1. Use of Molecular Signatures for in situ Detection of Organisms and Genes They Express The Next Generation of in situ Biological and Chemical Sensors in the Ocean July 14, 2003 Chris Scholin: scholin@mbari.org

2. Molecular Signatures and Biosensors “Molecular signatures” are basic building blocks of cells –carbohydrates, lipids, proteins, nucleic acids, metabolites, etc. Detection of molecular signatures is used in support of numerous research initiatives –phylogenetics/systematics –species identification/quantification –gene expression/function –food webs/symbioses –comparative genomics/“genomic prospecting” Development of biosensors rests fundamentally on characterization, detection and quantification of molecular signatures

3. Big Picture Questions What species are present? How are they related to one another? How do they respond to alterations in the chemical and physical environment? What role do they play in biogeochemical cycling, food webs and maintenance of global climate? Can we use specific organisms as indicators of environmental change?

4. Techniques for Detecting Molecular Signatures Share an Overlapping Set of Functional Requirements Sample collection Cell concentration, preservation and disruption Separation of molecules based on physical properties Use of intermolecular reactions to reveal target molecules –lectin/carbohydrate –antibody/antigen –nucleic acid hybridization –receptor/target –enzyme mediated processing Optical and electrochemical signal transduction common

5. Molecular Probes Molecules that detect molecules –nucleic acids, antibodies, lectins and biological receptors –used to detect organisms, genes they harbor and express, substances they produce, etc. No single type of probe is “best” –choice depends on question, organism, location, sample type, required lower limit of detection, speed of sample processing, user preference, etc. Whole cell and cell-free probe application formats possible –reveal different aspects of cells’ life cycle and ecology –provides a basis for understanding the utility of probes

6. Whole-Cell and Cell-Free Analytical Formats Whole-cell probingReal-time PCR DNA probe array

7. Whole-Cell and Cell-Free Assays are Highly Amenable to Automation Microfluidics –small scale reactions MEMS –miniaturized pumps, valves, etc. “Lab-on-a-chip” –integrated detection systems –cheap/disposable Wang 2002, Trends in Analytical Chemistry 21:226-32

8. Taking Cell-Free Assays “Nano” Technology Review May 2002 Nanofluidics

9. Taking Cell-Free Assays “Nano” Nano wire Technology Review May 2002 Nanosys

10. Probes applied in whole-cell and cell-free formats do not and should not agree always!

11. The ENVIRONMENTAL SAMPLE PROCESSOR  http://www.mbari.org/microbial/ESP

12. Some Functional Requirements of the ESP Support whole cell and cell homogenate formats Use different classes of probes (ab, nucleic acid, etc.) Take advantage of recurring sample processing themes –particle concentration –timed series of reagent exchanges –solid phase extraction chemistries –optical detection of reaction products –temperature control (etc.) Utilize array technology –multiple targets, and the list keeps growing…

13. Harmful algae Invertebrate larvae DNA Probe Arrays: Target organisms Balanus glandula (Acorn barnacle) Mytilus sp. (Shore mussels ) Pseudo-nitzschia (toxic and nontoxic) Heterosigma akashiwo (and other raphidophytes Alexandrium (and other dinoflagellates) Y. Fukuyo P. Miller

14. In situ ESP trials - Control Arrays Harmful algae Barnacle larvae Balanus glandula Tetraclita squamosa B915 B1066 U519 B1066 U519 All spot control Pseudo Alex Het Position = “who” Intensity = “how much” Y. Fukuyo P. Miller

15. 2G ESP System Components Core ESP –suitable for processing moderate sample volumes (e.g. 1L) –automates sample archival, DNA probe array development External Sampling Modules –developed for meeting special sampling requirements deep-water, large volume concentration, etc. Analytical Modules –Custom analytical devices fitted to the core ESP –require upstream sample collection and processing –ESP provides power, fluids and comms connections

16. Merging Molecular Diagnostics and Autonomous Sensor Systems in the Ocean Calibration of whole-cell and cell-free detection methods not trivial Capability for archiving samples is highly desirable Use data from high frequency measurements to trigger “molecular analytical event”

17. Merging Molecular Diagnostics and Autonomous Sensor Systems in the Ocean Microorganisms are the most logical targets Miniaturized analytical systems under active development and in some cases available now Environmental applications require development of appropriate sample collection and processing “front end” –requirements associated with environmental samples differ from biomedical samples