Post on 30-Dec-2015
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
Conclusions
• dsDNA target preparation
• Optimized conditions
• Demonstrated specific detection using ADK-functionalized sensor arrays
• Still to do: Compare ssDNA and dsDNA targets