The overall goal of this procedure is to functionalize an all solid state ion selective electrode for both cation and an ion detection First electro polymerize. A conductive polymer transducer layer on the working electrodes of the MAB spin coat, an ion selective membrane layer and condition the biochips overnight to activate the ion selective membrane. Now in the flow cell chamber, connect the contact pads to the BASSI three electrode potential stat push measurement solution into the chamber.
Removing unwanted bubbles, ultimately calibrations of the MAB chip to ions of interest can be performed and output signal from the MAB recorded in real time. Unlike traditional micro electrode and radio labeling probe technologies, all solid state ion selective electrodes are non-invasive and they can be multiplexed for real-time measurements of ion activities and model biological and physiological systems. We first had the idea to perform this method while participating in astrobiology research that requires limited space to perform physiological measurement.
June Hume Park, a grad student in a bourbon leaf physiological sensing facility, will assist me with a demonstration To form the electrochemical cell for electro polymerization. Use a Bassi C3 cell stand and an EC epsilon potentials stat galvan stat. Place the electro polymerization solution in the electrochemical cell, then bubble nitrogen for 20 minutes to remove dissolved oxygen.
Now clip a platinum gauze counter electrode to the electrochemical cell at the counter electrode position. Then clip the MAB at the working electrode position or center position of the electrochemical cell with the working electrodes facing the platinum gauze, adjust the MAB depth so that only the circular electrodes are submerged in the electro polymerization solution. Avoiding solution contact with the square electrical contact pads.
Next place a bassi saturated electrode at the reference electrode position of the electrochemical cell. Make sure that the reference electrode does not touch the working and counter electrodes. Then using the EC epsilon potentials Stat gal Vanos stat run a single cyclic gram from zero to 1.1 volts with a scan rate of 20 millivolts per second on a plus minus 100 micro ampu scale.
For calcium sensing, perform PDO calcium sulfate deposition on MAB bubble, the e. plus calcium sulfate solution for 20 minutes. To characterize the pdot surfaces, use cyclic telemetry of the pdot based polymer conjugates.
In two millimolar potassium cyanide run single cyclic grams from negative 653 millivolts to 853 millivolts with varying scan rates on a plus minus 10 microper scale center the multianalyte bio chipp on the vacuum spinner chuck deposit the 100 microliter membrane onto the center of the MAB and run the measurement. Next spin coat the ion selective membrane at 1, 500 RPM for 30 seconds With a five seconds ramp up and down, vacuum the spin coated MAB for 30 minutes. Then bake the chip in a 70 degree Celsius oven for 20 minutes.
Condition the MAB overnight in 10 micromolar sodium bicarbonate, and five millimolar potassium chloride in algal media. Now insert the MAB into the microfluidic flow cell chip holder. Inject five milliliters of test solution with the appropriate initial pH value or concentration.
Remove any bubbles from the flow cell chip holder and place the flow cell chip holder onto the flow cell electrical fixture. Next, open the EC epsilon software and enter open circuit potential mode. Set the time to 300 minutes the voltage scale to plus minus one volt the cutoff frequency to 10 kilohertz and also record the value every two seconds.
Let the MAB stabilize before continuing with the calibration process. Then flush the flow cell with test solution and inject the next concentration to be calibrated. Make sure that no bubbles are allowed to enter the flow cell.
Repeat measurements for the remaining samples of the calibration curve after the last run. Remove the MAB and dry it with nitrogen air. Place the MAB back into fresh conditioning solution until next.
Use for calibration of MAB and calcium chloride condition the MAB with pdot calcium sulfate conductive polymer conjugate and calcium selective membrane overnight in 0.1 molar calcium chloride and 10 micromolar sodium nitrate. Then replace the pH or carbonate test solutions with an initial concentration of 0.01 millimolar calcium chloride. Repeat for the other test solution concentrations.
These data show a cyclic volta of pdot PSS and its corresponding cathartic peak current versus the scan rate. Randall's subic analysis determine an effective surface area of 4.4 times 10 to the negative 11th centimeter squared for the solid contact without ion selective membrane. As a test of all solid state ISEs capability to acquire measurements in the actual cell culture environment.
ISEs were calibrated in Lgal media with pH ranging from four to nine after 20 days storage in the lgal medium. Here PDO PSS was calibrated in carbonate solution with a concentration range of 0.01 millimolar to one millimolar in both lgal biological medium and lgal biological medium buffered at pH 8.5. Note the change in concentration with a lowering of the slope with the buffered solution.
The carbonate selective electrode is pH dependent and an increment in voltage correlates to an increment in the carbonate species. Since measurements are made at pH 7.8, most species are in the bicarbonate form carbonate concentration measurements with biological model chlorella vulgaris in ambient light and dark conditions show a 30 millivolt change a control with only algal media shows no response indicative of a functional bio chipp. These MAB ISEs are based on PPSS in calcium chloride solution with increasing concentration, a near nian slope profile for divalent cation at 30 millivolts per decade.
Change in calcium concentration will be used to measure the calcium current levels in germinating fern. This approach has utility in biology, agriculture, and medicine for studying the biomolecular mechanisms involving ion transport and cell electrophysiology and signaling and plant and animal systems.
