Grantee Research Project Results
2009 Progress Report: Robust Piezoelectric-Excited Millimeter-sized Cantilever Sensors for Detecting Pathogens in Drinking Water at 1 cell/Liter
EPA Grant Number: R833007Title: Robust Piezoelectric-Excited Millimeter-sized Cantilever Sensors for Detecting Pathogens in Drinking Water at 1 cell/Liter
Investigators: Mutharasan, R.
Institution: Drexel University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2006 through September 30, 2009 (Extended to August 31, 2011)
Project Period Covered by this Report: October 1, 2008 through September 30,2009
Project Amount: $562,215
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Drinking Water , Nanotechnology , Water
Objective:
The goal of proposed research is to develop antibody-immobilized piezoelectric-excited millimeter-sized mechanically robust cantilever sensors (PEMC) for detecting pathogenic agents (PA) Cryptosporidium and Giardia in drinking water and source waters without a concentration or filtration step. The project has three main objectives. These are:
(1) Explore and establish experimentally piezoelectric-actuated millimeter-sized cantilever sensor suitable for detecting one pathogen in one liter of water using new cantilever oscillation and measurement modalities. The goal is to obtain higher resonance frequencies so that sensitive detection can be made.
(2) Develop flow cell-PEMC sensor detection assembly for testing sample volumes of 10-100 liters. The goal is to contact large-volume samples directly with the sensor instead of filtering and then testing. Characterize response of sensor to samples containing known number of Cryptosporidium and Giardia cells. Use E. coli O157:H7 as surrogate pathogen in methods development.
(3) Develop PEMC sensor for confirming pathogen identity by its DNA signature. Immobilize known 38-mer oligo (Gene Bank: L16997) and use DNA extracted from PEMC collected cells to verify the identity of pathogen (Cryptosporidium). The goal is to augment the primary antibody-based detection with DNA-based confirmation so that false readings could be reduced or eliminated. The following fourth objective was added (3/30/2006) in response to review panel comments.
(4) Evaluate detection method developed in objectives 2 and 3 in real matrix, river or source water.
Progress Summary:
During Year 1, the project centered on Objective 1 (sensor) and the large volume samples (Objective 2). Preliminary work was initiated on Objectives 3 and 4.
- We showed for the first time that a piezoelectric-excited millimeter-sized cantilever (PEMC) sensor can detect 5 C. parvum oocysts in background of proteins in PBS background in a flow format (1 mL/min). To improve sensitivity, a secondary antibody (murine IgM) was used to confirm the attachment of oocysts to the sensor. PEMC sensor is a resonant-mode cantilever sensor whose resonant frequency decreases when target analyte binds to its surface. The sensor was functionalized with Protein G, and then immobilized with goat polyclonal IgG anti-C. parvum for detection of oocysts. In the dynamic range of 50 to 10,000 oocysts/mL the sensor response is characterized by a semi-log relationship between resonant frequency response and C. parvum oocysts concentration. In proteinous background, binding kinetics was slower and total sensor response was lower (~45% ) than in water-like buffer. The details of this part of the study were submitted for publication.
- Current method for detecting waterborne parasite Giardia lamblia is tedious and requires a pre-concentration step. We show for the first time a piezoelectric-excited millimeter-sized cantilever (PEMC) biosensor immobilized with a monoclonal antibody against G. lamblia that exhibits selective and sensitive detection of G. lamblia cysts in several water matrices (buffer, tap and river water) at a detection limit of 1 ~ 10 cysts/mL without a pre-concentration step. PEMC sensor is a resonance-based device that functions at a high-order mode near 1 MHz. The antibody-immobilized sensor was exposed to 1 - 10,000 G. lamblia cysts/mL samples in a flow arrangement. When the cysts bound to the sensor, resonant frequency of the cantilever sensor decreased and was recorded continuously. Positive confirmation of sensor detection responses was obtained by environmental scanning electron microscope of sensor surface after detection experiments. Higher sample flow rates (0.5 to 5.0 mL/min) gave higher sensor detection response. Detection of as few as 10 cysts per mL was achieved in all three water matrices tested, and significant sensor response was obtained in 15 min. We also show the feasibility of analyzing at a low concentration of 1 cyst/mL in a 1 liter sample at a high flow rate of 5 mL/min. The details of this part of the study were submitted for publication.
- In order to simplify the measurement method, we examined fixed frequency impedance monitoring. We showed that monitoring impedance at a fixed frequency near resonant frequency of a piezoelectric excited millimeter-sized cantilever (PEMC) sensor provides equivalent measurement as the conventional approach of monitoring resonant frequency. Two sensing experiments are used to validate the proposed approach: density change and detection of a pathogen (E.coli O157:H7). The impedance approach is feasible because PEMC sensors exhibit modest Q-values of 30 to 60 at ~900 kHz and typical biosensing response falls well below 5 kHz, a frequency band near resonant frequency where impedance is a linear function of frequency. The simpler impedance approach lends itself to high throughput applications where a large number of sensor responses is to be monitored simultaneously. The details of this part of the study were recently published.
Future Activities:
- Examine feasibility of detecting live cells. (Objective 2)
- Design flow cell for large volume samples. The size should accommodate larger flow rate of 1 to 4 liters/min. (Objective 3)
- Initiate initial DNA-based detection of Cryptosporidium parvum oocysts. Use both signature DNA sequences and 16S-rRNA sequence. (Objective 3)
Journal Articles on this Report : 7 Displayed | Download in RIS Format
Other project views: | All 10 publications | 10 publications in selected types | All 10 journal articles |
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Campbell GA, Mutharasan R. A method of measuring Escherichia coli O157:H7 at 1 cell/mL in 1 liter sample using antibody functionalized piezoelectric-excited millimeter-sized cantilever sensor. Environmental Science & Technology 2007;41(5):1668-1674. |
R833007 (2007) R833007 (2009) |
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Campbell GA, Mutharasan R. Near real-time detection of Cryptosporidium parvum oocyst by IgM-functionalized piezoelectric-excited millimeter-sized cantilever biosensor. Biosensors and Bioelectronics 2008;23(7):1039-1045. |
R833007 (2007) R833007 (2009) |
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Maraldo D, Mutharasan R. Mass-change sensitivity of high-order mode of piezoelectric-excited millimeter-sized cantilever (PEMC) sensors:theory and experiments. Sensors and Actuators B: Chemical 2010;143(2):731-739. |
R833007 (2009) R833007 (Final) |
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Rijal K, Mutharasan R. PEMC-based method of measuring DNA hybridization at femtomolar concentration directly in human serum and in the presence of copious noncomplementary strands. Analytical Chemistry 2007;79(19):7392-7400. |
R833007 (2007) R833007 (2009) |
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Xu S, Mutharasan S. A novel method for monitoring mass-change response of piezoelectric-excited millimeter-sized cantilever (PEMC) sensors. Sensors and Actuators B: Chemical 2009;143(1):144-151. |
R833007 (2009) |
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Xu S, Mutharasan R. Detection of Cryptosporidium parvum in buffer and in complex matrix using PEMC sensors at 5 oocysts mL-1. Analytica Chimica Acta 2010;669(1-2):81-86. |
R833007 (2009) |
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Xu S, Mutharasan R. Rapid and sensitive detection of Giardia lamblia using a piezoelectric cantilever biosensor in finished and source waters. Environmental Science & Technology 2010;44(5):1736-1741. |
R833007 (2009) |
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Supplemental Keywords:
RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Water, Environmental Monitoring, Drinking Water, monitoring, pathogens, measurement method, piezoelectric microcantilevers, DNA, drinking water systemProgress 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.