Iridium Oxide-reduced Graphene Oxide Nanohybrid Thin Film Modified Screen-printed Electrodes as Disposable Electrochemical Paper Microfluidic pH Sensors

Summary

The study demonstrates the growth of iridium oxide-reduced graphene oxide (IrO₂-RGO) nanohybrid thin films on irregular and rough screen-printed carbon substrate through a green electrochemical synthesis, and their implementation as a pH sensor with a patterned paper-fluidic platform.

Abstract

A facile, controllable, inexpensive and green electrochemical synthesis of IrO₂-graphene nanohybrid thin films is developed to fabricate an easy-to-use integrated paper microfluidic electrochemical pH sensor for resource-limited settings. Taking advantages from both pH meters and strips, the pH sensing platform is composed of hydrophobic barrier-patterned paper micropad (µPAD) using polydimethylsiloxane (PDMS), screen-printed electrode (SPE) modified with IrO₂-graphene films and molded acrylonitrile butadiene styrene (ABS) plastic holder. Repetitive cathodic potential cycling was employed for graphene oxide (GO) reduction which can completely remove electrochemically unstable oxygenated groups and generate a 2D defect-free homogeneous graphene thin film with excellent stability and electronic properties. A uniform and smooth IrO₂ film in nanoscale grain size is anodically electrodeposited onto the graphene film, without any observable cracks. The resulting IrO₂-RGO electrode showed slightly super-Nernstian responses from pH 2-12 in Britton-Robinson (B-R) buffers with good linearity, small hysteresis, low response time and reproducibility in different buffers, as well as low sensitivities to different interfering ionic species and dissolved oxygen. A simple portable digital pH meter is fabricated, whose signal is measured with a multimeter, using high input-impedance operational amplifier and consumer batteries. The pH values measured with the portable electrochemical paper-microfluidic pH sensors were consistent with those measured using a commercial laboratory pH meter with a glass electrode.

Introduction

The determination of pH is ubiquitous in food, physiological, medicinal and environmental studies. Two most common tools for pH detection are pH strips and pH meters. Paper strips are impregnated with color-changing pH indicator molecules but the reading is sometimes limited in pH ranges, subjective and semi-quantitative with some deviations. On the other hand, a pH meter conventionally equipped with a glass electrode can measure pH accurately to the 0.01 level, and display by a digital-user interface. Lab-based pH meters not only need special care in maintenance and calibration, but also do not work well towards small sample volumes and often require a clean container such as a beaker to perform measurements. In spite of its sensitivity, selectivity and stability, glass electrodes suffer from acid/alkaline errors, high impedance, temperature instability and mechanical fragility¹. Therefore it is advantageous to have a pH measurement system that embodies the accuracy of pH meter and the simplicity and cost aspects of pH strips.

There is always an unmet need for such tools under limited resources conditions in many developing regions where expensive lab-based equipment or commercial laboratories are unaffordable. Also, the increasing role of new easy-to-use on-site sensing platforms is pushed by such a demand for point-of-care detection. Electrochemical detection is simple, easy to miniaturize and satisfactorily sensitive, as demonstrated by the commercialized low-cost SPEs and various glucose monitoring systems on the market. As a light, flexible and disposable porous material, paper can also have various controllable characteristics, such as different pore sizes, functional groups, and wicking rates.

As paper substrate barely affects analyte diffusion and electrochemical detection^2-4, combination of paper-fluidic devices and electroanalytical techniques has recently received extensive interests. An apparent advantage of such combinations is the tiny amount of sample volume used in the measurement which can potentially prevent interferences from vibration and convection during measurements. For instance, patterned microfluidic pads were applied to wick and deliver liquid samples to sensing area of SPEs for detection of heavy metal ions and glucose^2,5. Similar devices using paper microfluidic electrochemiluminescence were established to accomplish NADH detection⁴. More recently, simple electrochemical paper microfluidic devices can be built on a glass slide with pencil electrodes⁶ or using enzyme paper and SPEs³.

