CICEET Progress Report for the period 9/16/06 Through 3/15/07

Project Title: A Multichannel Handheld Sensor for Microbial Contaminants
Principal Investigator(s): John H. Paul
Additional Investigator(s): David P. Fries
Project Start Date: 10/01/06

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Tasks to meet objectives
These objectives encompass the entire project period. Our first task was to develop a design for the multichannel handheld analyzer. The second task was to begin construction of the analyzer. The third task was to compare our simplified nucleic acid extraction method (field method) with our lab method using natural bloom samples. An additional task has been to process data output from the handheld analyzer such that it is quantitative.

Progress on Tasks
Progress has been made on the construction of the first block of the multichannel analyzer (Figure 1). The first block has had the LED light source installed as well as the photodetector. We have changed the electronic configuration of the handheld analyzer as reflected in the new schematic (Figure 2). Figure 2 shows the updated schematics for the thermocontroller and the photodetection circuits. We are now using a different slave microcontroller from the one used in the previous design (MSP430F2013) due to a silicon bug in the previously selected microcontroller which was giving erroneous acknowledgement generation during the I2C multiprocessor communication with the master microcontroller (MSP430F169). Now the individual reaction blocks also use a MSP430F169 microcontroller. An integral part of the successful operation of the multichannel handheld analyzer is its capability to maintain a constant temperature (41+ 0.5 °C). Figure 3 shows a calibration curve of resistance vs temperature (left panel) and the capability of the instrument to maintain 41°C (right panel). The instrument has passed the test of the capability to maintain 41°C, required for isothermal NASBA amplification. This block is now ready for testing and side by side analysis using our bench top NASBA instrument, the BioMerieuex EasyQ analyzer. Testing has begun using purified transcripts of the Karenia brevis ribulose-1,5-bisphosphate carboxylase/oxygenase gene (rbcL). To date one such run has been completed and others are underway.

The additional task we undertook this reporting period involved data analysis of fluorescence output of the handheld analyzer. Using data collected with our dual channel analyzer (Casper et al., 2007) we realized that there may be a problem applying our calibration algorithm which was developed for the BioMerieux EasyQ Analyzer. This algorithm utilizes the ratio of the threshold cycles for the unknown (termed “Wild Type” or “WT”) and the internal control RNA (termed “IC-RNA” or “SC”; Figure 4 A & B). The threshold cycle (or Ct) is the time at which RNA amplification begins, and is inversely proportional to concentration. Because of the potential for amplification inhibitors to occur in samples and the tube-to-tube variability in NASBA amplification, we have included an internal control RNA in all our amplification samples. The IC-RNA is a synthetic RNA that contains the same primer binding sequences as the target unknown RNA but has a unique beacon binding site engineered in it. In a multiplex NASBA RNA reaction, a second molecular beacon is added that fluoresces in the second channel, such that target and IC-RNA amplification can be measured simultaneously in each reaction. With the EasyQ instrument, fluorescence of both target and IC-RNA are measured using a common excitation light source and photodetector. In the dual channel and multichannel handheld analyzers, each channel has its own light source and detector, operating in its own voltage range. This is illustrated in Figure 4 panel C, where the top graph shows amplification using the EasyQ analyzer, with both fluorescent signals having a similar range in scale, making it easy to choose a common threshold fluorescence. This is in contrast to the handheld instrument, which has the target and IC-RNA fluorescences on widely differing scales, making choice of a common threshold fluorescence impossible. To obviate this problem, we have investigated the first derivative of the curves (calculated as dy_n - dy_n-1 per time t) (see Figure 4D). The ratio of time of occurrence of the maxima for the target and the IC-RNA should be inversely correlated to target concentration.

The novelty of this method is that both fluorescence channels can be on entirely different output scales, as in Figure 4C, since the rate of change of signal as a function of time is the only critical measurement to be made. Additionally, this method will correct for inhibitors in the samples, because inhibition will also affect the kinetics of IC-RNA fluorescence formation.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal?
The results in this report have not changed the direction of the project.

Dissemination activities during this reporting period
Participant, Joint Center of Excellence in Marine Science, Mote Marine Lab and USF College of Marine Science, St. Petersburg, FL, December 13, 2006

Publications appearing
Casper, E.T., Stacey S. Patterson, Pragnesh Bhanushali, Andrew Farmer, Mathew Smith, David P. Fries, John H. Paul. 2007. A Handheld NASBA Analyzer for the Field Detection and Quantification of Karenia brevis. Harmful Algae 6:112-118.

Difficulties
Progress on the development of the Multichannel Handheld Analyzer is going a bit more slowly than anticipated

Data Generated to date
The data generated to date is in the attached figures and the publication,

Project Objectives for Next Reporting Period

Objectives
1) To complete construction of the Multichannel Handheld Analyzer; 2) To test the analyzer with standards and 3) Field test.

Work plan to Meet Objectives
Completion of construction of the Handheld Analyzer will include rapid prototyping of components, testing of individual reaction blocks, establishing a functional software interface, and other tasks. To analyze with standards means to perform NASBA amplification using the handheld and comparing results to our lab instrumentation (EasyQ analyzer).

Dissemination Objectives for next reporting period
This will be accomplished by participation in national, regional, and/or state meetings.

Overall Project Timeline Update
We are perhaps behind by one month’s time as compared to our original schedule.

Expenditures
The amounts expended coincide to the range anticipated for the work accomplished to date

End User Advisor Feedback
End User Advisor: Dr. David Heil
Organization: State of Florida SEAS program
Location: Tallahassee, FL
Phone number:
E-mail:

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
Potential applications for this research and uses of the technology unchanged from the previous end user advisor feedback. Estuarine monitoring of red tide concentrations in Molluscan shellfish growing waters. May allow for up to the minute information on presence / absence of red tide in estuarine environments. Once a red tide bloom is established there is less need for actual K. brevis water sampling (subsequent overnight shipping). May allows for faster response time to close shellfish harvesting areas. May determine more quickly when red tide is no longer in the water. Each of our five field offices would have the ability to aggressively monitor red tide in the water. Additionally, those engaged in beach monitoring for bathers could use the device from shore and would have to rely less on HAB flags and other sources for information.

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
Fabrication must produce an easily portable and durable sensor to withstand rigors of potential everyday use in saltwater environment. Ideally it would be capable of measurements throughout the water column (0 to 20 feet). Additionally, United States Food and Drug Administration (FDA) criteria for K. brevis are measured in cells/Liter. Therefore, conversion by the instrument (or inclusion of a conversion table for the user) from the NASBA result to an FDA definable result would be beneficial.

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
If successful, this would be a major advancement in red tide management / analysis for Molluscan shellfish growing waters. Review of a information provided shows excellent progress.

Questions/comments/ suggestions for the researchers?
None.

PI Response to End User Advisor Feedback
I need to clarify with the End User that this is not a sampling device but rather an analysis device. Sampling would occur independently. Otherwise I concur with all comments.