Transcript impedancefinal

Impedance Sensor Arrays
for Real Time and Label Free Bio-Affinity
Assay
Vena Haynes
Mariya Smit & Andrei Ghindilis
Holly M. Simon
Laboratories of America
Why Impedimetric Detection?
Functionalized sensor
– prior to analysis
Z
Target analyte species
bind to sensor
– impedance changes
in real time
Analyte
injection
Advantages:
• Label-free, real-time and rapid detection;
• Inexpensive devices;
• Local company (Camas, WA).
Project Aims: #1
Sensor array with reaction
chamber attached
New v3.0 sensor array format to test
Sensor array surface functionalization
New SLA READER
DNA oligonucleotide
• Measures 15 channels
simultaneously
Au
SH (thiol)
• Eight impedance
measurements
per second per channel.
• Sensor temperature
control.
Au
Ready for nucleic
acid assay
Project Aims: #2
Development of genomic assays
• Detection of all E. coli strains using probes for a housekeeping gene
adenylate kinase (ADK);
• Detection of enteropathogenic E. coli isolates using virulence genes,
hemolysin A (HlyA), and shiga toxin 2 (Stx2).
E. coli strains
• common commensal (laboratory K12) MG1655: adk gene only;
• uropathogenic clinical isolate CFT073: adk and hlyA genes;
• enterohemorrhagic (O157:H7, food poisoning) EDL 933: adk and stx2b
genes.
Project Aims: #2
Probes:
ADK probe:
TGGAGAAATATGGTATTCCG
HlyA probe:
TGAATTCCAGAAGCAAGTCT
Stx2b probe:
GCGGTTTTATTTGCATTAGT
Array functionalization
Target preparation:
1 Primer
2 Primer
Template E. coli genomic DNA
PCR
Amplicon
Probe binding part
Target preparation in detail:
Target types:
Double-stranded (dsDNA)
Single-stranded (ssDNA)
hlyA targets
ds ss
500
400
300
200
PCR amplicon
95oC for 5 min
PCR amplicon
l exonuclease digestion
extra purification
75
Simple and fast
Standard, more sensitive?
dsDNA test: Assay development
Goals: to optimize conditions and to compare to the ssDNA test in terms of
sensitivity (detection limit), specificity, and dynamic range.
Assay parameters:
Target concentration
Buffer (SSPE) concentration
Voltage (excitation potential)
Temperature
tested
2.5 and 0.5 µg/ml
1x, 2x, 4x
40, 75, 100, 150 mV
47, 52oC
selected
0.5 µg/ml
2x
75 mV
52 oC??
For almost ALL tests Stx negative control demonstrated negligible signal.
Hly negative control response minimization was the major challenge for
assay optimization.
Parallel Injection of Targets
= ADK; specific
= HlyA; neg.
= Stx2; control
Baseline
Sample injection
Sequential injection of targets to the same
sensor arrays: HlyA
buffer
Hly
ADK
Sequential injection results: Stx2
buffer
Stx
ADK
Conclusions
• dsDNA target preparation
• Optimized conditions
• Demonstrated specific detection using
ADK-functionalized sensor arrays
• Still to do: Compare ssDNA and dsDNA
targets
Acknowledgements
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Mariya Smit
Andrei Ghindilis
Holly Simon
Chris Brow
Simon/Haygood/Tebo Labs
Vanessa Green
CMOP
SLA: Kevin & Carmen
NSF