1. A Capillary Waveguide Biosensor for Marine Microbial Process Studies Kemp, P.F., Aller, J.Y., Dhadwal, H.S., Dantzler, M.M.

2. What is a biosensor? A biosensor is an instrument that couples a biological material to a transducer. A biosensor is an instrument that couples a biological material to a transducer. The transducer converts a change in the biological material into a measurable response. The transducer converts a change in the biological material into a measurable response.

3. Optical Hybridization Biosensors A monolayer coating of single-stranded DNA (probe) is bound at one end to the surface of an optical waveguide (e.g. an optical fiber) A monolayer coating of single-stranded DNA (probe) is bound at one end to the surface of an optical waveguide (e.g. an optical fiber) Hybridization of target RNA or DNA to the probe DNA induces a response (e.g. fluorescence) Hybridization of target RNA or DNA to the probe DNA induces a response (e.g. fluorescence) Hybridization biosensors can be used to detect the presence, abundance, and activity (growth, gene expression) of targeted microorganisms. Hybridization biosensors can be used to detect the presence, abundance, and activity (growth, gene expression) of targeted microorganisms.

6. Design of Capillary Waveguide Biosensors Harbans Dhadwal, Paul Kemp, Josephine Aller and Megan Dantzler

7. The measurement cycle consists of a series of hybridization reactions (signal) separated by denaturing reactions (background). Measurements are confounded by: power fluctuations, power fluctuations, tube-to-tube variations, tube-to-tube variations, hybridization time, hybridization time, target concentration. target concentration.

8. Power fluctuations: Target signal and background co-vary with power.

9. Tube-tube variations: The response of different sensor tubes to a given concentration of target varies, probably because of differences in probe coatings.

10. Time versus concentration: The time required to develop a maximum signal varies with target concentration. Time versus concentration: The time required to develop a maximum signal varies with target concentration.

11. What really happens To measure and compare concentrations, use the initial slope in a brief hybridization.

12. Uncorrected target and background responses

13. Signal = Target/ background response (correcting for variations in power)

14. Signal = Target/Background, corrected for tube-to-tube variations. Sensor response is very repeatable.

15. After correction, 95% confidence intervals for triplicate measurements are ±6%.After correction, 95% confidence intervals for triplicate measurements are ±6%. A small increase in signal above background is sufficient to detect target sequences.A small increase in signal above background is sufficient to detect target sequences. The lowest concentration we have tested to date is 31 pg DNA/mL.The lowest concentration we have tested to date is 31 pg DNA/mL. Another example Raw data Corrected data

16. At 31 pg DNA/ml, signal is measurable after 5 minutes. At 31 pg DNA/ml, signal is measurable after 5 minutes.

17. Concentration can be estimated from the initial slope of the hybridization signal.

18. What’s next? Real world tests are underway. We are evaluating the abundance of alpha- Proteobacteria in deep water, mid water, subsurface, surface microlayer, and aerosol samples. Real world tests are underway. We are evaluating the abundance of alpha- Proteobacteria in deep water, mid water, subsurface, surface microlayer, and aerosol samples. We are working on procedures for estimating target concentration from initial slope of the hybridization signal. We are working on procedures for estimating target concentration from initial slope of the hybridization signal. We are testing alternative detection protocols, focusing on a capture probe/detection probe strategy that allows for multiple fluorochrome labeling and enhanced signals. We are testing alternative detection protocols, focusing on a capture probe/detection probe strategy that allows for multiple fluorochrome labeling and enhanced signals.

19. With thanks to: NSF OCE-0083193 Optical Biosensor for Marine Microbial Process Studies: Development Phase II. (P.F. Kemp, J.Y. Aller, and H.S. Dhadwal)