Grantee Research Project Results
Final Report: Development of a New Microelectrode Array Biosensing System for Environmental Monitoring
EPA Grant Number: R825323Title: Development of a New Microelectrode Array Biosensing System for Environmental Monitoring
Investigators: Sadik, Omowunmi
Institution: The State University of New York at Binghamton
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1996 through September 30, 1999 (Extended to September 30, 2000)
Project Amount: $280,095
RFA: Analytical and Monitoring Methods (1996) RFA Text | Recipients Lists
Research Category: Water , Land and Waste Management , Air , Ecological Indicators/Assessment/Restoration , Environmental Statistics
Objective:
The objective of this research project was to develop, optimize, and carry out field demonstrations of electroactive polymer-based biosensing systems utilizing microelectrode arrays and individually addressable sensing chips in a multi-analyte sensing format for environmental monitoring.
Summary/Accomplishments (Outputs/Outcomes):
The successful coupling of suitable transducers with biomolecular systems is of great importance in the search for novel sensing technologies that are inexpensive, highly selective, and capable of generating early warning signals in the presence of toxic environmental chemicals. We have developed a new, generic multi-analyte detection technique that utilized conducting electroactive polymer (CEP) membranes. This novel signal generation technology made use of CEP as the sensing electrode, coupled with periodic, pulsed (or other transient) voltage waveforms. The voltage waveform induced changes in the CEP such that a detectable interaction with a target analyte was obtained in a reversible manner. This generic sensing strategy was successfully demonstrated for the detection of polychlorinated biphenyls (PCBs), halogenated phenols, toxins, microbes, and heavy metals. These sensors required only a simple immobilization scheme, in which the biomolecules were immobilized on a conducting matrix. The matrix provided for both the immobilization of bioreagents and the generation of analytical signals for the compounds of interest. The sensors have been tested using fixed laboratory methods such as flow-injection analysis, liquid and ion chromatographies, and gas chromatography. Alternatively, the method could be used for in situ field measurements when coupled to a low-cost, battery-operated electronic device.
During the first segment of this research project, we synthesized and characterized a range of functionalized pyrrole monomers. These pyrrole derivatives were polymerized using electrochemical oxidation to generate various conducting polypyrrole (PP) films. Twenty different polymers were employed in this study, and each one was different from the other by the choice of counterion and the nature of additives used in the polymerization solution. Several counterions were incorporated into the polymer during the application of electrical potential, including chloride (Cl-), dodecyl sulfate (DS-), and perchlorate (ClO4-). The polymers were designed so that their conducting properties could be controlled to produce an array of thin films, each having its own unique fingerprint for each analyte. The output signal obtained after the application of pulsed potential or exposure to vapors was fed into LabView™ software to generate a database for different analytes.
Once the necessary polymer templates were assembled, the first demonstration of the microsensor arrays was carried out using simple chemical sensors without any complex biomolecules. This template was used for detection of chlorinated phenols in a 32-array conducting polymer system, resulting in rapid measurement of volatile and semivolatile halogenated organic compounds of environmental interest. A mathematical expression for the microscopic polymer network model was developed using the classical, nonparametric, and unsupervised technique of cluster analysis. This provided a useful way of distinguishing polychlorinated organic phenol vapor response vectors in a 2-dimensional space (refer to Masila, et al., 1998). It also provided a way of identifying clusters, or groups, to which unknown vectors were likely to belong. Consequently, we were able to generate the characteristic pattern for each sample. These patterns were used to develop a database that was employed for the determination of Euclidean distances between two given patterns and the normalized sensor response. In addition, this mathematical expression was used to develop a 2-dimensional mapping from a multi-dimensional space that enabled us to quantify the distinctions between the samples. The resulting sensor arrays were capable of recognizing small molecules on the basis of their chemical structures, which were related to the nature of the chemical class, the type, and the position of the functional groups.
