Electrochemical, Multi-analyte Biosensor Array for
Neurotransmitter Detection
Anita Karegar
MS Thesis Presentation
8th March 2007
Committee Members• Advisor
– Dr. Tom Chen• Department of Electrical and Computer Engineering
• Committee members– Dr. Charles Henry
• Department of Chemistry– Dr. Stuart Tobet
• Department of Biomedical Sciences– Dr. George Collins
• Department of Electrical and Computer Engineering
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter
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Presentation Outline• Why do we need to detect neurotransmitter gradient?• Motivation of thesis• Review of existing sensors to detect neurotransmitters and their
limitations• Proposed approach• Basic electrochemistry fundamentals• Electrochemical sensor array
• Sensor array design• Electrochemical experimental Results• Observations and conclusions
• VLSI interface to sensor array• Preamplifier design• Simulation results• Summary of specifications
• Interface of sensor array to designed preamplifier• Future work
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter
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Significance Of Neurotransmitters• Nitric Oxide (NO)
– Endothelial-derived relaxation factor– Crucial role in neural communication in central and peripheral nervous
system– Physiological functions and behaviors regulated through hypothalamic
circuits• Dopamine (DA)
– Precursor of Norepinephrine (another major neurotransmitter)– Parkinson’s disease, Psychosis, Schizophrenia
• Serotonin (5-HT)– Biochemistry of depression, migraine, bipolar disorder and anxiety– Hormone and growth factor
• GABA– Inhibitory neurotransmitter– Spinal cord and cortical cell migration – Alteration of the cells movements in early brain development
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter
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Motivation of Thesis
• Objective– To detect diffusion profile of Nitric Oxide (NO)
that regulates physiological functions and behaviors
• To design a sensor array to provide high spatial resolution (~ sub µm) for capturing cell-to-cell interactions.
• To integrate sensor array with signal processing and storage circuits to allow continuous measurement in real-time.
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter
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Existing Work for Nitric Oxide (NO) Detection
• Electrochemical Methods– High sensitivity– Fast response time– Fabrication of electrodes with dimensions in micrometers– Analytical information in electrical domain– Continuous measurement
• Electrochemical method generally used– Amperometry– Cyclic voltammetry
• Limitations– Electrode fouling
• Cleaning of an electrode from time to time using cyclic voltammetry– Poor selectivity
• Electrode surface modification with conducting/non-conducting polymers
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter
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Existing Work for NO Detection • Electrode material ([Kim,98], [Pontie,99])
– Carbon fiber, Glassy carbon, Platinum, Gold, Pt/Ir alloy • Individual electrode dimensions ([Bedioui,03])
– Diameter: 0.8 µm – 500 µm– Length: 6 µm - 1 mm
• Electrode arrangement ([Naware,03], [Zhang,03])– Single electrode based– Multi-electrode array
• Multi-electrode array fabrication ([George,01], [Zhang,03])– Photolithography of graphite carbon on Si wafer– Screen printing of carbon electrodes on Si wafer
• Sensitivity ([Bedioui,03])– 2.05 nA/µM to 10.5 nA/µM
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter
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Existing Work for NO Detection • Linear range (Current Vs Concentration) ([Bedioui,03])
– 100nM to 20 µM• Detection limit (S/N = 3) ([Bedioui,03])
– 10 nM to 570 nM• Response time ([Bedioui,03])
– 10 ms to 4 s• Electrochemical Method ([Bedioui,03], [Kwang,03], [Mao,03])
– Two or three electrode systems– Cyclic Voltammetry, Differential pulse voltammtery, Amperometry– Oxidation potential of NO: +0.7 - +0.9 V Vs Ag/AgCl reference electrode
• NO calibration method ([Bedioui,03], [Zhang,03])– Dilution of saturated NO solution to prepare standards– S-nitroso-acetyl-DL-penicillamine (SNAP) to generate known
concentration of NO Electrochemical, Multi-Analyte
Biosensor Array for Neurotransmitter Detection
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Existing Work for NO Detection • Electrode surface modification ([Pontie,99], [Park, 98], [Bedioui, 03])
