Simultaneous Measurement of Simultaneous Measurement of Nonlinearity and Electrochemical Nonlinearity and Electrochemical Impedance for Protein Sensing Using Impedance for Protein Sensing Using Two Two-Tone Excitation Tone Excitation Jon Daniels, Ph.D. Candidate jon.daniels@stanford.edu Stanford Department of Electrical Engineering Stanford Genome Technology Center (SGTC) Co-authors: Erik P. Anderson – Stanford EE, SGTC Prof. Thomas H. Lee – Stanford EE Prof. Nader Pourmand – SGTC, UC Santa Cruz IEEE EMBC08 24-Aug-08 SuBT2.5
Affinity Affinity Biosensors Biosensors • Immobilized target probe selectively probe captures target • Target capture changes surface properties properties Incubation, Incubation, washing • Detecting change in surface properties = detecting target Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 2
Impedance for Affinity Impedance for Affinity Biosensors Biosensors Solution Probe Layer Probe Layer (Insulator) ( ) Electrode • Binding changes electrical properties of surface • Selectivity determined by probe layer => platform amenable to various applications by changing probe Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 3
Measuring Impedance Measuring Impedance I Z f Z DUT − V out V in + V V out = -V in (Z f /Z DUT ) = -V (Z /Z ) V Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 4
Electrode “ Electrode “Impedance Impedance” is Nonlinear ” is Nonlinear • Electrode-electrolyte impedance depends (quite strongly) on DC bias => nonlinear • ionic double layer capacitance: � φ � � � � � 2 � � � � C 0 1 + 1 228 µ F / cm 2 � � � � C dl ( φ ) = mol/L mol/L 8 8 V T V T I V Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 5
Question #1 Question #1 Can changes in nonlinearity be used to discriminate target binding? vs. ? Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 6
Question #2 Question #2 Can nonlinearity be quantified without extra measurement time? I I V Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 7
Measuring Nonlinearity w/ 2 tones Measuring Nonlinearity w/ 2 tones V in = A cos( ω A t ) + B cos( ω B t ) Z f Conventional Low frequency 1 + α 1 V in + α 2 V 2 � � Z DUT ( ω ) = Z 0 ( ω ) Z DUT − in V out Z f ( ω ) 1 V in + V out = − V in 1 + α 1 V in + α 2 V 2 Z 0 ( ω ) V out = -V in (Z f /Z DUT ) in − Z f ( ω A ) Z ( ω ) Small-signal impedance = Z 0 ( ω A ) A cos( ω A t ) V out 2 α 1 AB Z f ( ω A − ω B ) 1 + Z 0 ( ω A − ω B ) cos(( ω A − ω B ) t ) IM2 2 α 1 AB Z f ( ω A + ω B ) 1 + Z 0 ( ω A + ω B ) cos(( ω A + ω B ) t ) 4 α 2 AB 2 Z f ( ω A − 2 ω B ) 1 + Z 0 ( ω A − 2 ω B ) cos(( ω A − 2 ω B ) t ) IM3 4 α 2 AB 2 Z f ( ω A +2 ω B ) 1 + Z 0 ( ω A +2 ω B ) cos(( ω A + 2 ω B ) t ) + other terms far removed from ω A Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 8
System Diagram System Diagram PC with LabView Tone Outputs and ADC/DAC card FFT ADC Select DUT 0.1-100 kHz ω Α chip with 36 electrodes electrodes Z f Σ Z DUT,1 V in 16:1 − ω Β Z DUT,16 V out + 17 Hz PCB Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 9
PCB Implementation PCB Implementation Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 10
Validating Measurement Approach Validating Measurement Approach 12.5 1 k Ω 12.45 Capacitance [nF] Capacitance [nF] 12.4 10 nF 12.35 12.35 12.3 12.25 o = measured manually with custom setup � � � � = measured manually with commercial LCR meter 12.2 – = extrapolated from nonlinearity tones at 0 mV −100 −75 −50 −25 0 25 50 75 100 Bias [mV] Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 11
Making “Biology” Happen is Hard Making “Biology” Happen is Hard fluorescently-labeled BSA-biotin streptavidin BSA polymer multilayer Contact pads 300µm electrodes O-ring (holds solution) Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 12
