Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

Podsumowanie

All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.

Streszczenie

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media ^1-4. An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H⁺, Ca²⁺, and CO₃^2- ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal.

The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface ^5-7. To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) ^7-9 in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1).

Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO₃^2- (measured range 0.01 mM - 1 mM), and Ca²⁺ (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris.

The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space.

Biochip Design and Experimental Methods

The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 μm in diameter and is equally spaced at 1.4 mm from the REs, which are 480 μm in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2.

Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H⁺ and CO₃^2-, while one with sulphate (added to the solution as CaSO₄) is utilized for Ca²⁺. The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K₃Fe(CN)₆)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area ¹⁰. Spin-coating at 1,500 rpm is used to cast ~2 μm thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs).

The MAB is contained in a microfluidic flow-cell chamber filled with a 150 μl volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

Protokół

1. Preparation of Poly(3,4-ethylenedioxythiophene):Poly(sodium 4-styrenesulfonate) (PEDOT:PSS) Electropolymerization Solution for H⁺ and CO₃^2- Ions

1. Add 70 mg poly(sodium 4-styrenesulfonate) (Na⁺PSS^-) to 10 ml deionized (DI) water and vortex until completely dispersed (approx. 10 sec).
2. Add 10.7 μl 3,4-ethlyenedioxythiophene (EDOT) to the solution in 1.1 and vortex until solution is completely mixed.

2. Preparation of Poly(3,4-ethylenedioxythiophene):Calcium sulphate (PEDOT:CaSO₄) Electropolymerization Solution for Ca²⁺ Ions

1. Add 136 mg calcium sulphate (CaSO₄) to 10 ml DI water and vortex; the solution will not completely disperse and appears milky.
2. Add 10.7 μl EDOT to the solution in 2.1 and vortex until completely mixed.

3. Electropolymerization of PEDOT-based Conductive Polymer

1. A Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3) and an EC epsilon potentiostat/galvanostat are used to form the electrochemical cell for electropolymerization. Place the EDOT:PSS electropolymerization solution in the electrochemical cell and nitrogen bubble for 20 min to remove dissolved oxygen.
2. Now clip a platinum-gauze at the counter electrode position of the electrochemical cell. Then clip the MAB at the working electrode 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 PEDOT:PSS electropolymerization solution. Avoid solution contact with the square electrical contact pads.
3. Place a BASI saturated silver/silver chloride (Ag/AgCl) electrode in the reference electrode position of the electrochemical cell. Make sure that the reference electrode is not in between the working and counter electrodes.
4. For PEDOT:PSS deposition: Bubble the electrochemical cell for 20 min, and use the EC epsilon potentiostat/galvanostat to run a single cyclic voltammogram from 0V - 1.1V with a scan rate of 20 mV/sec on a ±100 μA scale.
5. For PEDOT:CaSO₄ deposition: Bubble the electrochemical cell for 20 min, and use the EC epsilon potentiostat/galvanostat to run chronopotentiometry at 814 nA for 30 min.

4. Cyclic Voltammetry of PEDOT-based Polymer Conjugates in K₃Fe(CN)₆

1. Perform steps 3.1- 3.3 above.
2. Use the EC epsilon potentiostat/galvanostat to run single cyclic voltammograms from -653 mV to 853 mV with varying scan rates of (25, 50, 75, 100, l25, 150, 175, 200) mV/sec on a ±10 μA scale.

5. Surface Functionalization Protocol

1. Deposit conductive polymer conjugate specific for the ions of interest as in Step 3.
2. Apply ion-selective membrane as in Step 6.

6. Application of Ion-selective Membrane

1. Center the MAB on the vacuum spinner chuck.
2. Deposit 100 μl membrane onto the center of the MAB and run.
3. Spin-coat ion-selective membrane with a spin coater at 1,500 rpm for 30 sec with a 5 sec ramp up and down.
4. Vacuum the spin-coated MAB for 30 min and bake the chip in an oven at 70 °C for 20 min.

