Multi-аналита биочипов (МАБ) на основе всех твердотельных ион-селективных электродов (пачка) для физиологических исследований
Summary
Все твердотельные ион-селективных электродов (ASSISEs), построенных из проводящего полимера (CP) преобразователь обеспечивают несколько месяцев функциональной жизни в жидких средах. Здесь мы описываем изготовление и калибровка процесса ASSISEs в лаборатории-на-чипе формате. Assise доказано, сохранили почти нернстовским профиля склона после длительного хранения в сложных биологических средах.
Abstract
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 CO32- 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), CO32- (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 CO32-, 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.
Protocol
Representative Results
Discussion
МАВ состоит из биочипа ASSISEs, которые построены из ISM вершине PEDOT на основе сопряженных CP трансдукции слой на электроде Pt, сочетание которых преобразовывает ионной концентрации интерес к измеримой электрический сигнал. Стабильный потенциал электрода определяется как слой CP и слой ISM. Об?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Мы хотели бы поблагодарить астробиологии NASA науки и технологий разработки приборов (ASTID) Программы финансовой поддержки (грантов номера 103498 и 103692), Гейл Локвуд из Birck Nantechnology Центр Университета Пердью для wirebonding из МАБ устройств и Хен Джун Парк САПР потока-клеток камеры.
Materials
| Name of the items | 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. |
References
- Migdalski, J., Bas, B., Blaz, T., Golimowski, J., Lewenstam, A. A Miniaturized and Integrated Galvanic Cell for the Potentiometric Measurement of Ions in Biological Liquids. J. Solid State Electrochem. 13, 149-155 (2009).
- Buehler, M. G., Kounaves, S. P., Martin, D. P. Designing a Water-quality Monitor with Ion-selective-electrodes. 1, 331-338 (2001).
- Adamchuk, V. I., Lund, E. D., Sethuramasamyraja, B., Morgan, M. T., Doberman, A., Marx, D. B. Direct Measurement of Soil Chemical Properties on-the-go using Ion-selective-electrodes. Journal Computers and Electronics in Agriculture. 48 (3), 272-294 (2005).
- Oelβner, W., Hermann, S., Kaden, H. Electrochemical Sensors and Sensor Module for Studying Biological Systems in Space Vehicles. Aerospace Science and Technology. 1, 291-296 (1997).
- Bobacka, J. Conducting Polymer-based Solid-state Ion-selective Electrodes. Electroanalysis. 18 (1), 7-18 (2006).
- Buck, R. . Ion Selective Electrodes in Analytical Chemistry. , (1980).
- Nam, H., Cha, G. S., Yang, V. C., Ngo, T. T. Chapter 18. Biosensors and their Applications. , (2000).
- Anatova-Ivanova, S., Mattinen, U., Radu, A., Bobacka, J., Lewenstem, A., Migdalski, J., Danielewski, M., Diamond, D. Development of Miniature All-solid-state Potentiometric Sensing System. Sensors and Actuators B. 146, 199-205 (2010).
- Michalska, A., Galuszkiewicz, A., Ogonowska, M., Ocypa, M., Maksymiuk, K. PEDOT Films: Multifunctional Membranes for Electrochemical Ion sensing. J. Solid State Electrochem. 8, 381-389 (2004).
- Bard, A. J., Faulkner, L. R., ed, 2. n. d. . Electrochemical Methods: Fundamentals and Applications. , (2000).
- Claussen, J. C., Artiles, M. S., McLamore, E. S., Mohanty, S., Shi, J., Rickus, J., Fisher, T. S., Porterfield, D. M. Electrochemical Glutamate Biosensing with Naanocube and Nanosphere Augmented Single-walled Carbon Nanotube Networks: A Comparative Study. J. Mater. Chem. 21, 11224-11231 (2011).
- Bobacka, J. Potential Stability of All-solid-state Ion-selective Electrodes using Conducting Polymers as Ion-to-electron Transducers. Anal. Chem. 71, 4932-4937 (1999).
- Lee, J. H., Yoon, I. J., Yoo, C. L., Pyun, H. J., Cha, G. S., Nam, H. Potentiometric Evaluation of Solvent Polymeric Carbonate-selective Membranes based on Molecular Tweezer-type Neutral Carriers. Anal. Chem. 72, 4694-4699 (2000).
- Song, F., Ha, J., Park, B., Kwak, T. H., Kim, I. T., Nam, H., Cha, G. S. All-solid-state Carbonate Selective Electrode based on a Molecular Tweezer-type Neutral Carrier with Solvent-soluble Conducting Polymer Solid Contact. Talanta. 57, 263-270 (2002).
