (materials, equipment, and protocols), results and discussion, and conclusions sections. The following points should be considered in the report: 1. Sketch the HDVs for APAP and pAP. Determine the optimal potential for each compound. 2. Determine the precision (% RSD, relative standard deviation) of the peak current for the compounds in the same (intra) and different (inter) chips. 3. Draft the calibration plots and determine the main analytical parameters (linear range, sensitivity, limit of detection, and limit of quantification) for each compound under optimal conditions. 4. Include the fiagrams for APAP and pAP using the wall-jet and microfluidic thin-layer approaches. For a better comparison, the peak current must be normalized taking into account the area of the working electrode. 5. Sketch the calibration curve of paracetamol obtained by the standard addition method considering the APAP additions. Determine the APAP concentration in the drug samples. Compare the results with those obtained by using the external calibration curve (using standard solutions of APAP).
16.7 Additional notes 1. Stock solutions should be kept on dark and stored at 4 C. 2. All working solutions/samples have to be filtered using a syringe filter of 0.1e0.45 mm to remove small particles, which can block the tubing/flow cell. 3. Be sure the flow cell add-on is correctly placed on the AIO platform to avoid the leakage of fluids. 4. Be sure there are no bubbles in the system (tubing and flow cell), especially on the electrode surface. Bubbles affect the accuracy, precision, and performance of the system.
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5. The electrode surface should be cleaned before starting first experiments. In this pretreatment the different particles adhered to the electrode surface are removed with hydrogen and oxygen gases generated. After the pretreatment, the electrodes could be used for several measurements depending on the sample. Thin-film electrodes could be reused after a new precleaning process. 6. The flow rate employed affect the analytical signals (shape and height of the peaks). A study of the flow rate can be accomplished. The peristaltic pump or syringe pump should be calibrated previously to the experiments performed in the FIA system. 7. In all the experiments, typically, three consecutive injections are performed to improve the precision and obtain better results. 8. Generally, pharmaceuticals state the paracetamol amount in the leaflet. It can be used to calculate the method recovery and also to select the most appropriate dilution of the sample (different dilutions should be performed depending of the pharmaceuticals). Be sure, after the dilution, the APAP concentration is in the lineal range (with and without the additions).
16.8 Assessment and discussion questions 1. 2. 3. 4.
What are the electrochemical processes involved for each compound? Draw the wall-jet and thin-layer flow cell configurations. What is the optimal detection potential for each compound? How is it chosen? Which flow cell approach shows a higher throughput for the determination of paracetamol? Why? 5. In real sample analysis, which methodology is more precise, the calibration curve with standard solutions or the standard addition method?
References [1] J. West, M. Becker, S. Tombrink, A. Manz, Micro total analysis systems: latest achievements, Anal. Chem. 80 (2008) 4403e4419. [2] Lab-on-a-Chip Technology, in: K.E. Herold, A. Rasooly (Eds.), Fabrication and Microfluidics, Caister Academic Press, 2009. [3] K.E. Herold, A. Rasooly (Eds.), Lab-on-a-Chip Technology: Biomolecular Separation and Analysis, Caister Academic Press, 2009. [4] M. Trojanowicz, Flow Injection Analysis: Instrumentation and Application, World Scientific, 2000. [5] The European Pharmacopeial Convention, the Sixth Edition European Pharmacopoeia, 2007, p. 0049. [6] The United States Pharmacopeial Convention, USP (the United States pharmacopoeia) 27-NF (The National Formulary) 22, vol. 27, 2004, p. 2494. [7] Editor Committee of National Pharmacopoeia, Chinese Encyclopedia of Medicines, vol. 2, Chemical Industry Press, Beijing, 2000, p. 206.
II. Electroanalysis and microfluidics
16 Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems Mario Castaño-Álvarez, Diego F. Pozo-Ayuso, Ana Fernández-la-Villa MicruX Technologies, Gijón, Asturias, Spain
16.1 Background Nowadays, new analytical devices that fulfill features such us portability, automation, user-friendliness, low cost, low power requirements, etc., are required. Therefore, a current trend in Analytical Chemistry is focused on the development of micro total analysis systems (mTAS) or lab-on-a-chip (LOC) platforms [1]. Microfluidics is the engineering or use of devices that applies fluid flow to channels smaller than 1 mm in at least one dimension. Microfluidic devices can reduce reagent consumption, allow well-controlled mixing and particle manipulation, integrate and automate multiple assays, and facilitate imaging and tracking. Electrochemical transducers offer also multiple advantages such as low cost, portability, and low power requirements. Moreover, they can be easily integrated on microfluidic devices because of their compatibility with the microfabrication technologies. Thus, the possibility of integrating both systems, electrochemistry and microfluidics, contributes to the development of true LOC platforms [2,3]. Microfluidic devices and electrochemical sensors can be coupled into a flow injection analysis (FIA) system to improve the automation and high throughput of the platforms (see also Chapters 5, 9 and 28 with other electrochemical FIA systems). Flow cells facilitate analyte delivery to electrode surfaces for a range of applications such as clinical diagnostics or food and pharmaceutical analysis. The most common flow cells used in an FIA system are wall-jet and thin-layer arrangements (Fig. 16.1) [4].
