Impedance Sensor Arraysfor Real Time and Label Free Bio-Affinity

Assay

Vena Haynes

Mariya Smit & Andrei Ghindilis

Holly M. Simon

Laboratories of America

Why Impedimetric Detection?

Z

Functionalized sensor – prior to analysis

Target analyte speciesbind to sensor – impedance changesin real time

Analyte injection

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

New v3.0 sensor array format to test

Project Aims: #1

SH (thiol)

Au

Au

Ready for nucleicacid assay

Sensor array surface functionalizationNew SLA READER

• Measures 15 channelssimultaneously

• Eight impedancemeasurements

per second per channel.

• Sensor temperaturecontrol.

DNA oligonucleotide

Sensor array with reaction chamber attached

• 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).

Project Aims: #2

Development of genomic assays

• 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.

E. coli strains

ADK probe: TGGAGAAATATGGTATTCCG HlyA probe: TGAATTCCAGAAGCAAGTCT

Stx2b probe: GCGGTTTTATTTGCATTAGT

Target preparation:

1 Primer 2 Primer

PCR

Amplicon

Template E. coli genomic DNA

Probe binding part

Project Aims: #2Probes:

Array functionalization

95oC for 5 min

PCR amplicon

Target preparation in detail:

Target types:

500400300200

75

hlyA targets

ds ss

Double-stranded (dsDNA) Single-stranded (ssDNA)

PCR amplicon

exonuclease digestion

Simple and fast Standard, more sensitive?

extra purification

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: tested selected

Target concentration 2.5 and 0.5 µg/ml 0.5 µg/mlBuffer (SSPE) concentration 1x, 2x, 4x 2xVoltage (excitation potential) 40, 75, 100, 150 mV 75 mVTemperature 47, 52oC 52 oC??

For almost ALL tests Stx negative control demonstrated negligible signal.Hly negative control response minimization was the major challenge forassay optimization.

= ADK; specific

= HlyA; neg.

= Stx2; control

Baseline Sample injection

Parallel Injection of Targets

Hlybuffer

ADK

Sequential injection of targets to the same sensor arrays: HlyA

Stxbuffer

ADK

Sequential injection results: Stx2

Conclusions

• dsDNA target preparation

• Optimized conditions

• Demonstrated specific detection using ADK-functionalized sensor arrays

• Still to do: Compare ssDNA and dsDNA targets

Acknowledgements

• Mariya Smit

• Andrei Ghindilis

• Holly Simon

• Chris Brow

• Simon/Haygood/Tebo Labs

• Vanessa Green

• CMOP

• SLA: Kevin & Carmen

• NSF