The perchlorate generated over decades has impacted our nation's waters; it is environmentally recalcitrant and potentially toxic. Perchlorate has significant effects on irrigated agriculture; it is detected in vegetables and dairy food products. Perchlorate has emerged as a significant threat to public health. Substantial efforts are devoted to perchlorate removal and also to its identification down to the lowest possible limit of detection (LoD). The US Environmental Protection Agency (EPA) has recently (in February 2011) announced its decision to set a first-ever national standard for perchlorate. Standard methods for perchlorate detection using ion chromatography or mass spectrometry are costly and time-consuming, and require professional laboratory operators. There is a strong need and opportunity to develop simple and inexpensive analytical methods for rapid field detection of perchlorate. The focus of this project is on development of an simple, inexpensive and highly sensitive nano-biosensor for rapid detection of perchlorate down to 1 ppb. This biosensor will suit field use, laboratory applications, and on-line monitoring; it facilitates cost-effective and convenient monitoring of perchlorate in groundwater, soil, drinking water, food and beverages. It will make important contributions towards improvement of public health. Three primary market segments have been identified for the technology: water quality testing, food safety evaluation, and remediation. In addition, the nano materials developed and investigated in the project for perchlorate biosensor would serve as a protocol for other species-selective bio-interfaces suiting detection of other environmental contaminates (e.g., nitrate). OBJECTIVES: The Phase I project developed a perchlorate biosensor platform based on immobilization of perchlorate-reductase on aligned nanowire/nanotube arrays. The Phase II project will build upon the Phase I accomplishments towards: (i) optimized cultivation of perchlorate-reducing bacteria, and purification of perchlorate reductase for improved enzyme activity, stability and selectivity; (ii) refined nanostructure and optimum immobilization of perchlorate-reductase to improve the sensor's performance, ruggedness and lifetime; (iii) further development of the bi-electrode biosensor for simultaneous detection of perchlorate and nitrate; (iv) thorough characterization of the perchlorate-sensitive biosensor, and validation of its advantages in real-life service environments; (v) design of a prototype portable perchlorate biosensor; and (vi) competitive evaluation of the perchlorate biosensor, and refinement of marketing strategies. APPROACH: Recent developments in nanotechnology are bringing about significant gains in the sensitivity, response speed and selectivity of biosensors. A portable perchlorate biosensor is being developed based on immobilization of perchlorate-sensitive enzymes (perchlorate reductase) on aligned nano-arrays, which feature high surface area-to-volume ratios, and nano-scale electrical contacts with working electrodes. As a result, electron transfer and biocatalytic reactions are risen significantly, boosting sensitivity by 10 times or more, and dramatically improving the LoD. In Phase II, the performance of perchlorate biosensor will be further improved by refined nanostructuring and optimized conditions for enzyme immobilization. The optimized biosensor will be thoroughly characterized, and will be evaluated in service environments relevant to monitoring of groundwater, milk and vegetable contamination by perchlorate. The test results generated using the new biosensor will be compared against those produced using the EPA standard ion chromatography method. The issue of nitrate interference will be resolved by a design of bi-electrode for simultaneous detection of perchlorate and nitrate. A prototype portable perchlorate biosensor will be designed incorporating an electrochemical station. PROGRESS: 2011/09 TO 2012/08 OUTPUTS: Perchlorate has emerged as a significant threat to public health. The focus of this project is on development of an inexpensive and highly sensitive biosensor for rapid detection of perchlorate using an enzymatic approach. The first year of Phase II project was focused on: (i) optimized cultivation of perchlorate-reducing bacteria, and purification of perchlorate reductase to realize improved enzyme activity, stability and selectivity; (ii) refined nanostructuring of conductive polymer, and optimum immobilization of perchlorate-reductase to improve the sensor's performance, ruggedness and lifetime; and (iii) further development of the bi-electrode biosensor for simultaneous detection of perchlorate and nitrate. Selection of perchlorate reductase was expanded to several available perchlorate-reducing bacteria, including Azospira oryzae (ATCC BAA-33), Dechlorosoma sp.KJ (BAA-592), Perc1ace (ATCC 202172), Dechloromonas aromatic strain RCB, and Dechloromonas agitata strain CKB. Perchlorate reductases extracted from Azospira oryzae and D. agitata str. CKB exhibited higher activities than others, which offer more promise than perchlorate reductase from Perclace, that was used in Phase I for production of perchlorate biosensors. The nanostructured conductive polymer array was further refined with the objective of improving its performance as electrode for the biosensor. The electropolymerization of poly(3,4 ethylenedioxythiophene) (PEDOT) nanotubes was optimized to maximize the performance of nitrate biosensor when nitrate reductase was immobilized in these highly ordered polymer nanotubes. Direct electron transfer of nitrate reductase was achieved with highly ordered PEDOT nanotubes; the resulting nitrate biosensor showed significantly improved capabilities that include enhanced sensitivity and rapid detection. The addition of methyl viologen was found to improve the amperometric response, and also contribute enhance the linear operational range of the biosensor. The perchlorate biosensors were produced with perchlorate reductases purified from various perchlorate-reducing bacteria. The one with perchlorate reductase from D. agitata str. CKB exhibited the highest sensitivity when compared with others, which is consistent with its high enzyme activity. N-methyl phenazine methosulfate (PMS) was noted to serve as a better mediator than methyl viologen (MV). The use of PMS improved the shutting of electrons, and thus enhanced the performance of perchlorate biosensors. Efforts were also initiated towards development of a portable prototype of bi-electrode biosensor system in order to facilitate field testing of biosensors. The instrument was designed to accommodate computer peripheral interfacing sensors as well as a computer communication port. This work was presented at CHEMS Research Forum, Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, May 2012; and AICHE Annual Meeting, Pittsburg, PA, Oct. 2012. PARTICIPANTS: Jue Lu, Senior Scientist, Technova Corporation Ilsoon Lee (Subcontract), Associate Professor, Michigan State University Benedict okeke (Consultant), Associate Professor, Auburn University, Montgomery John D. Coates (Consultant), Professor, University of California, Berkeley TARGET AUDIENCES: Three primary market segments: water quality testing, food safety evaluation, and remediation. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period. IMPACT: 2011/09 TO 2012/08 Persistent rise in generation of perchlorate over decades has caused significant contamination of groundwater and agricultural products. The end product of this development effort is a simple, low-cost and highly sensitive biosensor for perchlorate detection, which offers a rapid response with a low detection limit. This biosensor suits field use, laboratory applications, and on-line monitoring; it facilitates cost-effective and convenient monitoring of perchlorate in groundwater, soil, drinking water, food and beverages. It will make important contributions towards improvement of public health.We are targeting three primary market segments for the technology, including water quality testing, food safety evaluation, and remediation. PUBLICATIONS (not previously reported): 2011/09 TO 2012/08 Ankush A. Gokhale, Jue Lu, and Ilsoon Lee, Amperometric detection and quantification of perchlorate in groundwater supplies using a highly sensitive nanostructured electropolymerized PEDOT biosensor, AICHE Annual Meeting, Pittsburg, PA, Oct. 2012.