– Reasons• Selectivity against interferences e.g. Ascorbic acid, Nitrite, Dopamine• Sensitivity (catalytic electrochemical oxidation of NO)
– Coating materials• Conducting polymers (catalytic electrochemical oxidation)
– E.g. metal-porphyrin, metal-phthalocynine and M(salen) with central metal ion as Mn, Ni, Fe and Co
• Non-conducting polymers (Permselectivity, charge repulsion)– Resorcinol, o-phenylenediamine, m-phenylenediamine, nafion
• Combination of conducting and non-conducting polymers – E.g. Ni-porphyrin + nafion + o-phenylenediamine, M(salen) + nafion
– Methods• Electropolymerization• Dip coating• Spin coating
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter
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Limitations of Existing NO Sensors • Existing microelectrode diameter ranges from few microns to few
hundred microns. – Unable to provide enough spatial resolution for capturing cell-to-
cell interaction• Designed for single analyte detection• Experimental setup involves discrete components such as
potentiostat, data acquisition system, sensor array. – bulky – Costly– prone to noise
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter
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Proposed Approach• To create electrochemical microelectrode based sensor array integrated
with on-chip signal processing and storage circuits to allow multi-analyte analysis with high spatial and temporal resolution in real time
Existing ApproachProposed Approach
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Basic Electrochemistry• Electrochemical cell
– A set of two electrodes separated by one or more electrolyte phases
– Electrode-electrolyte Interfacial potential difference
– Working electrode– Reference electrode– Oxidation– Reduction
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter Detection 12
Two Electrode System• Working (WE) and
reference (RE) electrode• Redox potential is applied
across WE and RE and current is measured through WE
• Limitations– High IRs drop if current I or
solution resistance Rs is high
– Deviation of RE interfacial potential from equilibrium
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter Detection 13
Three Electrode System• Working, reference and
auxiliary/counter electrode
• Redox potential is applied across WE and RE and current is measured between WE and AE
• Advantages– Negligible current through
RE– Minimization of solution
potential drop by placing RE near WE
14Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter Detection
Four Electrode System• Two working electrodes
along with reference and auxiliary electrodes
• Oxidation potential at WEG (generator) and reduction potential at WEC (collector)
• Improved sensitivity – Redox cycling– WEs laid down in inter-
digitated manner
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter Detection 15
Electrochemical Experimental Setup
• The potentiostat senses potential between WE and RE and maintains it by controlling the potential between WE and AE
• The potential maintained at WE Vs RE can be constant potential as in amperometry or potential varying with time as in cyclic voltammetry
• Exists a unique relationship between current and potential
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter Detection 16
Cyclic Voltammetry• Potential across WE and
RE is swept linearly with time and i-E graph (voltammogram) is plotted.
• Involves reduction along with oxidation
• Cyclic voltammogram provides unique signature of a given compound
• Limitations– Background signal
correction– Poor detection limit
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter Detection 17
Amperometry• Potential step is applied
at WE Vs RE and i-t graph is plotted
• Redox potential applied is specific to analyte
• Improved detection limit• Fast response time• Limitations
– Selectivity– Electrode fouling
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter Detection 18
Sensor Array Design Goals• Minimum solution resistance Rs between WE and RE,
RE and AE, and WE and AE– To reduce solution resistance potential drop– To provide low impedance path between WE and AE
Cd: Double layer capacitance
R: Resistance of electrode
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Sensor Array Design Goals• Cd,RE >> Cd,WE and Cd,AE
– To maintain the constant RE potential – To change WE and AE potential to follow the applied potential
immediately • Biological tissue dimensions
– Electrode size – Pitch between electrode sites.
