Final Report: Rapid Detection of Algal Toxins

EPA Contract Number: EPD09019
Title: Rapid Detection of Algal Toxins
Investigators: Barrall, Geoffrey A
Small Business: Electronic Bio Sciences, LLC
EPA Contact: Manager, SBIR Program
Phase: I
Project Period: February 1, 2009 through September 15, 2009
Project Amount: $69,988
RFA: Small Business Innovation Research (SBIR) - Phase I (2009) RFA Text |  Recipients Lists
Research Category: Small Business Innovation Research (SBIR)


As water resources become scarcer and more stressed, rapid and inexpensive detection methods need to be developed to ensure the safety of the drinking water supply by determining the presence of cyanotoxins. This project aimed to make progress toward this goal by detecting a specific toxin with well-known harmful effects with a novel measurement system. Specifically, this project’s goal was to exploit the nicotinic acetylcholine receptors' (nAChRs) highly sensitive response to anatoxin-a as the basis for a reliable, rapid, and inexpensive ion channel-based detection system. 
The basis of this SBIR project was to adapt Electronic Bio Sciences’ (EBS) previously developed and demonstrated low-noise ion channel measurement platform utilizing the glass nanopore membrane (GNM) to allow for the incorporation of nAChRs into a bilayer. The robust platform and long bilayer lifetime would allow for a sample to be collected and flowed through the system to characterize the sample for the presence of anatoxin-a. The scope of this project involved obtaining the nAChRs, demonstrating successful incorporation of the channel on the glass nanopore platform, quantifying the effect of the anatoxin-a on the nAChR, and a preliminary evaluation of potential interferents. 

Summary/Accomplishments (Outputs/Outcomes):

EBS obtained nAChR proteins in their native membranes and performed numerous experiments to demonstrate functional nAChR channels. Initially, the nystatin/ergosterol vesicle fusion method was attempted and adapted to increase the likelihood of successful vesicle fusion. When these methods failed to yield successful demonstration of nAChR functionality, EBS altered the lipid type and composition. Despite numerous experimental protocols, channel functionality could not be demonstrated before the period of performance on the project ended.
In spite of the lack of successful nAChR incorporation, EBS made several significant achievements in relation to this project and EBS' low-noise ion channel measurement system. During the project, EBS successfully demonstrated the incorporation of nystatin channels into the small bilayers spanning the glass nanopore platform. This marked the first time vesicle fusion had been completed on the GNM. 
In addition, EBS adapted a flow system utilized with the ion channel measurement platform to achieve rapid flow in solutions for use with the detection system. This flow system is necessary because when an nAChR is exposed to a binding agonist for an extended period of time, the channel becomes desensitized and will no longer open in response to the presence of the agonist. This process, however, can be reversed by removing the agonist and allowing the channel to recover by exposing it to an electrolyte solution before re-exposing it to the agonist.


Although the goal of evaluating nAChR response to anatoxin-a on the GNM platform could not be realized during this project, EBS has every expectation that with additional research and further developed insertion protocols, the successful insertion of nAChR in the bilayer system can be accomplished. EBS already has begun to seek out new collaborative partners to develop these methods. Furthermore, this Phase I SBIR project allowed EBS to demonstrate the first successful channel and vesicle fusion to the small area bilayers on EBS' platform. This work has expanded the use of EBS' ion channel measurement system and allowed the development of insertion protocols to begin. 
The EBS low-noise measurement platform has been shown to provide lower noise measurements than previously possible with a planar lipid bilayer apparatus in addition to the robust and long lifetime bilayers achieved. In addition, the system was modified during this project to address key issues with the nAChR to allow fluid flow to allow the desensitization of the channel to be reversed. As a result, after new insertion protocols are developed and implemented for the nAChR, the detection system will be an ideal platform for anatoxin-a detection in water samples. 
After additional insertion protocols are developed, this system should provide a portable, rapid, and low-cost detection method for anatoxin-a. This is a critical need to quickly evaluate limited water resources. Thus, this type of detection system should be in high demand after it is fully demonstrated. In addition, the insertion protocols that have begun to be developed under this project can be further evaluated and applied to a wide range of ion channels for research purposes as well as sensor-based systems. EBS’ low-noise ion channel system allows for a lower noise planar lipid bilayer apparatus than has previously been available. By developing insertion protocols, a large number of channels previously difficult to evaluate at higher noise levels can be examined in depth. Thus, this project has provided a basis for expanding the reach of EBS' ion channel measurement system by potentially increasing the channel types that can be evaluated. 

Supplemental Keywords:

small business, SBIR, EPA, detection, algal toxins, anatoxin-a, contamination, drinking water, low-cost screening, neurotoxins, water processing facility, saxitoxin, rapid detection, measurement and monitoring