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

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

Tasks to meet objectives
These objectives encompass the first 14 months of the project. 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.

Progress on Tasks
Our engineering group came up with an elegant design for the handheld analyzer, consisting of 8 independent modules (“reaction blocks”) capable of analyzing one sample at a time (Fig. 1). Each one of these modules contains its own LED light source and detector. The reaction blocks are designed to snap in and out of a holder, like LeggoR blocks.

The schematic in fig.2 represents the individual modular detection block. The MSP430F2013 (U1) microcontroller acts as one of the slaves to the master controller. This slave microcontroller is responsible for the controlling the optics as well as the heat regulation in the individual reaction block. The MSP430F2013 has three 16 bit Sigma-Delta A-D converters which are used to measure the signal from the two photodiodes and the resistive heater. Pins 10 & 11 are used to program the microcontroller and Pins 8 and 9 are used by the I2C interface to communicate with the master microcontroller.

The prototype heater (R4) is a resistive heater made by etching the copper film on a flexible LCP (Liquid Crystal Polymer) substrate to a desired thickness and length to achieve a predetermined resistance value. Fig.1 shows the actual LCP heater placed next to the reaction block. The microcontroller controls the amount of heat generated in the heater by varying the duty cycle of its Pulse Width Modulated (PWM) signal. The resistive heater also substitutes as a temperature detector in the configuration showed in the schematic. PWM output generated by the microcontroller is periodically turned off to measure the voltage across the heater, which represents the resistance value of the heater at a particular temperature. As a result, the temperature inside the heater block is determined and is used as a control parameter to regulate the duty cycle of the PWM signal which drives the heater. The temperature inside the reaction block is thus maintained at a desired temperature, 41oC (+/-0.5) in our case.

The outputs of photo detectors with integrated optical filters of desired wavelength drive the other two A-D converters on the microcontroller after appropriate signal conditioning. Pin 12 which is a general purpose I/O pin is used to control the LED light sources. Only the light source and photodetector pair associated with a fluorophore is active while detecting that particular fluorophore. This helps reducing the crossover detection between the two different fluorophores used in the reaction.

The optical components used in the assembly are as follows:

	Excitation Wavelength LED/Filter (_) nm	Emission Wavelength Photodetector/Filter (_ ) nm
Fluorophore 1(FAM)	490/10	520/10
Fluorophore 2(ROX)	578/10	600/10

We have completed the testing of the field RNA extraction protocol. The salient features of the RNA extraction field kit is a syringe filtration assembly consisting of a 60 cc syringe, and adapter, and a commercially available RNeasy (Qiagen) purification column (Fig. 3, left panel). A comparison of the results obtained with the field extraction kit compared to the standard lab protocol for extraction of RNA from K. brevis in culture and field samples appears in Fig 3 (right panel). There was excellent agreement between the two methods.

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
Conferences and presentations
Paul, J.H. Smith, M.C. Fries, D.P. Casper, E. Patterson, S. Farmer, A.S. 2006. Handheld And Autonomous Nasba-Based Sensors For Red Tide Detection. ASLO Summer Meeting in Victoria, British Columbia June 4-9 J.H. Paul. 2006. Molecular Detection of Red Tides in the Coastal Zone. Florida Coastal Ocean Observing Systems Caucus Meeting 5. Mote Marine Lab, April 3, 2006.

J.H. Paul. 2006. Handheld and autonomous sensors for microbial detection in the oceans. Seminar at Plymouth Marine Lab, July 28th, Plymouth, UK.

Participant, State of the Research on Red Tide in the Gulf of Mexico, Mote Marine Lab, Sarasota, FL, July 17-20, 2006

Publications submitted
Casper, E.T., Stacey S. Patterson, Pragnesh Bhanushali, Andrew Farmer, David P. Fries, John H. Paul. A Handheld NASBA Analyzer for the Field Detection and Quantification of Karenia brevis Submitted for publication

Difficulties
The only major difficulty encountered in the project was that a student hired for the summer decided to resign from the project in August. I will move a different person to the project

Data Generated to date
The data generated to date is in the attached figures and the manuscript submitted

Project Objectives for Next Reporting Period

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

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 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

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
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 draft manuscript for publication indicates shows excellent statistical comparisons.

Questions/comments/ suggestions for the researchers?
None.

PI Response to End User Advisor Feedback
I agree.