Grantee Research Project Results
Final 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 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 the 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 1 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 a surrogate pathogen in methods development. (3) Develop PEMC sensor for confirming pathogen identity by its DNA signature. Immobilize the known identifying gene and use DNA extracted from PEMC-collected cells to verify the identity of pathogen. 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.
Conclusions:
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 oocysts detection. 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 exhibits selective and sensitive detection of G. lamblia cysts in several water matrixes (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. Detecting as few as 10 cysts per mL was achieved in all three water matrixes 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 (Escherichia 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.
Detection of viable pathogenic bacteria is important in the context of drinking water. Antibody-based methods require a growth step which limits time-to-results performance. In this study, we use a mass-change sensitive cantilever biosensor and a probe, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), that accumulates only in live cells inducing a mass-change response to determine the cell viability in a short time. A poly-L-lysine coated sensor immobilized with live E. coli JM101 (a surrogate for a pathogenic target) at various concentrations was exposed to BCECF-AM in a flow arrangement. Larger resonant frequency decrease to 100 µL 60 µM BCECF-AM was observed when sensor surface cell concentration was increased from 1,090 ± 580 to 3,960 ± 370 cells/mm2 (n = 5). A log-linear relationship between the sensor surface cell concentration and frequency response was obtained in the range of 1,000-4,000 cells/mm2 and as low as ~2,000 viable E. coli cells was rapidly detected (<1 h).
A simpler method for measuring sensor response was developed so that the technique developed in this study could be applied in the field. Through a series of studies, we establish that monitoring impedance at a fixed frequency near the initial resonant frequency of piezoelectric-excited millimeter-sized cantilever (PEMC) sensors is superior to using resonant frequency or impedance measurements at resonance for monitoring sensor response. Density change experiments were conducted, and the three monitoring approaches were compared with respect to the obtained signal, noise level and signal-to-noise ratio. Monitoring impedance at fixed frequency approach exhibited a significantly larger response and superior signal-to-noise ratio than resonant frequency or impedance at resonant frequency monitoring approaches. The impedance approach is simpler compared to the conventional resonant frequency monitoring method and is effective for applications that require simultaneous monitoring of multiple sensors.
In the first phase of the study, only IgM against Cryptosporidium was available, and this paper reported on its use with the sensor for detecting oocysts in water and buffer. Piezoelectric-excited millimeter-sized cantilever (PEMC) biosensors were fabricated and functionalized with immunoglobulin M (IgM) for the detection of Cryptosporidium parvum oocyst in a flow configuration at 1 mL/min. The detection of 100, 1,000, and 10,000 oocysts/mL was achieved with a positive sensor response in less than 1 min. Bovine serum albumin (BSA) was used as a blocking agent in each experiment and was shown to eliminate non-specific binding. The sensor's resonance frequency response correlates with C. parvum oocyst concentration logarithmically. The oocyst attachment rate was found to increase by an order of magnitude in increasing concentration from 100 to 10,000 oocysts/mL. The significance of these results is that IgM-functionalized PEMC sensors are highly selective and sensitive to C. parvum oocyst and therefore, have the potential to accurately identify and quantify C. parvum oocyst in drinking water.
One of the main goals of the project is to detect pathogens in large volumes of samples. In this study, we develop a method for 1 liter sample that can be scaled up further, if a design calls for a larger volume. Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors immobilized with antibody specific to E. coli (EC) O157:H7 is used to detect EC at 1 cell/mL in 1 mL and 1 L samples in a batch and flow mode, respectively. Two sensor designs were used. The first design (PEMC-a) has both the piezoelectric and non-piezoelectric layer anchored, while in the second design (PEMC-b) had only the piezoelectric layer anchored. PEMC-a, used in batch mode with 1 mL sample, showed limit of detection at 10 cells/mL using the second bending mode at 85.5 kHz in air. PEMC-b exhibited resonant frequencies at 186.5, 883.5, and 1778.5 kHz in air and 162.5, 800.0, and 1725.5 kHz in sample flow conditions. A 1 liter sample containing 1,000 EC cells was introduced at 1.5, 2.5, 3, and 17 mL/min, and the change in resonant frequency was monitored. The total frequency change observed for the mode at 800 kHz and sample flow rates of 1.5, 2.5, 3, and 17 mL/min were 2230 +/- 11, 3069 +/- 47, 4686 +/- 97, and 7188 +/- 52 Hz, respectively. Each detection experiment was confirmed by exposing the sensor to a low pH solution followed by a phosphate buffered saline (PBS) rinse, which caused the release of the attached EC. The final frequency change observed was nearly identical to the value prior to EC attachment. Kinetic analysis showed that the observed binding rate constant at 1.5, 2.5, 3 mL/min were 0.009, 0.015, and 0.021 min(-1), respectively. The significance of these results is that very low concentration of pathogens in large sample volumes can be measured in a short time period without the need for filtration or enrichment.
