This study investigated using nanoparticles of different sizes conjugated to biomarkers to tune electrochemical impedance spectroscopy (EIS) signals and resolve signal aliasing. The researchers found that conjugating smaller 5nm gold nanoparticles to IL-12 biomarkers caused a shift in the peak frequency response compared to unconjugated IL-12. Larger 20nm nanoparticles caused a negative frequency shift. This relationship could be used to distinguish between similar biomarker signals in simultaneous measurements with EIS.

1. Background
Tuning Electrochemical Impedance Spectroscopy
Chi Lin1, David Probst1, Aldin Malkoc1, 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
- Aliasing is the overlap of two output signals that cause noise to one
another
- Once a signal is aliased it can not be undone or rectified
- Electrochemical impedance spectroscopy (EIS) has become a more
prominent technique for analyzing biomarkers over the last few years
- The largest advantage with EIS is its possibility to measure two or more
biomarkers simultaneously and accurately
- One road block to this achievement is the aliasing of similar biomarker
signals
Functionalization and Techniques
Figure 1: This figure shows the aliasing of two
biomarkers, 1,5 AG with glucose. This is slope
(Ohm/(mg/dl)) versus frequency. The overlap of
signal occurs from 10 Hz up to 1000Hz.
Immobilization onto GDE’s using Il-12 and Nanoparticles
Bare Electrode
MHDA treatment
EDC/NHS application
Biotinylated Il-12 Antibody
Streptavidin nanoparticle
Ethanolamine
Il-12 Antigen
Figure 2: Schematic of immobilization process, the first step is a self assembling
monolayer, MHDA. Then MHDA is activated by EDC/NHS, which in turn antibody binds
to the MHDA. Using streptavidin nanoparticles the nanoparticles were conjugated to the
antibody using biotin-strepaviden interactions
Quantitative Results
Figure 6: The conjugation of quantum dots
to Il-12 antibody shifted the characteristic
peak a magnitude larger, but had low
sensitivity, as well has a very large full width
at half maximum. As compared to untuned,
the quantum dots loss nearly an entire
magnitude of sensitivity.
Figure 3: Overlays of all 4 Nanoparticles
and untuned Il-12. The untuned IL-12
seems to peak at 17 Hz, where all 4
Nanoparticles shift the peak either up or
down. This shift seems to have a direct
correlation to the size of nanoparticle. own.
Discussion
- By analyzing the imaginary impedance rather then complex the
shape of the Slope vs Frequency graph is no longer a low/high
pass filter, but a band pass exhibiting a particular peak
characteristic to that measured marker
- By conjugating nanoparticles to Il-12 the optimal frequency has
shifted
- There was a consistent inverse correlation between the diameter
of the nanoparticle and its effect on the protein properties
- This may be attributed to the surface area of the particle, with
5nm there is a much lower surface area, giving the IL-12 the
ability and room to bond with more particles
- With the bonding to more particles, there is a greater magnitude
of full width at half-maximum
- Quantum dots showed a large shift in frequency, but a much
lower response and sensitivity
Future Work
- Further exploring the electrochemical effects of conjugating
nanoparticles to different types of biomarkers (enzymes)
- Conjugating different types of nanoparticle's to several markers and
measuring them simultaneously by applying several frequencies
unique to a particular analyte to measure
Figure 8: Schematic
of three biomarkers
conjugated to three
different
nanoparticles
References
Acknowledgements
Many thanks to the entire La Belle Group Lab for support, especially the TEIS teams.
[1] J.T. La Belle, et al Methods 61 (2013), 31-59
[2] J.T. La Belle, et al. Analyst, 2011, 136, 1496.
[3] T.L. Adamson, et al. Journal of Diabetes Science and Technology. 8 (2014), 350-355.
Figure 5: This graph shows the relationship
between the diameter of the nanoparticle
and its affect on frequency as well as full
width at half maximum (FWHM). As seen
above the smaller the diameter in
nanoparticle, the greater the shift in
frequency the equation for the relationship
is: y = 259.85e-0.247x. This also holds true
for the full band width, as the diameter
decreases, the full band width increases
the relationship between these two can be
best fit by the following line: y = 805.55e-
0.218x. Both R^2 correlations were about .98
showing a strong relationship between the
size and electrochemical attributes. N=3 for
diameter 5 nm and 20 nm, and N=5 for
diameter of 10 nm.
Figure 4: Overlays of the complex islope
versus frequency. Unlike imaginary
impedance, there is no obvious distinct
peak. Also every frequency below 75 Hz
has aliasing, covering other wanted
signals.
Impedance of AC Circuit
- Z(w) = Z’(w)+Z’’(w)
𝑍 𝑤 = 𝑍°(cos ∅ + 𝑗𝑠𝑖𝑛(∅)
- Z(w) is the Complex
Impedance
- Z’’(w) is the Imaginary
Impedance or is equal to
 𝑍° 𝑗𝑠𝑖𝑛 ∅
- Looking at imaginary
versus total complex
impedance gives us
unique peak previously
not observed
Nano Particle Frequency Shift (Hz) Full Width at Half Maximum
5 Nm Gold 64.2 295.06
10 Nm Gold 4.05 79.946
20 Nm Gold -14.09 10.726
20 nm QD 441.82 2001.5
Table 1: (below) Shows the frequency shift
caused by the addition of each
nanoparticle, as well as the full band
width. As the diameter of the nanoparticle
increases, the shift decreases, to the point
where there is a negative shift, relative to
untuned IL-12
Figure 7: Applying
several frequencies
to measure
simultaneous
markers
0
100
200
300
400
500
600
700
1 10 100 1000 10000 100000
Slope(Ohm/(pg/ml))
Frequency (Hz)
Slope Verses Frequency Il-12 unconjugated
Quantum Dot Slope Versus Frequency
10 Nm GolD Nanoparticle Slope Versus
Frequency
5 Nm Gold Nanoparticle Slope Versus
Frequency
20 Nm Gold Nanoparticle Slope Versus
Frequency
Unconjugated Il-12 Slope Versus
Frequency
-140
-120
-100
-80
-60
-40
-20
0
1 10 100 1000 10000 100000
Slope(Ohm/(pg/ml))
Frequency (Hz)
Imaginary Slope Versus Frequency
10 nm Gold Nano Particle n=4
20 nm Quantum Dot n=3
5 nm Gold Nano Particle n=3
20 nm Gold Nano Particle n=2
IL-12 Untuned Imaginary Slope
-20
-10
0
10
20
30
40
50
60
70
0
50
100
150
200
250
300
350
5 10 20
FrequencyShift(Hz)
FullWidthatHalfMaximum
Diameter of Nanoparticle (nm)
Comparison of Nanoparticel Daimeter and Frequency
Gold Nanoparticle Diameter Versus
Full Band Width
Gold Nano Particle Diameter Versus
Frequency Shift
-2
-1.8
-1.6
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
-120
-100
-80
-60
-40
-20
0
1 10 100 1000 10000 100000
Slope(Ohm/(pg/ml))
Slope(Ohm/(pg/ml))
Frequency
Imaginary Slope Versus Frequency
IL-12 Untuned Slope
20 Nm Quantum Dot
Slope