A nanohybrid thin film material composed of IrO₂ and RGO was prepared using a facile and efficient electrochemical approach. We found that on the irregular and rough SPE graphitic carbon surface, anodically electrodeposited IrO₂ thin film cannot be smooth and stable without the aid of RGO. The resulting IrO₂-RGO SPE was integrated into a paper microfluidic device which has patterned hydrophobic barriers for pH sensing. The assembled device showed excellent analytical performances in pH sensing with a slightly super-Nernstian behavior. The results are comparable to a conventional lab-based pH meter with glass electrodes. Lastly, cost-effective miniaturized pH meters were built on a breadboard to measure open circuit potential output signal with a digital multimeter. The measurements of the portable pH meter correlates well with those of a commercial laboratory pH meter. 

Protocol

1. µPAD and Apparatus Preparation

1. Engrave a 500 µm groove on the bottom plastic holder to house SPE with an ABS or compatible plastic sheet by three-dimensional (3D) milling machine and milling bit which has 1.6 mm of diameter. Hold SPE and µPAD firmly in place during testing with the holder (Figure 1A).
2. Make a stamp and a vacuum cover using synthetic resin tablet or compatible plastic sheet with convex and concave patterns, respectively, by the 3D milling machine, in order to pattern hydrophobic PDMS barriers on paper pads.
   1. Prepare a mixture of PDMS pre-polymer and cross linker at the ratio of 10:1 or as suggested by manufacturer, mix with spatula and apply appropriate amount onto the convex surface of the PDMS stamp.
3. Place the stamp on top of a filter paper pad pre-cut to desired size and then the vacuum cover on the opposite side of the stamp across the paper. Apply vacuum for up to 30 sec by a hand-operated vacuum pump. Remove the paper pad from the stamp and vacuum cover, and bake in a convection oven for 10 min at 80 °C to harden the patterned PDMS (Figure 1B). The resulting paper pad has an approximately 0.2 cm² sensing region and 1 cm x 0.4 cm hydrophilic sample wicking region.
   Note: Take special cautions on the amount of applied PDMS and vacuum time to avoid any possible PDMS contamination in the inner hydrophilic region of the filter paper where the liquid samples are transferred.

2. Modification of SPEs with IrO₂-RGO Nanohybrid Thin Films

1. Drop cast 3 μl of as-prepared 1 mg∙ml^-1 GO solution on the graphitic carbon working electrode of SPE with a micropipette and let it dry at room temperature in a Petri dish. Purge a pH 5.0 PBS buffer with N₂ for 20 min, dip the SPE in the 10 ml deaerated PBS buffer while keeping N₂ flowing, and conduct 100 cycles of repetitive cathodic potential cycling from 0.0 to -1.5 V to electrochemically reduce GO into RGO. Rinse the SPE with DI water in a squirt bottle and dry at room temperature.
   Note: Well-exfoliated GO sheets, stabilized by electrostatic repulsion, are from graphite powder using modified Hummer's method as reported elsewhere⁷. The homogeneity of as-synthesized RGO film is important, because it serves as the carbon support for further growth of IrO₂ thin films.
2. Make 100 ml IrO₂ deposition solution composed of 0.15 g iridium tetrachloride (IrCl₄), 0.6 ml 50% (w/w) hydrogen peroxide (H₂O₂) and 0.5 g oxalic acid dehydrate by adding them in DI water. Gradually add small amount of anhydrous potassium carbonate while stirring until the pH reached 10.5, verified by a lab-based pH meter. Then, solution turned yellowish. Aging the solution for 48 hr at room temperature, then its color is eventually turning pale blue.
3. Put the RGO-SPE in the above deposition solution and apply a constant potential of +0.6 V for 5 min. The thickness of IrO₂ thin films can be precisely controlled by the deposition potential and time.
4. Confirm the structure of the sensing area by SEM. Acquire SEM images following instructions at the Materials Science Center in University of Wisconsin-Madison, as we did before⁷.

