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

The overall goal of this method is to use the electrochemical synthesis of graphene iridium oxide hybrid thin films to fabricate a portable electrochemical paper-fluidic sensor for point of care pH measurements. This method provides a new way to perform point of care pH measurements by combining the synthesis of antartically electrodeposited iridium oxide thin films, assisted by reduced graphene oxide scaffolds. Combined with paper microfluidic pads and digital automatic display, it truly provides a portable and disposable pH sensor.

This technique combines the features of pH paper strip and solid state pH meters using paper microfluidics and electrochemical detection to offer quick, accurate, and point of care measurements of the pH outside labs using low sample volume. We first had the idea for fabrication of such devices because we wanted to measure the relatively small pH variations of lake water in different months in Madison, Wisconsin. In fact, the pH strip cannot tell such a small pH difference and it can hardly be read in the chart.

It is also not practical to use a laboratory based pH meter. To begin, secure a 7cm x 4 cm x 0.6 cm sheet of ABS or other compatible plastic into the holder of a three dimensional milling machine that is equipped with a 1.6 mm milling bit. Use the milling machine to engrave a dog bone shaped groove 500 micrometers deep into the surface.

Create the shape by milling two 1 cm radius circles connected by a 1 cm x 5 mm wide channel. This pattern will be used as the stamp. Next, place a separate sheet of plastic into the mill to be used as the vacuum cover and mill out a groove that is large enough to contain the entire stamp pattern.

Next, prepare a mixture of PDMS prepolymer and cross linker at ratio of 10 to one and mix the two parts with a spatula. Then, place the mixture into a vacuum chamber for five minutes to degas the mixture. Apply an appropriate amount of degassed PDMS to the channel groove of the stamp so that it fills the entire groove.

Scrape away the excess material with a flat piece of plastic. Then, place a piece of filter paper pad, pre-cut to the desired size, on top of the PDMS filled stamp. Set the vacuum cover on the opposite side of the stamp, across the paper, and apply a light finger pressure on the stamp.

Apply vacuum for up to 30 seconds using a hand-operated vacuum pump. When finished, remove the paper pad and place it in a convection oven for 10 minutes at 80 degrees celsius to harden the pattern to PDMS. First, obtain a screen-printed three electrode array consisting of a graphitic carbon working electrode, a counter electrode, and a silver silver chloride palette reference electrode.

Then, use a micropipette to drop cast three mcL of a 1 mg per ml graphene oxide solution onto the graphitic carbon working electrode. Let the solution dry at room temperature. In the meantime, nitrogen purge 10 ml of a pH 5.0 PBS buffer for 20 minutes.

When the screen-printed electrode is dry, dip it into the buffer while the nitrogen continues to flow. Next, connect the electrode to an electrochemical work station and conduct 100 cycles of repetitive cathodic potential cycling from 0.0 to 1.5 volts to electrochemically reduce the graphene oxide. Then, rinse the screen-printed electrode with deionized water and dry it at room temperature.

The homogeneity of the reduced graphene oxide film is important because it serves as the carbon support for further growth of iridium oxide thin films. Next, make 100 ml of iridium oxide deposition solution in deionized water. Gradually add a small amount of anhydrased potassium carbonate while stirring until the pH of the solution reaches 10.5 and the solution turns a yellowish color.

Age the solution for 48 hours at room temperature. During this time, observe as its color turns from yellow to a pale blue. Place the reduced graphene oxide screen-printed electrode into the iridium oxide deposition solution and use the electrochemical work station to apply a constant potential of 0.6 volts for five minutes.

Confirm the structure of the sensing area by scanning electron microscopy using standard SEM sample preparation and imaging techniques. To begin construction of the portable digital pH meter, collect the required components as shown here, which are listed in the accompanying text protocol under the list of materials. Then plug in the INA 111 high speed field effect transistor input instrumentation amplifier onto the breadboard to achieve sufficiently high internal impedance for stable measurements.

Next, connect two grounded nine volt Alkaline consumer batteries in series to power the pH meter. Connect the cathode to pin seven, and the anode to pin four. Then, connect a 10 meg-ohm resistor between pins one and eight.

Also, connect the positive probe of a digital multimeter to pin six and a negative probe to pin five. Set up the multimeter to measure and display the output voltage. Finally, connect the reference end of the modified screen-printed electrode to pin two and the working electrode to pin three.

Prepare 100 ml mixtures of Britton-Robinson buffers by combining varying amounts of 0.2 molar sodium hydroxide with a 0.04 molar acid solution composed of equimolar amounts of phospheric acid, acetic acid, and boric acid combined. Next, assemble the microfluidic pH meter by placing the modified screen-printed electrode into the milled plastic holder and then setting the microfluidic paper on top of the electrode so that one of the hydrophilic circles is located over the sensor. Using a pipette, add 60 mcL of one of the pH samples directly onto the hydrophilic area of the micropad so that it will wick towards the sensor.

Then, begin to measure the voltage signal with an assembled portable digital pH meter. Once the open circuit potential becomes steady, record the value. Repeat this process with each of the prepared standards to create a standard curve using a new microfluidic paper each time.

The electrochemical reduction technique described for the screen-printed electrodes will produce a defect free, homogeneous reduced graphene oxide thin film that is visible under scanning electron microscopy. After iridium oxide modification, a uniform and smooth film should appear without any observable cracks. Only a small amount of fluid is necessary as the sample wicks along the microfluidic paper towards the sensor.

In the image shown here, a sample of blue dye solution was placed at the base of the paper and was drawn towards the sensor location, dyeing the entire piece of paper. The time required to reach a steady reading varies depending on the pH of the sample. As with any pH meter, it is important to wait until a steady state is achieved before taking the reading.

Once steady state is reached, the values acquired with a microfluidic pH sensor linearly correlate with those acquired from a typical pH meter. The graphene iridium oxide hybrid thin film pH meter has a small hysteresis, fast response time, reproducible performances, and good correlation with the commercial glass electrode of laboratory based pH meter. Once mastered, the pH measurements can be carried out rapidly on the order of a few seconds.

Following this procedure, other pH responsive carbon batter oxide hybrid thin films can be synthesized in a similar way on any conducting, or semi-conducting substrates, such as class E carbon, indium tin oxide coated glass, and silica. The electrochemical micrfluidic sensor combines advantages of both pH meters and pH strips. And is a promising platform for future in field and point of care pH measurements in resource limited settings.