Measured Biological Data Measured Biological Data |Z dut | @ 1 kHz [k Ω ] CPE Magnitude ["nF"] CPE phase parameter 15 30 0.925 29 before 0.92 14 28 0.915 13 27 0.91 12 26 0.905 25 11 0.9 24 10 0 50 100 150 0 50 100 150 0 |Z dut | @ 1 kHz [k Ω ] 50 100 150 CPE Magnitude ["nF"] CPE phase parameter Electrode Bias [mV] Electrode Bias [mV] Electrode Bias [mV] 15 30 after addition of 0.925 29 14 0.92 1 µg/mL avidin 28 0.915 13 27 0.91 12 26 0.905 11 25 0.9 24 10 0 50 100 150 0 50 100 150 0 50 100 150 Electrode Bias [mV] Electrode Bias [mV] Electrode Bias [mV] Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 13
Nonlinearity Indicates Binding Nonlinearity Indicates Binding change after 0.06 addition of 0.04 250 ng/mL 0.02 avidin α 1 [V −1 ] 0 ∆ α −0.02 −0.04 −0.06 −0.08 BSA−biotin (positive) BSA (negative) n=4 n=8 Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 14
Conclusions Conclusions • Two-tone approach allows simultaneous measurement of small-signal impedance and nonlinearity – No extra measurement time – Cost is redesigning measurement apparatus • Nonlinearity can indicate target binding • Nonlinearity can indicate target binding • Nonlinearity appears to be influenced by surface charge – Surrogate for field-effect sensor? Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 15
Questions? Questions? Suggestions? Suggestions? Suggestions? Suggestions? Jon.Daniels@stanford.edu Jon.Daniels@stanford.edu Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 16
Backup Slides Backup Slides Backup Slides Backup Slides Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 17
What is Impedance? What is Impedance? • impedance = measure of opposition to a sinusoidal alternating electric current, generalizing Ohm's law to AC circuit analysis • impedance = ∆ voltage / ∆ current Z = 1000 + 15915 j � 15915 j � 10 nF 1000 � |Z| = 15947 � θ Z = -86.4° Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 18
Impedance Spectroscopy Impedance Spectroscopy Source: http://www.gamry.com/App_Notes/EIS_Primer/EIS_Primer.htm Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 19
Affinity Detection Affinity Detection Target concentration K d = k off affinity k on , probe coverage Chemical Target surface coverage Selective Probe Readout/transducer type Electrical Electrical Transducer Transducer Surface property change Surface property change readout readout Amplifier Instrumentation Data Acquisition Post-processing Acquired Measured change (a) (b) (c) Daniels/Pourmand, Electroanalysis 19:1239-1257 (2007) Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 20
Typical Measurement Approach Typical Measurement Approach $10-30k • Single electrode measurement Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 21
Custom Measurement Apparatus Custom Measurement Apparatus Contact pads 300µm electrodes Computer with LabView R known O-ring (holds solution) V in DUT DUT V out Socket Z DUT = -(V in /V out )*R known PCB with circuitry Chip Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 22
Double Layer Capacitance Double Layer Capacitance � φ � � � 2 � C 0 1 + 1 228 µ F / cm 2 � � C dl ( φ ) = mol/L 8 V T • α 2 ~ 188 V -2 for double layer capacitance according to Gouy-Chapman model • Predict α ~ 14 V -2 in our measurement based • Predict α 2 ~ 14 V -2 in our measurement based on voltage across ionic layer (vs. interface) – expect less due to Stern modification • We observe | α 1 | ~ 0.1-1 V -1 and | α 2 | ~ 1-10 V -2 in all our measurements Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 23
Next Steps Next Steps • Custom measurement IC design (in progress) and testing • Explore nonlinear effects with constant phase element? • Try “real biology”? • Graduate and get a “real job”! Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 24
Acknowledgements Acknowledgements • Nader Pourmand, Prof. Ron Davis (Biochem) • Erik Anderson, Prof. Tom Lee (ElecEng) • Heng Yu (surface chemistry) • Other SGTC personel • NIH and NSF for direct and indirect funding • NIH and NSF for direct and indirect funding Jon.Daniels@stanford.edu IEEE EMBC08 24-Aug-08 SuBT2.5 25
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