Background

Tuning Electrochemical Impedance SpectroscopyChi 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

AcknowledgementsMany 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

Slo

pe

(Oh

m/(

pg/

ml)

)

Frequency (Hz)

Slope Verses Frequency Il-12 unconjugated

Quantum Dot Slope Versus Frequency

10 Nm GolD Nanoparticle Slope VersusFrequency

5 Nm Gold Nanoparticle Slope VersusFrequency

20 Nm Gold Nanoparticle Slope VersusFrequency

Unconjugated Il-12 Slope VersusFrequency

-140

-120

-100

-80

-60

-40

-20

0

1 10 100 1000 10000 100000

Slo

pe

(Oh

m/(

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

Freq

uen

cy S

hif

t (H

z)

Full

Wid

th a

t H

alf

Max

imu

m

Diameter of Nanoparticle (nm)

Comparison of Nanoparticel Daimeter and Frequency

Gold Nanoparticle Diameter VersusFull Band Width

Gold Nano Particle Diameter VersusFrequency 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

Slo

pe

(Oh

m/(

pg/

ml)

)

Slo

pe

(Oh

m/(

pg/

ml)

)

Frequency

Imaginary Slope Versus Frequency

IL-12 Untuned Slope

20 Nm Quantum DotSlope