One of the important research objectives is to show experimentally that the sensitivity obtained in pathogen and parasite detection is demonstrable in a calibrated method. In this study, we show that mass-change sensitivity predicted from first principles model of piezoelectric-excited millimeter-sized cantilever (PEMC) sensors is in reasonable agreement with previously reported experimental values. We introduce a new attogram-level mass addition calibration method using 11-mercaptoundeconoic acid that enables easy measurement of mass-change sensitivity of PEMC sensors. The finite element model of PEMC sensors predicts resonant frequency values that agree within 2 percent of experimental values. However, the model gives mass-change sensitivities that are within one order of magnitude of experimentally measured values. Simulations show two interesting properties of PEMC sensors that are observed experimentally. First, the sensor response is log-linear and thus it is most sensitive at low attached mass or low analyte concentration. Second, the sensor becomes less sensitive with increased attached mass or at high analyte concentration, which gives the sensor the highly desirable property of wide dynamic range of 6-logs.
In order to enhance detection accuracy, we examined the possibility of detecting pathogens via their genes. This was a two-stage development; first to show that DNA detection is feasible, and then to examine its application for E. coli O157:H7. In this first study, we showed it is feasible. This study was ongoing when the EPA grant was approved, and thus partial credit is given to the STAR grant in the acknowledgement. The second and more detailed study is currently being written as a paper for submission later in spring 2012. The second stage of gene detection involved many new methods to be developed. Piezoelectric-excited, millimeter-sized cantilever (PEMC) sensors having high-mode resonance near 1 MHz are shown to exhibit mass change sensitivity of 1-300 ag/Hz. Gold-coated PEMC sensors immobilized with 15-mer single-stranded DNA (ssDNA) were exposed to 10-mer complementary strands at concentrations of 1 fM, 1 pM, and 1 pM, both separately and sequentially at 0.6 mL/min in a sample flow cell housing the sensor. Decrease in resonance frequency occurred as complementary strands hybridized to the immobilized probe DNA on the sensor surface. Hybridization in three background matrixesbuffer, buffer containing 10,000 times higher noncomplementary strands, and 50 percent human plasma, were successfully tested. Sensor hybridization responses to 1 fM, 1pM, and 1 gamma M complementary strand were nearly the same in magnitude in all three matrixes, but the hybridization rates were different. In each case, the sensor detected the presence of 2 amol of complementary 10-mer strand. The extent of hybridization calculated from resonance frequency change did not decrease in serum. The findings suggest ssDNA can be detected at 2 amol without a sample preparation step and without the use of labeled reagents.
We were invited to contribute a chapter to a monograph being prepared by Dr. Sen Keya, EPA laboratories, Cincinnati, OH. This chapter which summarizes the bulk of the progress in STAR 83382901 will be in a chapter authored by Sen Xu and I. The abstract of it is the following: environmental monitoring. In this chapter, we review the application of piezoelectric millimeter-sized cantilever (PEMC) sensor for the detection of waterborne parasites. Cantilever physics and working principle, sensor fabrication and surface functionalization, flow experimental design, as well as examples of detecting C. parvum and G. lamblia are presented. PEMC sensor is a mass-sensitive biosensor whose resonant frequency decreases when the mass of the sensor increases due to target binding. PEMC sensor functionalized with a specific antibody was exposed to target parasites in a flow apparatus. When the parasite binds to the surface-immobilized antibody, mass of the sensor increases and causes decrease of sensor resonant frequency. Real-time monitoring of resonant frequency changes was used for low concentration parasite detection.
We show for the first time detection of ~700 E. coli O157:H7 (EC) in both buffer and proteinous sample using the virulent gene stx2 as a sensing molecule immobilized on piezoelectric-excited millimeter cantilever (PEMC) sensors. Genomic DNA of EC suspended in buffer or beef wash was extracted using a 30-minute procedure, or using a commercial extraction kit, and then was exposed to the sensor immobilized with a 19-mer probe for stx2 gene. As hybridization occurred, resonant frequency of the cantilever sensor decreased due to increased attached mass indicating the presence of stx2 gene in the sample. Wild strain JM101 subjected to the same preparation and procedure did not induce a hybridization response; nor did the genomic extract of EC with a bare sensor. PEMC sensor responses to diluted genomic extracts indicate a much lower concentration of 700 cells is detectable. In order to compare the results with antibody-based detection, samples with EC at 2,500 cells/mL were exposed to antibody-immobilized PEMC sensors which gave reproducible responses confirming previous experiments with such samples.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 10 publications | 10 publications in selected types | All 10 journal articles |
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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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Supplemental Keywords:
Water, environmental monitoring, drinking water, monitoring, pathogens, measurement method, piezoelectric microcantilevers, DNA, drinking water system, 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.