CICEET Progress Report for the period 9/02/07 Through 3/01/08

Project Title: A Multichannel Handheld Sensor for Microbial Contaminants
Principal Investigator(s): John H. Paul
Additional Investigator(s): David P. Fries
Project Start Date: 10/01/05

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Tables

Table 1

Tasks to meet objectives
The success of the project is contingent upon development of the multichannel handheld analyzer. In September 07 we totally changed design because our prior format did not meet the optical sensitivity needed. We have upgraded the components of the multichannel handheld to meet these criteria. To meet objective 2, we have successfully completed the development for field RNA extraction. Although not part of our proposed objectives, we are refining the methodology we would employ to successfully complete a field NASBA assay. This includes replacing mRNA standards and internal controls with oligonucleotides, to improve field stability of reagents.

Progress on Tasks
Development of the Handheld Analyzer.
The portable hand-held multiplexed heat regulated fluorometer capable of performing and detecting real-time (RT)-NASBA has gone through continual advancement. This system uses custom a developed thermal heater with integrated sensor circuit. The various subsystems of thermal, electrical, optical mechanical and systems layout have all been designed and completed and ordering of all parts are nearly finished. Upon receipt of ordered parts the final build of prototype and subsequent testing will occur.

We anticipate the complete prototype to be ready for testing by May 2008. The multiplexed system utilizes the concept of flexible printed circuit board (PCB) substrates, planar fabrication techniques and component placement, with subsequent folding to produce a 3-dimensional reaction chamber in the form of a compact heated fluorometer. The mini-fluorometer subsystem uses an inserted heater/detection chamber manufactured from polyimide printed circuit material. Circuits for a planar resistive heater, integrated LED and filtered photo-detector have been completed. The heater/detection chamber is manufactured as a planar circuit and LED and photo-detector attached. The entire construct is then folded in space to form a cylindrical and distributed reaction/detection flex unit that is inserted inside the (ABS) acrylonitrile-butadiene-styrene block. Heat is both supplied and regulated to the heater section by measuring the resistance of the heater circuit at a constant voltage and varying current. Figure 1, and Figure 2, exhibit the 2D-3D transform concept and the final opto-electro-thermal block that forms the basis for the multiplexed system. We have two blocks fully completed and ready for integration. Microcontroller developments have been completed and described in a previous report.

Replacement of transcript standard and internal control RNA with oligonucleotide DNAs
In attempt to make the hand held sensor NASBA assay quantitative for K. brevis in the field without further lab analysis, we propose the use of lyophilized DNA oligonucleotide internal control standards for use in quantifying cell numbers. In vitro transcribed RNA used for this purpose would seem optimal given that our target for the field assay is mRNA expressed by K. brevis. However, RNA is unstable outside strict cold storage conditions and is not able to be lyophilized without significant degradation, making it less than ideal for use in the field as an internal control standard. Unlike RNA, DNA oligonucleotides are significantly more stable and are able to lyophilized and stored at ambient temperatures without degradation making them ideal for internal control standard field applications. Our goal is to equate oligonucleotide target copy number to K. brevis cell number using NASBA for quantifiable field applications of the hand held sensor.

Oligonucleotide targets and internal control molecules for rbcL were designed with the exact base sequences as their respective mRNA targets. NASBA assays were performed targeting equal copy numbers of both oligonucleotides and in vitro RNA transcripts at various ranges of titers. In vitro RNA transcripts were used in the comparison instead of whole cell K. brevis mRNA extracts due to constant availability at times when borrowed K. brevis cultures were not readily available. Previous research by our lab has shown a highly reproducible correlation between in vitro RNA transcript copy numbers and mRNA extracted from known numbers of K. brevis cells. This correlation allows us to equate oligonucleotide copy number to inferred in vitro RNA transcript copy number and then to inferred K. brevis cell number.

Table 1 shows the performance of oligonucleotides in three such cross calibration experiments. The TTP ratio (time to positivity ratio) is the ratio of the rbcL target time to positivity divided by the internal control target time to positivity. As can be seen, although TTP ratio increases with a decrease in concentration for transcripts or oligoucleotides, the actual TTP values are significantly greater for the oligonucleotides. Strictly speaking, on a molecule-per-molecule basis, the RNA transcripts were significantly more sensitive than the DNA oligonucleotides.

Using the RNA transcripts to serve as a standard curve, the oligonucleotide TTP ratios were used to generate apparent transcript concentrations (Figure 3). As can be seen from the slope of the line, the oligonucleotides give about _ the signal (or twice the TTP ratio) as in vitro transcribed mRNA. Why the amplification primes RNA better than synthetic oligonucleotides is not known. It may be that the length of the oligonucleotides (87 bp and 92 bp for rbcL target and internal control, respectively) may push the limit of oligonucleotide synthesis, and that a portion of our oligonucleotides are shorter than expected, and therefore cannot be amplified. This would account for the overestimation of the actual oligonucleotide copy number. Alternatively, NASBA might prime RNA more efficiently than ssDNA. In either case, we need to use roughly twice as much oligonucleotide as transcript to achieve the same dynamic range.

Using regression analysis from the equation derived from the in vitro transcript RNA standard curve, inferred RNA copy numbers were calculated for each DNA oligonucleotide titer. Previous studies in our lab have shown there to be approximately 10,000 rbcL mRNA copies per K. brevis cell. Given the linear relationship between TTP ratios from the RNA transcript and DNA oligonucleotide comparison (Figure 3), we can now equate DNA oligonucleotide copy number to cell number using TTP ratio regression analysis (Figure 4). This will allow us to produce lyophilized rbcL DNA oligonucleotide internal control standards that equate directly to a target cell number for a quantitative NASBA assay using the hand held sensor. Our intention is to calibrate each oligonucleotide lot against whole K. brevis cell abundance to ensure precise quantification in the field.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal? Please explain.
The project has not had a change in direction but our design of the handheld sensor has occurred.

Dissemination activities during this reporting period (please include the number of participants where applicable).
Conferences and presentations
Paul, J.H. 2007. Development of sensors for the molecular detection of red tide blooms. Coastal Zone 07, July 25th, Portland OR.

Publications appearing
None this period

Difficulties
Our least expensive design for the handheld device did not meet the performance criteria for optical detection. We have discussed the cost of the revised design handheld with our end user and have decided that a $25,000 ceiling for the instrument would enable us to produce a functional module.

Data Generated to date
The data generated to date is in the attached figures.

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
Specific ordering of the PCB backplane/motherboard for the eight units is in process. The additional 6 ABS blocks are being immediately rapid prototyped. We are awaiting receipt of the additional optical filters for the additional blocks. Upon receipt of all of the piece parts the final prototype will be final assembled and tested. The prototype will be enclosure in a commercially available enclosure so no delay in transfer of the system to the field is anticipated. We anticipate the complete prototype to be ready for lab testing by May 2008. Field testing should be available in June 2008.

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 about one year behind from our projected timetable and we are currently working on a no-cost extension.

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.
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 agree and hopefully we can overcome the technical hurdles in the next reporting period