Laboratory Methods in Dynamic Electroanalysis https://doi.org/10.1016/B978-0-12-815932-3.00016-4
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Copyright © 2020 Elsevier Inc. All rights reserved.
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16. Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems
(A)
(B) Inlet Outlet
Electrode surface
Top view
FIGURE 16.1
Inlet
Outlet
Electrode surface
Top view
Sketch of typical (A) wall-jet and (B) thin-layer flow cells.
In a wall-jet cell, the axis of the solution stream is normal to the electrode surface and the solution is drained away completely from the electrode vicinity after contacting the electrode. In a thin-layer cell, the solution flows through a thin flat channel parallel to the electrode surface, which is embedded in the channel wall. The use of microfluidics improves the design and performance of thin-layer flow cells. Thus, the solution layer thickness is easily controlled through the dimensions of the microfluidic channels. Microfluidics enable a perfect control of the dimensions of microchannels and the positioning of electrodes, decreasing the cell volume, the sample, and reagents requirements and enhancing the efficiency and sensitivity of the system. Therefore, integration of microfluidics and electrochemical detection on a single chip allow enhancing the control of fluids over the electrode surface. The use of a thin-layer-based flow cell in FIA systems is ideal for (bio)chemical sensors development. Acetaminophen, N-acetyl-p-aminophenol, or paracetamol (APAP) is a common analgesic and antipyretic drug formulated in a variety of dosage forms. APAP is used for the relief of fever, headaches, and other minor aches and pains. Their determination in pharmaceuticals is of paramount importance because an overdose of APAP can cause fulminating hepatic or renal necrosis and other toxic effects. Hepatic toxicity begins with plasma levels of APAP in the 120 mg/mL range 4 h after the ingestion and an acute damage is presented with plasmatic levels up to 200 mg/mL 4 h after the ingestion. p-aminophenol (pAP), the primary hydrolytic degradation product of APAP, can be present in pharmaceutical preparations of APAP as a synthetic intermediate or as a degradation product. pAP is limited to the low level of 50 mg/mL (0.005% w/w) in the drug raw material and 0.1% w/w in tablet formulations by the European [5], United States [6], and Chinese [7] pharmacopoeias. The low level ensures APAP drug safety because pAP has significant nephrotoxicity and teratogenic effects.
II. Electroanalysis and microfluidics
16.2 Flow injection analysis system setup
163
Therefore, establishment of simple, economical, and accurate analytical methods for determination of APAP and pAP would be useful to medical manufacturers, etc., for investigation of the stability of APAP, pharmaceutical analysis, and quality control. This lab experiment is focused on undergraduate and postgraduate students of different areas (Chemistry, Pharmacy, Biology, etc.) and can be performed in two/three sessions of 3 h. The students will be able to acquire practical skills in the use of microfluidic devices integrating electrochemical sensors in an FIA system.
16.2 Flow injection analysis system setup The FIA system setup for using the microfluidic electrochemical sensor and thin-film electrodes is shown in Fig. 16.2.
FIGURE 16.2 Flow injection analysis system setup for microfluidic sensors and thin-film electrodes. AIO, all-inone; PC, personal computer.
Microfluidic electrochemical sensors (Fig. 16.3A) consist of a three-electrode system fabricated on gold (150 nm) or platinum (150 nm) deposited on a glass substrate (10 6 mm). The working electrode has a geometrical area of 0.3 mm2. An SU-8 resin layer is used for building the microfluidic channel on the glass substrate with the electrodes. The channel is 40 mm height with a width of 250 mm and 1 mm for the electrochemical cell. The single channel
FIGURE 16.3 (A) Microfluidic electrochemical sensor and (B) thin-film single-electrode layouts. AE, auxiliary electrode; RE, reference electrode; WE, working electrode.