7. Calibration of PEDOT-PSS Conductive Polymer Conjugate with pH and Carbonate (CO₃^2-) Ion-selective Membrane

1. Condition the MAB overnight in 10 μM sodium bicarbonate (NaHCO₃) and 5 mM potassium chloride (KCl) in algal media.
2. Insert the MAB into the microfluidic flow-cell chip holder.
3. Inject 5 ml test solution with initial pH value or concentration (e.g. pH 4 or 10 μM for CO₃^2-). Remove bubbles from the flow-cell chip holder.
4. Place the flow-cell chip holder onto the flow-cell electrical fixture.
5. Open the EC epsilon software and enter open-circuit potential (OP) mode. Set the time to 300 min, the voltage scale to ± 1V, and the cutoff frequency to 10 kHz, and record the value every 2 sec.
6. Let the MAB stabilize (look for a flat line) before continuing with the calibration process.
7. Once the MAB is stabilized, flush the flow cell with test solution and inject the next concentration to be calibrated (pH 5 or 25 μM CO₃^2-). Make sure that no bubbles are allowed to enter the flow cell. Repeat steps 7.5 and 7.6 for pH 6, 7, 8, and 9 or CO₃^2- concentrations of 50, 75, 100, 250, 500, 750, and 1,000 μM.
8. After the last concentration has run, remove the MAB and dry with nitrogen air.
9. Place the MAB back into fresh conditioning solution until next use.

8. Calibration of PEDOT:CaSO₄ Conductive Polymer Conjugate in CaCl₂

1. Condition the MAB overnight in 7 ml of 0.1 M CaCl₂ and 10 μM NaNO₃.
2. Follow steps similar to 7.2 - 7.10. In step 8.3, replace carbonate test solution with an initial concentration of 0.01 mM CaCl₂. Repeat for test-solution concentrations of 0.05, 0.1, 0.5, 1 and 10 mM.

Wyniki

An example of a cyclic voltammogram (CV) result of PEDOT:PSS and its corresponding cathodic peak current (i_p) vs. the scan rate (v_1/2) are shown in Figures 5a and 5b respectively. PEDOT:CaSO₄ at various scan rates and its cathodic peak current are not shown. Using Randles-Sevcik analysis ¹⁰, the effective surface areas of the solid contact PEDOT:PSS and PEDOT:CaSO₄ without ion-selective membrane were found to be 4.4...

Dyskusje

The MAB biochip consists of ASSISEs that are constructed from an ISM atop a PEDOT-based CP conjugate transduction layer on a Pt electrode, the combination of which transduces the ionic concentration of interest to a measurable electrical signal. A stable electrode potential is defined by both the CP layer and the ISM layer. Both layers also determine the working lifetime of the MAB and the quality (noise, drift) of the measured electrical signal.

PEDOT is especially attractive as a transductio...

Materiały

Name	Company	Catalog Number	Comments
3,4-Ethylenedioxythiophene	Sigma-Aldrich	483028
Poly(sodium 4-styrenesulfonate)	Sigma-Aldrich	243051
EC epsilon galvanostat/potentiostat	Bioanalytical Systems Inc.	e2P
Saturated Ag/AgCl reference electrode	Bioanalytical Systems Inc.	MF-2052
Pt gauze	Alfa Aesar	10283
Potassium ferricyanide	Sigma-Aldrich	P-8131
Potassium nitrate	J.T. Baker	3190-01
Sodium bicarbonate	Mallinckrodt/ Macron	7412-12
Sodium carbonate	Sigma-Aldrich	S-7127
Calcium chloride	J.T. Baker	1311-01
Potassium chloride	Sigma-Aldrich	P9541
Calcium sulphate	Sigma-Aldrich	237132
C3 cell stand	Bioanalytical Systems Inc.	EF-1085
Flow-cell chip holder			Custom, courtesy of NASA Ames
Flow-cell electrical fixture			Custom, courtesy of NASA Ames
Table 2. Specific reagents and equipment.