• High sensitivity for NO– Concentration of NO in nanomolar range
• Design of analog circuitry– Double layer capacitance of AE and RE in the range of 5 - 40
µF/cm2
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Three Electrode System• Electrode site
– WE, individual or common RE and AE
• Size of electrodes– 1 µm– 1.5 µm– 2 µm
• Spacing between electrodes– 2 µm– 2.5 µm– 3 µm (minimum feature size in
the lift-off process)• Pitch between electrode sites
– 15 µm– 20 µm– 25 µm
WE AE
RE
Pitch
Spacing
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Four Electrode System• Electrode site
– WE, individual or common RE and AE
• Diameter of electrodes– 8 µm– 10 µm– 12.25 µm
• Spacing between electrode fingers– 2 µm– 2.5 µm– 3 µm
• Pitch between electrode sites– 15 µm– 20 µm– 25 µm
RE
AE
WE1
WE2
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Overall Sensor Array Dimensions
• Overall size 9 mm X 9 mm• Electrode sites arranged in 5 X 5 grid• Pad size 160 µm X 160 µm• Interconnect width 6 µm
9 mm
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Sensor Fabrication Steps
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Electrochemical Experiment setup
• Three types of electrochemical sensor– Sensor array fabricated by standard CMOS process (Avago Inc)– Polydimethylsiloxane (PDMS) electrodes using twisted and
separated platinum (Pt, dia 25 µm) and gold (Au, dia 75 µm) wires– Sensor array fabricated on glass substrate
• Three electrode system– Pt/Au as working and auxiliary electrode – External Ag/AgCl reference electrode
• Two electrochemical methods– Cyclic Voltammetry
• Potential range = -0.4 V to +1.0 V, scan rate = 0.1, 1, 10, 100, 333 and 500 V/s, Segments = 2, Sampling interval = 0.001, Potentiostat Sensitivity = 1e-7
– Amperometry• Applied potential = 0.1 V to 1V in steps of 0.1 V, Sampling interval = 0.01, Run
time = 1.5e3, Potentiostat Sensitivity = 1e-6• Chemical compounds
– Dopamine (DA), NONOate (liberates NO), Ascorbic acid (AA)
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Verification of Electrochemical Behavior of PDMS Electrode
Commercial Pt Electrode (10 µm)
PDMS electrode with twisted Pt wires (25 µm)
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Amperometry Results of NONOate (NO)
• PDMS electrode with separated Au (dia 75 µm) wires
• Applied potential varied from 0.1 V to 1 V in steps of 0.1 V
• 3 injections of 10 µL of NONOate in PBS (pH 7.4) buffer near electrodes
• 1 µL of NONOate liberates 75 nMoles of NO
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Amperometry Results in NONOate (NO)
I-V plot of PDMS electrode with separated Au (dia 75 µm) wires
I-V plot of PDMS electrode with separated Pt (dia 25 µm) wires
23.14nA
67.48nA
83.32nA
294.4nA
689.8nA
4.06µA
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Amperometry Results in NONOate (NO)
I-V plot of PDMS electrode with twisted Au (dia 75 µm) wires
I-V plots of PDMS electrode with twisted Pt (dia 25 µm) wires
29Electrochemical, Multi-Analyte
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50.49nA
163.7nA
209.4nA
238.3nA
2.45 µA
5.04 µA
Amperometry Results of Ascorbic Acid
I-V plot of PDMS electrode with twisted Au (dia 75 µm) wires
I-V plot of PDMS electrode with twisted Pt (dia 25 µm) wires
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330.9nA
447.9nA
519.1nA
2.101µA
4.717µA
6.135µA
Observations From Electro-chemical experiments
• In all I-V graphs, the oxidation current increases linearly with applied potential till Eapp = 0.8 V – 0.9 V and then stabilizes at diffusion current as it enters diffusion limited region
• Between Pt and Au electrode materials, Pt is selected as electrode material for sensor array because of its high current density (Pt current density = 0.237 +/- 0.117, Au current density = 0.149 +/- 0.02 [Allen,04])
• The electrochemical behavior of separated electrodes (distance between electrodes in mm range) is similar to that of twisted electrodes (distance between electrodes is um range). This shows that sensor array with micrometer spacing is feasible system to detect neurotransmitters.