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16. Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems
(A)
(B)
Bo om view
Top view
FIGURE 16.4
Top view
Bo om view
(A) Thin-layer and (B) wall-jet flow cell add-ons.
has a length of 3 mm. Finally, an SU-8 film containing the inlet/outlet is used as cover for closing the microfluidic channel. The basic thin-film single electrodes (Fig. 16.3B) also consist of a three-electrode system fabricated on gold (150 nm) or platinum (150 nm) deposited on a glass substrate (10 6 mm). In this case, an SU-8 layer is simply used for delimiting the electrochemical cell (2 mm diameter). The working electrode has a diameter of 1 mm with a geometrical area of 0.8 mm2. Two different flow cell add-ons are used with the all-in-one (AIO) platform depending on the sensor. For the microfluidic sensors, the flow cell add-on (Fig. 16.4A) consists of two independent inlet and outlet isolated with two O-rings enabling a thin-layer approach. For the thin-film single electrodes, the flow cell add-on (Fig. 16.4B) consists of two inlets/outlets embedded in the same O-ring enabling a wall-jet approach. Both approaches integrate standard fluidic ports (1/4 00 -28 UNF) with inlet channel of 0.5 mm ID.
16.3 Chemicals and supplies Reagents: Analytes: APAP and pAP. Preparation of electrolytes: Sulfuric acid (H2SO4), phosphoric acid (H3PO4), hydrochloric acid (HCl), and sodium hydroxide (NaOH). Solutions: Background electrolyte: The carrier used in the FIA system is 0.1 M H2SO4 and 0.1 M phosphate buffer (PB) pH 7.4. Prepare 1000 mL of each solution. Stock and standard solutions: 10 mM stock solutions of APAP and pAP are prepared in 10 mM HCl. Prepare 100 mL of each stock solution. For the FIA system, standard solutions of APAP and pAP (1, 10, 50, 100, 250, and 500 mM) are prepared by diluting the stock solutions in 0.1 M H2SO4 or 0.1 M PB pH 7.4. Prepare 25 mL of each standard solution.
II. Electroanalysis and microfluidics
16.5 Experimental procedure
165
Instrumentation and materials:
Potentiostat AIO platform (drop-cell and thin-layer/wall-jet flow cell). Thin-layer microfluidic single electrodes (TL-SE1-Pt/Au). Thin-film single electrodes (ED-SE1-Pt/Au). Additional instrumentation for the FIA system:
Peristaltic pump or syringe pump. Six-port injection valve (20 mL sample loop). Tubing and fittings. Volumetric material (flasks, pipettes, vessels, micropipettes, etc.) and all the materials necessary for the preparation of solutions, which should be of analytical reagent grade. Syringes and syringe filters (0.1e0.45 mm), used for removing particles of working solutions and samples.
16.4 Hazards Acid and alkaline solution preparations should be carried out under a fume hood. Protective garment and gloves should be worn at all times.
16.5 Experimental procedure 16.5.1 Electrochemical procedures 16.5.1.1 Electrode precleaning The metal surface of thin-film electrodes integrated into microfluidic chips should be cleaned to get the best electrochemical signals. The electrode surface is cleaned by a simple electrochemical pretreatment. With this aim: 1. Using the AIO cell with thin-layer (for TL-SE1 sensors) or wall-jet (for ED-SE1 sensors) flow cell add-on, drive the carrier throughout the FIA system (be sure the solution covers all the electrode surfaces in the cell without any bubble). 2. Stop the flow and perform a cyclic voltammetry experiment between 1.5 and þ1.5 V with a scan rate of 0.1 V/s (at least 10 cycles) for platinum-based electrodes and between 1.0 and þ1.0 V with a scan rate of 0.1 V/s (at least 12 cycles) for gold-based electrodes. 16.5.1.2 Amperometric measurements A flow cell (thin-layer or wall-jet) is used in the FIA system for obtaining the amperometric measurements in dynamic conditions. To get the signals: 1. Connect the flow cell to the FIA system and to the potentiostat. 2. Perform the electrode precleaning, as commented in Section 16.5.1.
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3. Drive the carrier continually throughout the system using the peristaltic pump (be sure no bubbles are formed in the tubing or flow cell). 4. Apply a constant detection potential to the electrode and record the baseline on the corresponding software window. 5. When the current of the baseline is stable, inject the standard solution (or sample) by using the six-port injection valve of the FIA system. This valve enables the injection of a sample volume in a very fast and reproducible way without disturbing the flow of carrier. In all the experiments, a sample volume of 20 mL is used. Record the signals on the software window.
