1. Electrochemical Quantification Discussion
Future Work
References
Acknowledgements
Background
Towards the Development of a Dry Eye Point of Care Diagnostic
Brittney A. Haselwood1, Chi Lin1, Aaron Meidinger2, Brian Kalen1, Garrett Repp1, Mark L. Spano1, Jennifer Blain-Christen1,3, Pierce Youngbar4, Ken
Greenburg4, Marcus Smith4 and Jeffrey T. La Belle¹,²,5
1Tempe, Arizona, Arizona State University, School of Biological and Health Systems Engineering; 2 Tempe, Arizona, Arizona State University, School for Engineering Matter, Transport and Energy; 3 Tempe, Arizona, Arizona
State University, School of Electrical, Computer and Energy Engineering;4 Raleigh, North Carolina, Advanced Tear Diagnostics, LLC;5Scottsdale, Arizona, Mayo Clinic Arizona, School of Medicine
• Over 4.9 mil. people over the age of 50 in the US
suffer from dry eye syndrome.
• However, dry eye symptoms such as itching, burning,
and running eyes are very similar to that of an allergy
• Goal: simple, rapid and accurate Point-of-Care
Technology (POCT) to quantify clinically relevant
biomarkers and rule out allergy.
• Two biomarkers used:
1. IgE: a biomarker for allergic conjunctivitis.
Clinical cutoff of 80 ng/mL
2. Lactoferrin: a biomarker for aqueous deficient
dry eye. Clinical cutoff of 0.9 mg/mL
Figure 1: Prevalence of dry eye
syndrome by age between men
and women. Numbers refer to
sample sizes [1]
Special thanks to: the entire team for the work; Dr. Spano for the electronics and programming; Dr. Blain-
Christen for hardware support; Dr. Youngbar and Dr. Greenburg for providing ophthalmological expertise. Also a
big special thanks for the Advance Tear Diagnostics and its CEO, Markus Smith, for funding the project.
[1]S.E Moss, et al. Arch Ophthalmol. 122;3 (2004), 369-373
[2]J.T. La Belle, et al. Biosens. Bioelectron. 23 (2007), 428–431.
[3]T.L. Adamson, et al. Analyst. 137 (2012), 4179-4187.
[4]S.I. Gonzales, et al. Biosen. J. 1 (2012), 1-5.
Functionalization and Techniques
Electrochemical Impedance Spectroscopy (EIS)
• AC frequency sweep yields change in φ and |current|
• Measured current and applied voltage are used to calculate impedance (figure 3)
• Femtomolar sensitivity; impedance can be related to concentration of analyte
Device Prototyping and Fabrication
Immobilization onto GDEs for EIS Characterization of Proteins & Targets [4-6]:
Figure 2. General
schematics of
protein
immobilization
process onto a
GDE for EIS
measurements
Bare Au Disk Electrode
1 mM 16-MHDA Linker
EDC/NHS Coupling Ethanolamine Blocking
MRE Immobilization
Figure 3: EIS input and output signals . Bode plots result in Nyquist plots.
Time
Potential(V)
Input AC Voltage
Output Measured Current
Φ= Change in phase
~Δ |Z|
-100
-80
-60
-40
-20
0
0.0E+0
2.0E+4
4.0E+4
6.0E+4
8.0E+4
1.0E+5
1 1000
Phase(deg)
|Impedance|(ohm)
log Frequency (Hz)
-3.5E+4
-2.5E+4
-1.5E+4
-5.0E+3
0.0E+0 5.0E+4
Z"(ohm)
Z' (ohm)
Figure 4 (left): shows the
different layers of the proposed
screen printed electrode.
Figure 6: The graph above shows the raw EIS data in
the form of a Nyquist curves for 1 ng/mL IgE in
simulated tear (dashed gray), and in purified (solid
gray) solutions. The black lines show the response of
10,000 ng/mL IgE in simulated tear (dashed black) and
purified (solid black) solutions.
• Figure 4&5: Filter paper will serve as tear collection mechanism and
miniaturization will be performed to limit the sample volume to 0.5 μL.
Filter paper was chosen over PU foam based on FDA approval in dry eye
diagnostics.
• Figure 6: Generally, the difference between simulated tears and purified solution
responses becomes more apparent at lower concentrations of IgE. With
enough replication, this effect can be minimized in the calibration curve
equation.
• Figure 7: The sensitivity and EIS signal seem to increase drastically in simulated
tear fluid. This will expand the limits of detection of lactoferrin
• Figure 9: The calibration curves would be used to program the future handheld
device so the measured complex impedance could be converted into the
concentrations of IgE and lactoferrin, which would appear on the handheld
screen.
