Transcript The ocean as regional enabler A Pacific Northwest success

Biosensor technologies for monitoring and
prediction of microbial community response to
environmental change.
Mariya Smit and
Holly Simon
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.
2
Methods: DNA microarrays
3
• Biosensor-based technologies for nucleic acid analysis.
Biosensors*
*Biological recognition of a target analyte by a
specific probe with subsequent physicochemical
detection of the target:probe interaction.
DNA Microarrays= High throughput biosensors based
on nucleic acid hybridization
Water sample
RNA
Labeled targets
Hybridization
Microarray Data
Microarray Probes
• Well-established for simultaneous analysis of thousands of genes;
• High cost (hundreds of dollars per experiment);
• Fluorescent detection requiring lab conditions and large equipment.
Other types of biosensors
4
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.
Oligonucleotide DNA microarray format
Commercial from CombiMatrix
Microelectrodes
5
• Oligonucleotide probes
between 25 and 40
nucleotides in length.
• Custom synthesis: userdefined probe content.
• Re-usable up to 4-5
times.
45 mm
Semiconductor
• 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).
Oligonucleotide probe design
6
1. Selection of 5000 group-specific, or “common” probes from 11
species present in Columbia River
Actinobacteria
Proportion of probes specific for
taxonomic groups
Bacteroidetes
g
a
Proteobacteria
b
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
Fe-S 25
enzymes
Carbon
27
metabolism
Nitrogen
metabolism
48
Microarray Hybridization
Hybridization Image
7
• 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.
Differential gene expression between fresh
and salt water habitats
8
Salt water: Log2(Intensity)
• Scatter plot: for each probe, X-axis is Log2(Intensity) in Fresh, and
Y-axis is Log2(Intensity) in Salt water pools.
2-fold cut-off
Up-regulated in Salt water
Up-regulated in Fresh water
Fresh water: Log2(Intensity)
• 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.
Clustering of gene expression patterns
9
• 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.
Upregulated in Fresh and/or Mix FS
Upregulated in Salt
Log2(Ratio)
-1.6
0
–3
0
Fold change
Probes
Comparisons
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.
1.6
3
Towards development of a deployable
biosensor
A novel impedance-based biosensor from Sharp
10
Labs of America (SLA)
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.
electrode
solution
probes
Impedance Z
dielectric layer
1550
electrode
1540
1530
Z, Ohm
Advantages:
• Label-free, real-time and
rapid detection;
• Inexpensive devices;
• Local company (Camas, WA).
Z at 2510 Hz
1520
1510
1500
1490
1480
0
500
1000
Sample
Injection 30 pM
1500
2000
2500
v
3 nM
3000
3500
4000
Acknowledgements
CMOP:
• Holly Simon
• Lydie Herfort
• Jon Schnorr
• Mikaela Selby
• Pete Kahn
• Dan Murphy
• Peter Zuber
• Bill Howe
• Antonio Baptista
11
• Sharp Labs of America:
Andrei Gindilis
John Hatrzell
David Evans
Paul Schuele
• CombiMatrix Corporation:
Dominic Suciu