Grantee Research Project Results
Final Report: Near-real Time, Highly Sensitive and Selective Field Deployable Biosensor for Cyanotoxins and Cyanobacteria Using both Antibodies and DNA-signatures
EPA Grant Number: R833829Title: Near-real Time, Highly Sensitive and Selective Field Deployable Biosensor for Cyanotoxins and Cyanobacteria Using both Antibodies and DNA-signatures
Investigators: Mutharasan, R.
Institution: Drexel University
EPA Project Officer: Aja, Hayley
Project Period: June 10, 2008 through March 31, 2011 (Extended to September 30, 2013)
Project Amount: $599,999
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens and Cyanobacteria and Their Toxins in Drinking Water (2007) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The overall goal of the proposed research is to develop piezoelectric-excited millimeter-sized cantilever sensors (PEMC) for cyanotoxins in source, finished and system waters that measure rapidly so that cyanotoxin(s) hazard and management decisions can be made in a timely fashion. The cantilever sensors are of unique design and are the result of development in the PI’s laboratory over the past 5 years. PEMC’s significant advantage is the use of label-free reagents and a proposed simple measurement format. PEMC sensors are mechanically robust millimeter-sized resonant mode cantilevers that exhibit mass changes at femtogram (10-15 g) level under liquid immersion and flow conditions.
Therefore, a PEMC immobilized with an antibody against a cyanotoxin may be able to measure the toxin presence in water rapidly. Similarly, the presence of cyanotoxin producing microorganism can be detected using its identifying gene(s) with a complementary sequence immobilized on the sensor.
There are two objectives centered on the cyanotoxin and cyanobacteria detection. These are:
- Develop a cyanotoxin measurement method.
- Develop a rapid DNA-based assay for cyanobacteria.
Summary/Accomplishments (Outputs/Outcomes):
We have successfully completed both of the major objectives: detection of cyanotoxin and toxin-producing cyanobacteria using DNA.
(1) For detecting cyanotoxin, both batch and in continuous flow method was demonstrated for the detection of microcystin LR (MC-LR) at 1 picogram/mL (equivalent to one part per trillion) in a buffer and in tap water, and in both cases without any sample preparation by exposing the sensor prepared with a suitable antibody to one 2 mL water sample containing the MC-LR standard. Second, the method was extended to application in river water. Thirdly, a new method (not originally contained in the proposal) of using a sandwich assay on PEMC sensor was demonstrated which enhanced detection as well as reduced false negatives. It is to be pointed out that this is the first time such a low concentration was measured experimentally without a sample preparation step, and in a very short assay time of an hour. ELISA assay is capable of detecting at 1 ng/mL (equal to ppb). The results of this study were reported in Environmental Science & Technology 2010;45(4):1490-1496; see reference noted below.
(2) Monitoring of cyanotoxins in source waters is currently done through toxin-targeting assays, which suffer from low sensitivity due to poor antibody avidity. A biosensor-based method was shown as an alternative for detecting toxin-producing cyanobacteria Microcystis aeruginosa via species-selective region of 16S rRNA at concentrations as low as 50 cells/mL, and over a five-log dynamic range (second main objective of the research program). The cantilever biosensor was immobilized with a 27-base DNA strand that is complementary to the target variable region of 16S rRNA of M. aeruginosa. The cantilever sensor detects mass-changes through shifts in its resonant frequency. Increase in the biosensor’s effective mass, caused by hybridization of target strand with the biosensor-immobilized complementary strand, showed consistent and proportional frequency shift to M. aeruginosa concentrations. The sensor hybridization response was verified in situ by two techniques: (a) presence of duplex DNA structure post-detection via fluorescence measurements, and (b) secondary hybridization of nanogold-labeled DNA strands to the captured 16S rRNA strands. The biosensor-based assay, conducted in a flow format (~ 0.5 mL/min), is relatively short, and requires a post-extraction analysis time of less than two hours. The two-step detection protocol (primary and secondary hybridization) is less prone to false negatives, and the technique as a whole can potentially provide an early warning for toxin presence in source waters. (It is worthy to note that the method of double-hybridization developed above was not in the original proposal, and evolved as a technique for minimizing or even eliminating false negatives; the nanogold labeled DNA approach not only enhanced detection sensitivity, but also served to confirm detection; this strategy was not in the original proposal). The entire results of DNA-based detection was recently published in Environmental Science & Technology 2013;47(21):12333–12341.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
| Other project views: | All 2 publications | 2 publications in selected types | All 2 journal articles |
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Ding Y, Mutharasan R. Highly sensitive and rapid detection of microcystin-LR in source and finished water samples using cantilever sensors. Environmental Science & Technology 2011;45(4):1490-1496. |
R833829 (2011) R833829 (Final) |
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Johnson BN, Mutharasan R. A cantilever biosensor-based assay for toxin- producing cyanobacteria Microcystis aeruginosa using 16S rRNA. Environmental Science & Technology 2013;47(21):12333-12341. |
R833829 (Final) |
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Supplemental Keywords:
Resonant frequency, ultrasensitive, immuno-binding, biosensors, cantilever sensors, cyanobacteria, cyanotoxinsProgress 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.