Aptamer Based E-coli Detection in Waste Waters by Portable ...
About OMICS Group OMICS Group International is an amalgamation of Open Access publications and worldwide international science conferences and events. Established in the year 2007 with the sole aim of making the information on Sciences and technology Open Access, OMICS Group publishes 400 online open access scholarly journals in all aspects of Science, Engineering, Management and Technology journals. OMICS Group has been instrumental in taking the knowledge on Science & technology to the doorsteps of ordinary men and women. Research Scholars, Students, Libraries, Educational Institutions, Research centers and the industry are main stakeholders that benefitted greatly from this knowledge dissemination. OMICS Group also organizes 300 International conferences annually across the globe, where knowledge transfer takes place through debates, round table discussions, poster presentations, workshops, symposia and exhibitions. About OMICS Group Conferences OMICS Group International is a pioneer and leading science event organizer, which publishes around 400 open access journals and conducts over 300 Medical, Clinical, Engineering, Life Sciences, Pharma scientific conferences all over the globe annually with the support of more than 1000 scientific associations and 30,000 editorial
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ELISA used antibodies, not stable for long time. Over the past decade, many biosensor systems have been developed for sensitive and reliable detection protocol. 1. Dufour, A.P. (1977) Escherichia coli : the fecal coliform. In Bacterial Indicators/health Hazards Associated with Water (eds A.W. Hoadley and B.J. Dutka), pp. 4858, American Society for Testing and Materials, PA. 2. Bej, A.K., R. Steffan, J. DiCesare, L. Haff, and R.M. Atlas. 1990. Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Appl. Environ. Microbiol. 56: 307-314. Introduction SWCNTs Modified Biosensor System The flexible biosensor was fabricated by directed assembly using reusable template. Flexible template: polyethylene-naphthalate (PEN) film. A plain gold templates were fabricated as two electrode system. The device was immersed into SWNTs suspension (0.001 wt%, 90 semiconducting SWNTs). DC power supplier was used to apply the potential (2~2.5V) between the two electrodes. Negatively charged SWNTs were attracted onto positive conductive patterns in template. Yang Zhang et al 2013 Nanotechnology 24
IV Measurement Procedure Conductance and resistance changes were measured between two gold electrodes. IV measurement was conducted before and after each experiment. Introduction contd Aptamers Aptamers are single-stranded DNA, RNA, or modified nucleic acids. Specifically recognize target molecules. Obtained from a combinatorial library via an in vitro selection process known as the systematic evolution of ligands by exponential enrichment (SELEX) method. There are several advantages of aptamers over antibodies; much smaller than antibodies, more easily modified at terminal sites with several functional groups, more stable than antibodies. E-coli Aptamer DNA aptamer for E-coli; can specifically distinguish the pathogen E. coli O157:H7 from other pathogens (Bruno et al. 2010).
DNA library was first incubated with the E. coli K12 strain and the DNAs binding to the strain were discarded. The precluded DNAs were then used for the selection of O157:H7-specific aptamers. After 6 rounds of the subtractive cell-SELEX process, the selected aptamer was found to specifically bind to the O157:H7 serotype, but not to the K12 strain. Bruno, J.G.; Carrillo, M.P.; Phillips, T.; Andrews, C.J. A novel screening method for competitive FRET-aptamers applied to E. coli assay development. J. Fluoresc. 2010, 20, 12111223. Biosensor Systems for E-coli Detection Detection method Recognition element/target Detection range Detection time Enrichment or DNA-protein
extraction Regeneration Ref. DNA aptamer-based impedance biosensor DNA-aptamer/ E. coli 110-7 to outer 210-6 M membrane proteins 90 minutes E. coli OMPs extraction 15- 45% of regeneration (Queirs et al. 2013) DPV based
electrochemical biosensor DNA-aptamer/ E-coli surface lipopolysaccharide 2 x 102 to 2 x 3.5 hours 107 CFUml1 NO NO (Luo et al. 2012) Real-time PCR DNA template of the RNA-aptamer/ E-coli surface protein 101 to 107 CFUml1
60 minutes incubation + 40 minutes detection DNA extraction NO (Lee et al. 2009) DNA aptamer-based colorimetric detection DNA aptamer/ whole 104 to 108 E-coli cell CFUml1 2 hours NO NO (Su et al. 2012)
DNA aptamer-based colorimetric detection DNA aptamer/ whole 105 to 108 E-coli cell CFUml1 20 minutes NO NO (Wu et al. 2012) Antibody modified microfluidic chip and real-time qPCR Antibody/ whole Ecoli cell 101 to 106 CFUml1
Incubation + 74 minutes detection DNA extraction NO (Dharmasiri et al. 2010) Label free polyaniline based impedimetric biosensor antibody 102 to 107 CFUml1 10 minutes binding + few minutes detection NO
NO (Chowdhur y et al. 2012) Aspects of Improvement Using E-coli specific DNA aptamer with high specificity and affinity without any reactivity loses in the harsh conditions DNA-aptamer directly binds to the surface lipopolysaccarides, so eliminate protein extraction step. SWCNTs modified biosensor system with using features of the electrochemical systems such as; low voltage need, small, cost effective and easy to use equipment. Improve sensitivity to detect the U.S. EPA allowable levels of E. coli and less than 1cells/ml infectious dose. Detection Procedure Pumping to the sample cell
Hybridization of the aptamer and probe DNA pyrene-butyric acid Probe-DNA immobilization onto the SWCTs surface Regeneration with 0.5 % SDS I-V Measurement Curret (uA) Mixing E-coli cells and aptamer Filtration of the mixture with 0.22 m m pore size filter 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 1 15 29 4 3 5 7 7 1 85 99 Voltage (mV) Dose-response Measurements of the Sensor Each data value is the average of three independent experimental results. The linear range between 4 cfu/ml and 105 cfu/ml since we got negligible signal with the E-coli concentration smaller than 4 cfu/ml. Assessment of Sensor Specificity All bacteria strains were applied at 2000 cfu/ml. Still have 10 % to 15 % of signal decrease with other pathogens. May come from just non-specific binding to the pathogens or loosing aptamers during filtration
Regeneration and Sensor Stability The sensor system is stable over 80 % with E-coli detection in30 days period. Analysis of Spiked Wastewater Treatment Effluent Samples plant 1 plant 1 tap water Spiked (cfu/ml) Found (cfu/ ml) cv % Recovery % 10 8.7
101.5 10 9.2 2.6 92.7 104 8687.7 5.5 86.8 Two duplicate experiments were performed for all samples. The recovery of all measured samples was between 93 and 118 %, and the parallel tests showed that the relativity coefficient was within 4.3-21.8 %. Conclusion
An aptamer-based biosensor for rapid and selective E-coli detection was developed with using a SWCNTs modified system. Low detection limit; 4 CFU/ml. When compare with the present studies, the new system is cost-effective, rapid (less than one hour), easy to use and reusable. The system is selective for E-coli O157:H7 . The biosensor evaluated in spiked wastewater samples. APRIL Z. GU Associate Professor and College of Engineering Faculty Scholar Department of Civil and Environmental Engineering 471 Snell Engineering Center 360 Huntington Ave. Boston, MA 02115
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