Biosensor technologies for monitoring and Biosensor technologies for monitoring and prediction of microbial community response to prediction of microbial community response to environmental change.environmental change.

Mariya Smit and Holly Simon

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Goals

• Identify metabolic processes that differentiate microbial communities across aquatic habitats;• Characterize microbial metabolic responses to chemical and physical gradients.

Challenges: • Limited knowledge about composition of microbial communities; • High complexity and temporal changes of microbial metabolism;• Paucity of genomic sequence information for environmental microbes.

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Methods: DNA microarrays

• Biosensor-based technologies for nucleic acid analysis.

Water sample RNA Labeled targets Hybridization Microarray Data

Microarray Probes

Biosensors*

DNA Microarrays= High throughput biosensors based on nucleic acid hybridization

*Biological recognition of a target analyte by a specific probe with subsequent physicochemical detection of the target:probe interaction.

• Well-established for simultaneous analysis of thousands of genes;• High cost (hundreds of dollars per experiment);• Fluorescent detection requiring lab conditions and large equipment.

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Other types of biosensors

Requirements that are not fulfilled with DNA microarrays: • Deployment in field conditions: - Portability and tolerance of mechanical disturbances; - Low costs of operation to enable analysis of thousands of samples;• Label-free and real-time procedure to decrease the assay time. Formats: • A novel impedance-based biosensor from Sharp Labs of America (SLA).

Our approach:• First, DNA microarrays to select the analytes of interest from thousands present in the samples.• Second, low-cost biosensors to assay the selected analytes in time series across chemical and physical gradients in different habitats.

Like planes and helicopters: both are used to fly, but have very different applications.

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Oligonucleotide DNA microarray format

Microelectrodes

45 m

Semiconductor

Commercial from CombiMatrix • Oligonucleotide probes between 25 and 40 nucleotides in length.

• Custom synthesis: user-defined probe content.

• Re-usable up to 4-5 times.

• 12,000 spots.

Microarray Probe Design:• CombiMatrix software to select probes from a user-defined gene list; • Probe specificity check against a user-defined sequence dataset (in our case, rDNA sequences).

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Oligonucleotide probe design

1. Selection of 5000 group-specific, or “common” probes from 11 species present in Columbia River

Actinobacteria Bacteroidetes

Proteobacteria

Proportion of probes specific for taxonomic groups

Nitrogenmetabolism

Carbonmetabolism 48

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25Fe-S enzymes

2. Selection of 7000 “function signature” probes specific for particular genes involved in nitrogen and carbon metabolism. 251 species of environmental bacteria.

Proportion of probes specific for functional categories of genes of interest

Total: 12,000 probes

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Microarray Hybridization

Hybridization Image • 12K format with 12,000 different probes;• Hybridization with: - Pool of salinity gradient samples (6-28 PSU);- Fresh water sample pool (0 PSU);- Salt water sample pool (32-33 PSU).

• Goals: - Selection of probes that show significant (above background) signals;- Selection of probes that are differentially expressed among the sample pools.

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Differential gene expression between fresh and salt water habitats

Fresh water: Log2(Intensity)

Salt

wate

r: L

og2

(Inte

nsi

ty)

• The majority of probes are expressed at similar levels in both habitats;• Approximately 1300 probes are differentially expressed (>2-fold) between fresh and salt water.

Up-regulated in Fresh water

Up-regulated in Salt water

2-fold cut-off

• Scatter plot: for each probe, X-axis is Log2(Intensity) in Fresh, and Y-axis is Log2(Intensity) in Salt water pools.

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Clustering of gene expression patterns

Fresh/Salt

FS/Salt

FS/Fresh

Upregulated in Mix FS vs both Fresh and Salt pools

UP in Fresh and Mix FS vs Salt pool.

UP in Salt vs Mix FS and Fresh pools.

DOWN in Mix FS vs Salt and Fresh pools.

Upregulated in Fresh and/or Mix FS Upregulated in SaltLog2(Ratio)-1.6 0 1.6

–3 0 3 Fold change

Probes Comparisons

• 2D clustering of 641 probes that are differentially expressed in at least one comparison of sample pools.• Well-defined gene expression patterns correlating with different habitats.

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Towards development of a deployable biosensor

A novel impedance-based biosensor from Sharp Labs of America (SLA)

Advantages: • Label-free, real-time and rapid detection; • Inexpensive devices;• Local company (Camas, WA).

electrode

solution

dielectric layer

electrode

probes

30 pM 3 nM

Impedance Z

SampleInjection

Principle: Formation of target-probe hybrids on the electrode surface causes changes in parameters of alternating current

Proof of concept: detection of a species of Archaea in a mixed culture using probes against 16S rDNA.

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Acknowledgements

CMOP: • Holly Simon• Lydie Herfort• Jon Schnorr• Mikaela Selby• Pete Kahn • Dan Murphy• Peter Zuber• Bill Howe• Antonio Baptista

• Sharp Labs of America:Andrei GindilisJohn HatrzellDavid EvansPaul Schuele

• CombiMatrix Corporation:Dominic Suciu