16.5.2 Influence of the electrode material, carrier solution, and detection potential The electrode material, carrier (composition, ionic strength, pH, etc.), and detection potential affect the analytical signals obtained in the FIA system. The optimal detection potential is determined by performing the hydrodynamic voltammogram (HDV, see Chapters 5, 9 and 28). Using the microfluidic sensors in the FIA system with the thin-layer flow cell, 1. Study the influence of the detection potential for APAP by varying it between 0.0 and þ1.0 V, for a 100-mM solution, with microfluidic gold- and platinum-based electrodes using different carrier solutions (0.1 M H2SO4 and 0.1 M PB pH 7.4). 2. Study the influence of the detection potential for pAP by varying it between 0.0 and þ1.0 V, for a 100-mM solution, with microfluidic gold- and platinum-based electrodes using different carrier solutions (0.1M H2SO4 and 0.1 M PB pH 7.4). For all the experiments, fix a flow rate of 1.0 mL/min. The flow rate in the system can also affect analytical signals (shape and height of the peaks). Then, a study of the flow rate can be also accomplished. After these studies, the most appropriate detection potential, carrier, and electrode material is selected for the detection of each analyte.
16.5.3 Analytical parameters Using the optimal detection potential, electrode material, and carrier: 1. Study the precision (repeatability) for successive injections (at least 15 measurements) of standard samples of 10 and 100 mM APAP solutions as well as 10 and 100 mM pAP solutions. Evaluate the intrachip (in the same chip) and interchip (in different chips) precision (Fig. 16.5). 2. Study the effect of the compound concentration on the analytical signals (calibration plot). Prepare solutions of different concentrations of APAP and pAP (values ranging from 1 mM to 1 mM) to be injected in the flow system. Perform the calibration plot, starting first by the lowest concentration to the highest. Then, repeat injections from the highest concentration to the lowest one. This study also allows checking the durability of the electrodes without spotting an electrode fouling effect (Fig. 16.6). II. Electroanalysis and microfluidics
16.5 Experimental procedure
FIGURE 16.5
167
Fiagrams for successive injections in (A) the same and (B) different microfluidic chips.
16.5.4 Comparison of flow injection analysis systems: wall-jet versus microfluidic thin-layer flow cells Microfluidic sensors enable the use of a thin-layer approach in an FIA system. Thus, they enable an excellent control of fluids through the electrode surface, decreasing the volume of sample and reagents as well as enhancing the sensitivity with very low dead volume. To check the high performance of the thin-layer approach, a comparison study between the microfluidic sensors with the thin-layer approach and a thin-film single electrode with a wall-jet configuration is accomplished. Perform successive injections of standard samples of 100 mM APAP and 100 mM pAP (separately) in the FIA system with the microfluidic thin-layer sensors using the optimal
FIGURE 16.6
Amperometric response for increasing/decreasing concentrations of acetaminophen.
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FIGURE 16.7
16. Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems
Comparison of analytical signals for (A) wall-jet and (B) microfluidic thin-layer approaches.
conditions. Repeat the same experiments but using the thin-film single electrodes with the wall-jet approach. In this case, as the electrode area is different, for a better comparison, the analytical signals are normalized (current density, J) considering the area of the electrodes (Fig. 16.7).
16.5.5 Paracetamol determination 16.5.5.1 Sample preparation 1. Pulverize finely a tablet of paracetamol in a mortar. 2. Weigh accurately three samples. Powder can be directly weighed. 3. Solve each weighed sample directly in deionized water and transfer quantitatively to a 100-mL volumetric flask, making up with deionized water. 4. Filter the solutions using a syringe filter (0.1e0.45 mm). 5. Dilute the sample to the desired concentration with the background electrolyte. Prepare 25 mL of each diluted sample. 6. Inject directly into the FIA system. 16.5.5.2 Sample measurement The analysis of the paracetamol samples is performed using the microfluidic thin-layer sensors under optimal conditions. APAP determination in drug samples is carried out using two different methodologies: 1. Calibration with standard solutions: The calibration plot accomplished in Section 16.7 is used for the determination of the paracetamol concentration in the sample. Thus, the sample (paracetamol tablet/powder dissolved in 100 mL of deionized water) diluted in the carrier is directly injected into the FIA system using the six-port valve. Perform at least three replicates of the sample. The mean current intensity of the peaks is used to calculate the paracetamol using the calibration curve.