• NO shows significant oxidation between 0.7 V - 1 V. Hence 0.8 - 0.9 V is selected as redox potential range in further amperometry experiments.
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Observations From Electro-chemical experiments
• By comparing I-V curves for NONOate and AA, it observed that AA shows significant oxidation in the same potential range as that of NO. Hence, electrode surface need to modify with polymers to improve selectivity against AA . Nafion is selected as coating material because of its inherent negative charge.
• Dynamic range of current is derived as 0.2184 nA to 1.092 µA from extrapolated NO calibration curve for concentration range of 1 nM to 5 µM
• The electrochemical behavior of sensor can be verified by performing cyclic voltammetry at various scan rates and plotting Ip Vs sqrt(scan rate) (linear graph).
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Electronic Circuitry for Signal Amplification and Conditioning
• Preamplifier Specification– Bandwidth and gain
– The smallest sensor current – The dynamic range of sensor current– The input voltage range and voltage resolution of ADC– Frequency components of cyclic voltammetry and amperometry response
• Summary of Preamplifier specifications
Gain >= 110 dBBandwidth >= 40 KHzInput dynamic range pA - µAStability phase margin > 45Low equivalent input noise pA/sqrt(Hz)
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Preamplifier Block Diagram• Common gate stage
– Low input impedance and hence, improve bandwidth
– Decouples sensitive feedback node from external capacitances
– Converts bidirectional sensor current into unidirectional
– Crucial from noise point of view• Transimpedance amplifier
– Cascode amplifier• Provides high gain• Reduces the miller effect of Cgd on
input• Shares Ibias and hence, provide
high gain at low power consumption– Source follower
• Converts high impedance to low impedance
• Improves voltage swing• Negative feedback
– stabilizes the gain against supply, temperature and device parameters variation
34Electrochemical, Multi-Analyte
Biosensor Array for Neurotransmitter Detection
NMOS Preamplifier• Simulation
– DC analysis – AC analysis– Transient analysis– Noise analysis– Monte Carlo analysis
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NMOS Preamplifier Simulation Results
• AC analysis– Gain =
110.2008 dB– Bandwidth =
808.5534 KHz– Phase margin =
204.5654 degree
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter Detection 36
NMOS Preamplifier Simulation Results
• Transient Analysis– Iminp-p = 34
pA, Voutminp,p = 11.381 uV
– Imaxp,p = 3.6 uA, Voutmaxp,p = 1.2119 V
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NMOS Preamplifier Simulation Results
• Noise Analysis– Total output
noise = 859.1171 uV/sqrt(Hz),
– input referred noise = 2.6547 nA/sqrt(Hz)
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NMOS Preamplifier Simulation Results
• Monte Carlo Simulation– Vtnmos, Vtpmos, Minimum
feature size • 5 % variation with gaussian
distribution– VDD, temperature
• 10 % variation with uniform distribution
– Gain • Std deviation = 5.5824 dB
– Bandwidth• Std deviation = 17.0877
KHz– Phase margin
• Std deviation = 22.0352 degree
Gain
Bandwidth
Phase margin
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NMOS Preamplifier Simulation Results
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Summary NMOS preamplifier performanceAC Analysis
Gain 110.2008 dBBandwidth 808.5534 KHzPhase margin 204.5654 degree
Transient AnalysisIminp,p = 34 pA 11.381 µVImaxp,p = 3.6 µA 1.2119 V
Noise AnalysisTotal Output Noise 859.1171 µV/sqrt(Hz)Input referred noise 2.6547 nA/sqrt(Hz)
Monte Carlo AnalysisGain std deviation 5.5824 dBBandwidth std deviation 17.0877 KHzPhase margin std deviation 22.0352 degree
PMOS Preamplifier• Simulation
