User Needs Assessments UNA

1
User Needs Assessments (UNA)
India Completed Aug 2006
Kenya Pending
Brazil Completed Aug 2007
2
Potential DxBox End-Users

• Developing world
• Small urban or rural clinics
• Healthcare workers
• Military
• NGOs
• Disaster/crisis healthcare workers
• Developed world
• Physicians office laboratories
• Elder-care facilities
• Small urban or rural clinics
• Military
• Public healthcare labs
• First responders
• Individuals

3
Initial Target for the DxBox PlatformFever-Causi
ng Pathogens in Whole Blood

• A Challenge Sample to be 2-3 drops of blood from
  a finger stick

4
Nailing the diagnosis with protein and NA
detection

• Percentage of 57 serum specimens positive for
  dengue virus-specific IgM or viral RNA, plotted
  according to when the samples were collected, in
  relation to the number of days post onset of
  disease

From Optimized diagnosis of acute dengue fever
in Swedish travelers by a combination of reverse
transcription-PCR and immunoglobulin M detection.
Lindegren, et al., J. Clin. Microbiology,
43(6), 28502855 (2005)
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When the DxBox could be useful
Pathogen proliferation, infectiousness
IgM titersand symptoms
IgG titers
Bacteremia,viremia
infection
NA detectability
IgM detectability
IgG detectability
treatment
epidemiology
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Operational Design
Finger stick for 2-3 drops blood
Add blood to disposable card
Insert card in reader
Sample conditioning
Immunoassays
NA assays
Absorbance imaging
Fluorescence differences
Analyze images
Analyze values
Determine presence of pathogen(s)
Report presence of pathogen(s)
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Piezoelectric Microdispensing System for
Patterning Capture Reagents on Surfaces
Recently patterned(drying) droplets

• Based on 4 piezoelectric actuators (and controls)
  from MicroFab Technologies, Inc.
• Single drop control
• Custom-built control of T, r.h., drop firing
  pattern and rate

8
Flexible Membrane Spotting
Orifice diameter 50 µm Drop diameter 30
µm Droplet volume 15 pL

• Drops of HRP-labeled anti-aldolase IgG spotted
  onto nitrocellulose membranes
• Dried overnight
• Developed w. precipitating TMB
• Spot surface area proportional to volume
  dispensed
• Spot intensity function of many factors

lt Single Drops Multiple Drops gt
Yager group
9
IA Eggs in 2 Baskets
Strength signal amplification Weaknesses unproven
storage potential two-step detection
Strengths proven storage stability single-step
detection Weakness no signal amplification
Yager group
10
Microfluidics can speed up sandwich immunoassays!
Flow-through Membrane Assay
Conventional Well-based ELISA
sample flow (2 min)
secondary Ab Incubation (hour)
secondary Ab flow (2 min)
development (2 min)
development (10 min)
sample Incubation (hour)
total assay time lt10 min
total assay time gt2 hours
Membrane assay gives similar results to
conventional ELISA in a fraction of the time
Yager group
11
Current Membrane IA Performance
without IgG mop-up
Yager group