• Figure 10A&B: Result suggests that the sensor built on the filter paper is functional
and probable for biomarker detection after fine-tuning. With the screen
printer in the future, a much smaller design with great consistency can be
manufactured.
• Perform Impedance Time (Z(t)) on both Lactoferrin and IgE at their corresponding
frequencies and optimize the calibration curves accordingly.
Prototype Electrochemical Characterization
Figure 11: proposed meter and test strip design.
The ergonomic feature of the meter allows the
physician to collect 0.5 μL of tear fluid in a steady
and natural manner, avoiding reflex tearing.
Target Capture
Figure 9: The graph (left) shows the simulated tears
calibration curves of IgE (dashed line, characterized by Z
=184.94*ohms/ln[IgE] + 1901.9) with R2 of 0.96 at 312.5
Hz, and Lactoferrin (solid line, characterized by Z = 2614.37
* ohms/ln[lactoferrin] + 3800.4) with R2 of 0.99 at 175.8
Hz. The error bars shown are based on N=3 standard error.
Figure 10A&B - 10A (left): CV of a hand-made screen printed electrode prototype (dashed line) versus the
CV of a Zensor (solid line) using 100 mM ferricyanide sweeping from 1V to -1V. Resulting currents
resemble similar pattern to that of Zensor. 10B (right): Bare electrode EIS of the said sensor (dashed line)
versus the bare electrode EIS of Zensor (solid line) using 100 mM ferricyanide. Impedance patterns are
similar to that of Zensor.
Figure 7: The graph above shows the raw EIS data in
the form of a Nyquist curves for 0.5 mg/mL
Lactoferrin in purified solution (solid grey) and in
simulated tear fluid (dashed grey). The black lines
show the response of 2.5 mg/mL Lactoferrin in
purified solutions (solid black) and in simulated tear
(dashed black).
Figure 8: (right) Shows how optimal frequency of
lactoferrin is determined. The logarithmic slopes
(dashed line) of all concentrations in the gradient
are overlaid with the R2 (solid line) across the
frequency range. Typically, there is a trade-off
between high sensitivity and reproducibility.
Figure 5 (right): shows a sensor
prototype
1 10 100 1000 10000
1500
2000
2500
3000
3500
4000
1500
2500
3500
4500
5500
6500
0.1 1 10
IgE Concentration (ng/mL)
AverageComplexZforIgE(Ω)
AverageComplexZforLactoferrin(Ω)
Lactoferrin Concentration (mg/mL)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
1 10 100 1000 10000 100000
R-square
Slope(ohm/ln[Lactoferrin])
Frequency (Hz)
-3000
-2500
-2000
-1500
-1000
-500
0
0 2000 4000 6000
ImaginaryZ(Ω)
Real Z (Ω)
-10000
-9000
-8000
-7000
-6000
-5000
-4000
-3000
-2000
-1000
0
0 5000 10000 15000 20000
ImaginaryZ(Ω)
Real Z (Ω)
0.E+00 1.E+03 2.E+03 3.E+03 4.E+03 5.E+03
-1.E+03
-9.E+02
-8.E+02
-7.E+02
-6.E+02
-5.E+02
-4.E+02
-3.E+02
-2.E+02
-1.E+02
0.E+00
-2.E+02
-2.E+02
-2.E+02
-1.E+02
-1.E+02
-1.E+02
-8.E+01
-6.E+01
-4.E+01
-2.E+01
0.E+00
0.E+00 2.E+02 4.E+02 6.E+02 8.E+02 1.E+03
Screen Printed Sensor Z' (Ω)
ScreenprintedSensor-Z"(Ω)
Zensor-Z"(Ω)
Zensor Z' (Ω)-6.E-04
-4.E-04
-2.E-04
0.E+00
2.E-04
4.E-04
6.E-04
8.E-04
1.E-03
1.E-03
1.E-03
-6.E-03
-4.E-03
-2.E-03
0.E+00
2.E-03
4.E-03
6.E-03
-1 -0.5 0 0.5 1
ScreenPrintedElectrode'scurrent(A)
Zensor'scurrent(A)
Voltage (V)
• Optimize mesoporous carbon ink with
IgE and Lactoferrin antibodies.
• Ergonomic focus-group testing on the
handheld.
• Miniaturize electronic components to
fit in the handheld and enable its
rapid-manufacturing
• Optimize sensor strip manufacturing
for efficiency and reproducibility
• Develop production pipeline