3. Construction of Inexpensive and Portable Digital pH Meters

1. Build an inexpensive and miniaturized pH meter with digital display by plugging in either a series of two single LF356N operational amplifiers (OpAmps) or one INA111 high speed field-effect transistor (FET)-input instrumentation amplifier (high input impedance > 10¹² Ω) on breadboard to achieve sufficiently high internal impedance for stable measurements.
   Note: All the parts are easily accessible from electronic stores and can be easily assembled.
2. Use the IrO₂-RGO-SPE as the pH probe and OpAmps as the unity gain buffer. Connect two grounded 9 V alkaline consumer batteries in series to power the pH meter and plug in the wires into the breadboard based on the pin layout of OpAmps.
3. Connect the cathode and anode to pins 7 and 4. Also connect the positive and negative probes of a digital multimeter to pins 6 and 5 of OpAmps respectively to measure the output voltage and display readings. Reference and working electrodes of SPE are connected to pins 2 and 3 correspondingly. Detailed connections are shown in Figure 1D.

4. pH Measurements

1. Prepare 100 ml B-R buffers with 0.04 M equimolar phosphoric acid, acetic acid and boric acid and mix with different volumes (5, 25, 42, 60, 78 and 98) of 0.2 M sodium hydroxide (NaOH) to achieve different pHs from 2-12 for calibration.
2. Locate patterned μPAD on top of the sensing area. Mount 60 µl liquid samples directly by a micropipette into the hydrophilic area of the μPAD for wicking. The μPAD can be held in place with or without ABS cover, when it is wetted.
3. Measure the voltage signal between the IrO₂-RGO working electrode and the Ag/AgCl reference electrode over time with either a lab-based CHI 660D electrochemical analyzer or the portable digital pH meter, when the open circuit potentials (OCP) become steady (potential variations < 5%).
4. Keep the sensing region wet by immersing the paper pad in liquid samples to be tested, if needed, to achieve better electrical contact as well as stable and reproducible readings in long-term operation. Recorded steady-state OCP values are averaged at each pH value to determine a calibration curve.

Results

The setup of the electrochemical IrO₂-RGO-SPE pH sensor incorporating paper microfluidics is shown in Figure 1A. The patterned paper pad with PDMS hydrophobic barriers was placed on top of the sensing area of IrO₂-RGO-SPE which located on the ABS plastic holder. The sensing zone of paper pad was carefully aligned with electrode surface. An aqueous methylene blue dye solution was used to test the patterned paper pad and as observed, samples wick into ...

Discussion

Device Setup

The pH sensor works by measuring the OCP between the working and reference electrodes, since it changes proportionally to the negative logarithm of H⁺ concentration. The measurements can be achieved both by a lab-based potentiostat such as CHI 660D and simple pH meter constructed on breadboard with reading by multimeter. Two different portable pH meters were built similarly on breadboards using two 9 V alkaline batteries, a digital multimeter, as-synthe...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Screen-printed electrodes	Zensor	TE100	3-electrode integrated
Acrylonitrile butadiene styrene (ABS)
Polydimethylsiloxane (PDMS) prepolymer and cross linker mixture	Dow-Corning Co.	Sylgard 184	10:1 mixture w/w
Whatman No. 1 filter paper	GE Healthcare Co.
3D milling system	Roland DGA Co.	iModela IM-01
PDMS stamp and vacuum cover	Roland DGA Co.	Sanmodur	Synthetic resin tablet
Hand-operated vacuum pump	Cole-Parmer Co.
Electrochemical workstation	CH Instruments	CHI 660D
LF356N operational amplifiers	Texas Instruments Inc.
INA111 high speed field-effect transistor (FET)-input instrumentation amplifier	Burr-Brown Inc.
DMM914 digital multimeter	Tektronix Inc.	70979101
From Fisher or Sigma:
Iridium tetrachloride (IrCl4)
50% (w/w) hydrogen peroxide (H2O2)
Oxalic acid dihydrate
Potassium carbonate (K2CO3)
Phosphoric acid
Acetic acid
Boric acid
Sodium hydroxide (NaOH)
Na2HPO4
NaH2HPO4