Background Tuning Electrochemical Impedance Spectroscopy Chi Lin 1 , David Probst 1 , Aldin Malkoc 1 , Jeffrey T. La Belle¹ , ² ,5 1 Tempe, 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; 5 Scottsdale, 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) = ° (cos ∅ + (∅) - Z(w) is the Complex Impedance - Z’’(w) is the Imaginary Impedance or is equal to ° ∅ - 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 Frequency Shift (Hz) Full Width at Half Maximum 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