We then reported the design and development of immunosensors for low molecular weight analytes typically encountered in environmental matrices, including PCBs, s-triazines, and heavy metals. Although simple in operation, direct immunosensors previously have been difficult to develop. The problems encountered include difficulties of fabrication, immobilization of antibody onto the transducer, and efficient generation of analytical signals as a result of the binding of antibody-antigen. The electrochemical immunosensor was based on the measurement of the current because of the specific binding between PCB and anti-PCB antibody-immobilized conducting polymer matrix. Several experimental variables affecting sensor sensitivity and selectivity were investigated and optimized, including the effects of applied potential, flow rate, film thickness, and interfering substances. This resulted in an immunosensor having linear dynamic range of 0.3-100 ng/mL and a correlation coefficient of 0.997 for Aroclor 1242. Some selective electrodes for phenols (for details of the PCB sensors refer to Benda, et al., 1998, and to Masila, et al., 2000 for details of the atrazine sensors).
To demonstrate the feasibility of immunosensors for heavy metals, we used different strategies using nonantibody-based sensing. In this case, we prepared new bioconjugates using a well-known chelator (i.e., 4-[2-pyridylazo] resorcinol, referred to as PAR). These conjugates were synthesized by linking PAR to proteins and enzymes. From these studies, we found that PAR bioconjugates had very low sensitivity to lead, mercury, and cadmium. It was discovered, however, that these conjugates could be used to detect gallium in biological matrices when used in nonantibody-based sandwich assay format. This technique resulted in a detection limit of 5 x 10-8 M for the metal. The selectivity for gallium (III), relative to nearly 30 other metal ions that were investigated, was very remarkable (refer to Xu et al., 1999 for details).
To design a robust sensor, it is important to understand the nature of analyte interactions at the interface. This will enable a rational design of sensor templates and provide for correlation between sensor response and subtle structural features for untested compounds or sensor interfaces. We have studied the mechanism of signal generation at sensor-analyte interfaces using impedance spectroscopy and other analytical techniques. Using impedance spectroscopy, results have shown that we can control the binding of an analyte to antibody-coated sensors by applying pulsed potential (details in Sargent, et al., J Electroanal Chem, 1999; Ana Chim Acta 1998; and Electrochimica Acta, 1999). The insights gained in the mechanistic studies provided a way to generate novel strategies. Our new generic sensing technique was used for the identification and quantitation of toxins. Under this project, we developed a competitive optical sensor for determination of microcystin-LR using microcystin bioconjugate synthesized in our laboratory by coupling this microcystin with fluorescein (details are reported in Yan, et al., 2000).
Another central goal of this project was to conduct a field demonstration of our newly developed sensors to demonstrate their applicability and to correlate these findings with U.S. Environmental Protection Agency (EPA) test methods for evaluating solid wastes (SW-846). During our initial attempt to use the new biosensors for field monitoring at selected areas of Binghamton, NY, we encountered difficulties regarding sampling and preconcentration of analytes. As a result, further studies were carried out to couple the new sensors to gas chromatographic (GC) modules. This led to the design of an electronic switch box that removed problems of condensation and temperature fluctuation at sensor chamber. In a recent publication, we described how the multiarray sensor was coupled to GC and how the analytical utilities of the arrayed sensors were improved to, at least, two orders of magnitude compared to stand-alone types of sensors (refer to Masila, et al., ACS Symposium Series). Consequently, we tested the utility of these sensors for real-life applications and obtained comparable results with EPA Method 8081 for PCBs, chlorinated phenols, and PAHs.
Journal Articles:
No journal articles submitted with this report: View all 70 publications for this projectSupplemental Keywords:
innovative technologies, biosensors, multianalyte detectors and systems, rapid identification and quantitation techniques, continuous monitoring., RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Physics, Chemistry, Monitoring/Modeling, Electron Microscopy, Engineering, environmental monitoring, environmental measurement, field portable monitoring, microelectrode, biomonitoring, biosensing system, environmental engineering, spectroscopic, electrochemical analysis, solid waste, groundwaterRelevant Websites:
http://www.chem.binghamton.edu/SADIK/sadik.htm Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.