– DC analysis – AC analysis– Transient analysis– Noise analysis– Monte Carlo analysis
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter Detection 41
NMOS Design PMOS Design
PMOS Preamplifier Simulation Results
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Summary PMOS preamplifier performanceAC Analysis
Gain 110.2994 dBBandwidth 166.4917 KHzPhase margin 190.6952 degree
Transient AnalysisIminp,p = 36 pA 12.159 µVImaxp,p = 2.6 µA 0.8587 V
Noise AnalysisTotal Output Noise 742.6503 µV/sqrt(Hz)Input referred noise 2.2689 nA/sqrt(Hz)
Monte Carlo AnalysisGain std deviation 1.5891 dBBandwidth std deviation 31.8187 KHzPhase margin std deviation 12.3940 degree
Comparison of NMOS and PMOS Preamplifier
Electrochemical, Multi-Analyte Biosensor Array for Neurotransmitter Detection 43
Preamplifier performance NMOS Preamplifier PMOS PreamplifierAC Analysis
Gain 110.2008 dB 110.2994 dBBandwidth 808.5534 KHz 166.4917 KHzPhase margin 204.5654 degree 190.6952 degree
Transient AnalysisIminp,p Voutminp,p 34 pA 11.381 µV 36 pA 12.159 µVImaxp,p Voutmaxp,p 3.6 µA 1.2119 V 2.6 µA 0.8587 V
Noise AnalysisTotal Output Noise 859.1171 µV/sqrt(Hz) 742.6503 µV/sqrt(Hz)Input referred noise 2.6547 nA/sqrt(Hz) 2.2689 nA/sqrt(Hz)
Monte Carlo AnalysisGain std deviation 5.5824 dB 1.5891 dBBandwidth std deviation 17.0877 KHz 31.8187 KHzPhase margin std deviation 22.0352 degree 12.3940 degree
Layout of PMOS Preamplifier
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Summary of Preamplifier Design• PMOS preamplifier specifications after including extracted parasitic,
sensor electrical model and load
Preamplifier specificationsGain 110.1192 dBBandwidth 166.6680 KHzPhase margin 173.1397 degreeDynamic range Iinp-p = 36 pA - 2.6 uA,
Voutp-p = 11.909 µV - 0.8308 VInput referred noise 2.2833 nA/sqrt(Hz)
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Summary from Electrochemical Experiment Results
• Between Pt and Au, Pt is finalized as electrode material of sensor array because of its high current density
• The sensor array with micrometer spacing is feasible system to detect neurotransmitter
• NO shows significant oxidation at 0.7 V - 1 V• Electrode surface modification is necessary to improve selectivity
against AA for NO detection. Nafion is selected as coating material.• Dynamic range of preamplifier is derived as 0.2184 nA - 1.092 µA
for NO concentration range of 1nM to 5 µM.
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Interface of Preamplifier to Sensor
• Smallest current detected by the designed preamplifier • 6.8499 nA with SNR = 3
• Dynamic range derived from extrapolated NO calibration curve• 0.2184 nA – 1.092 µA
• Detection limit of integrated system • 31.364 nM of NO
• The noise of preamplifier needs to reduce
• The transient response of preamplifier to signal emulating the sensor current is linear.
47Electrochemical, Multi-Analyte
Biosensor Array for Neurotransmitter Detection
Sensor Signal (Ip) = 50 nA
Preamplifier output (Vp) = 14.643 mV Gain = 285,759 V/A (109.12 dB)
Future Work• Sensor array
– Electrode material– Reference electrode on array itself– Electrode surface modification with Conduction/non-conducting
polymers – GABA detection using enzymes coating – Simultaneous detection of DA, 5-HT, GABA and NO using a
combination of electrochemical methods• VLSI interface
– Noise reduction to improve the detection limit– Different topologies e.g. integrate with programmable integration time– Wireless telemetry modules
• Integrated system– Study of signal analysis features in existing potentiostats, additional
feature requirement such as compression and DSP algorithms and circuits to implement them
– USB interfaceElectrochemical, Multi-Analyte
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Questions ?
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