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16.7 Additional notes
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2. Standard addition method: It is used in instrumental analysis to determine the concentration of a substance (analyte) in an unknown sample by comparison to a set of standards of known concentration included in the sample matrix. Standard addition method can be applied to most analytical techniques and is used instead of a calibration curve to solve matrix effect problems and get a better precision. Prepare four drug samples (paracetamol tablet/powder dissolved in 100 mL of deionized water) in separated flasks. One of the samples is directly diluted with the carrier to the desired concentration (as in the previous methodology). In the other samples, increased volumes of an APAP standard solution (solution of known concentration of analyte) are added. For obtaining the standard addition curve, the intensity of current is plotted against the concentration of standard added. The intercept in the x-axis corresponds to the concentration of APAP in the sample.
16.6 Lab report At the end of the experiment, write a lab report including an introduction, experimental (materials, equipment, and protocols), results and discussion, and conclusions sections. The following points should be considered in the report: 1. Sketch the HDVs for APAP and pAP. Determine the optimal potential for each compound. 2. Determine the precision (% RSD, relative standard deviation) of the peak current for the compounds in the same (intra) and different (inter) chips. 3. Draft the calibration plots and determine the main analytical parameters (linear range, sensitivity, limit of detection, and limit of quantification) for each compound under optimal conditions. 4. Include the fiagrams for APAP and pAP using the wall-jet and microfluidic thin-layer approaches. For a better comparison, the peak current must be normalized taking into account the area of the working electrode. 5. Sketch the calibration curve of paracetamol obtained by the standard addition method considering the APAP additions. Determine the APAP concentration in the drug samples. Compare the results with those obtained by using the external calibration curve (using standard solutions of APAP).
16.7 Additional notes 1. Stock solutions should be kept on dark and stored at 4 C. 2. All working solutions/samples have to be filtered using a syringe filter of 0.1e0.45 mm to remove small particles, which can block the tubing/flow cell. 3. Be sure the flow cell add-on is correctly placed on the AIO platform to avoid the leakage of fluids. 4. Be sure there are no bubbles in the system (tubing and flow cell), especially on the electrode surface. Bubbles affect the accuracy, precision, and performance of the system.
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16. Integrated microfluidic electrochemical sensors to enhance automated flow analysis systems
5. The electrode surface should be cleaned before starting first experiments. In this pretreatment the different particles adhered to the electrode surface are removed with hydrogen and oxygen gases generated. After the pretreatment, the electrodes could be used for several measurements depending on the sample. Thin-film electrodes could be reused after a new precleaning process. 6. The flow rate employed affect the analytical signals (shape and height of the peaks). A study of the flow rate can be accomplished. The peristaltic pump or syringe pump should be calibrated previously to the experiments performed in the FIA system. 7. In all the experiments, typically, three consecutive injections are performed to improve the precision and obtain better results. 8. Generally, pharmaceuticals state the paracetamol amount in the leaflet. It can be used to calculate the method recovery and also to select the most appropriate dilution of the sample (different dilutions should be performed depending of the pharmaceuticals). Be sure, after the dilution, the APAP concentration is in the lineal range (with and without the additions).
16.8 Assessment and discussion questions 1. 2. 3. 4.
What are the electrochemical processes involved for each compound? Draw the wall-jet and thin-layer flow cell configurations. What is the optimal detection potential for each compound? How is it chosen? Which flow cell approach shows a higher throughput for the determination of paracetamol? Why? 5. In real sample analysis, which methodology is more precise, the calibration curve with standard solutions or the standard addition method?
References [1] J. West, M. Becker, S. Tombrink, A. Manz, Micro total analysis systems: latest achievements, Anal. Chem. 80 (2008) 4403e4419. [2] Lab-on-a-Chip Technology, in: K.E. Herold, A. Rasooly (Eds.), Fabrication and Microfluidics, Caister Academic Press, 2009. [3] K.E. Herold, A. Rasooly (Eds.), Lab-on-a-Chip Technology: Biomolecular Separation and Analysis, Caister Academic Press, 2009. [4] M. Trojanowicz, Flow Injection Analysis: Instrumentation and Application, World Scientific, 2000. [5] The European Pharmacopeial Convention, the Sixth Edition European Pharmacopoeia, 2007, p. 0049. [6] The United States Pharmacopeial Convention, USP (the United States pharmacopoeia) 27-NF (The National Formulary) 22, vol. 27, 2004, p. 2494. [7] Editor Committee of National Pharmacopoeia, Chinese Encyclopedia of Medicines, vol. 2, Chemical Industry Press, Beijing, 2000, p. 206.
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