@

University of Cincinnati

A Disposable On-Chip Phosphate Sensor with Planar Cobalt Microelectrode on Polymer Substrate
Zhiwei Zou, Jungyoup Han, Am Jang*, Paul L. Bishop*, and Chong H. Ahn
MicroSystems and BioMEMS Lab Department of Electrical and Computer Engineering and Computer Science *Department of Civil and Environment Engineering

University of Cincinnati, Cincinnati, OH 45221-0030, USA

University of Cincinnati

Outline

Introduction Device Concept and Design Fabrication Experiment Results Conclusions

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

Toronto, Biosensors 2006

University of Cincinnati

Introduction
Environment

Phosphate is the major source of eutrophication of rivers and lakes.

Agriculture

Phosphate is an essential nutrient for all plants; phosphate fertilizer has been extensively used.

Clinical diagnostics

Phosphate concentration in human body is directly related to the diagnosis of several diseases.

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

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Introduction

Spectrophotometry

Atomic Emission Spectrometry

Standard phosphate measurement methods

High accuracy Expensive instrument and long analysis time

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Research Motivation
Environmental applications

Large scale field deployment Mass data collection

Miniaturized Inexpensive Simplified Accurate Rapid

Clinical applications

Single-use and disposable Rapid detection

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Previous Approach
Enzyme Based Biosensors Enzyme materials

Alkaline phosphatase, pyruvate oxidase, maltose phosphorylase, et al.

Advantages

High selectivity and sensitivity

Limitations

Enzyme materials are relative expensive and unstable. The sensor structure is relative complicated.

H. Nakamura, M. Hasegawa, Y. Nomura, Y. Arikawa, R. Matsukawa, K. Ikebukuro, and I. Karube, Development of A Highly Sensitive Chemiluminescence Flow-Injection Analysis Sensor for Phosphate-Ion Detection using Maltose Phosphorylase, Journal of Biotechnology, Vol. 75, pp. 127-133, 1999.

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Previous Approach
Cobalt-Wire Electrode

Sensitivity

Xiao et al., (1995) first introduced cobalt metal as a phosphate-sensitive electrode material. Simplicity They showed that the metallic Co-wire has a selective potential response toward dihydrogen phosphate (H2PO4-1) in the aqueous medium.
Selectivity

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Previous Approach
Cobalt-Wire Electrode

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

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Previous Work
Microfabricated On-Chip Biosensors on Polymer Substrate
Glucose permeable membrane Immobilized glucose oxidase
Lactate Sensor Oxygen Sensor Glucose Sensor

0.7 V R Reference electrode Counter electrode OH R O + H+ + e-

RE

WE
Tyrosine Tyrosyl

CE
e-

Glucose, lactate, and pO2 sensors

200 m

RE
Mn+ CE RE M M Hg Mercury droplet WE Mn+

WE CE

Insulin sensor

Heavy metal ion sensor

C. Gao, H.L.R. Rilo, P. Myneni, and C.H. Ahn, A New On-Chip Insulin Biosensor for Monitoring Dynamic Response of Human Islet Cells, Proceedings of the 8th International Conference on Miniaturized Systems in Chemistry and Life Sciences (microTAS 2004), Malmo, Sweden, Sep. 26-30, 2004 X. Zhu, C. Gao, J.-W. Choi, P.L. Bishop, and C.H. Ahn, On-Chip Generated Mercury Microelectrode for Heavy Metal Ion Detection, Lab on a Chip, Vol. 5, pp. 212-217, 2005.

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

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Previous Work
Polymer Lab-on-a-Chip Cell Monitoring
Glucose Consumption

Chemical/Culture media loading channel On-Chip Insulin Biosensor

1 0.8 0.6 0.4 0.2 0 1 2 3

Cultured Islet Cells

Unhealthy Cell

Days

Healthy Cell

On-Chip Glucose Biosensor

PDMS microfluidic system

Islet
Peak Current /nA

Captured islet

1200 1000 800 600 400 200 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Filtering pillars

Insulin Concentrations /uM

C. Gao, H.L.R. Rilo, P. Myneni, and C.H. Ahn, A New On-Chip Insulin Biosensor for Monitoring Dynamic Response of Human Islet Cells, Proceedings of the 8th International Conference on Miniaturized Systems in Chemistry and Life Sciences (microTAS 2004), Malmo, Sweden, Sep. 26-30, 2004

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Previous Work
Polymer Lab-on-a-Chip Clinical Diagnostics
Pouch Waste chamber Calibration pouch

POLYMER SUBSTRATE
Biosensor array
Biochemical sensor

Optical transparency Biocompatibility Mass production Very low cost

Spray and screen printing

150 um

Integration of pouch

AIBN

Microneedle Lateral metallic Integration of Metal needle microneedle AIBN heater sPROMs (passive valve) Solid-propellant (AIBN)

Pressure source Screen printing 200 um Mold injection Rapid injection molding

Techniques for MASS-PRODUCTION Cyclic Olefin Copolymer (COC)

C.H. Ahn, J.-W. Choi, G. Beaucage, J. Nevin, J.-B. Lee, A. Puntambekar, and J.Y. Lee, "Disposable Smart Lab on a Chip for Point-of-Care Clinical Diagnostics" Proceedings of the IEEE, Special Issue on Biomedical Applications for MEMS and Microfluidics, Vol. 92, pp. 154 - 173, 2004.

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

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Research Objectives
Miniaturized on-chip phosphate sensor using planar cobalt microelectrodes on polymer substrate Measurement of both inorganic and organic phosphate in aqueous solutions Integrated with polymer microfluidic system to achieve disposable lab-on-a-chips Benefits

Low cost, mass production, less analyte consumption, rapid detection, easy-to-use, long storage time, et al. High sensitivity and selectivity
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Device Concept and Design

Microfluidic chip

Sensing chip

Integrated biochip

Electric contact
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Device Concept and Design

Microfluidic chip
Inlet

Outlet

Automatic system using Pump-33 fluidic dual syringe pump micro-pump and micro-vales

Sensing chip

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

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Device Concept and Design

P

Microfluidic chip
P

O2

O2

O2

3CoO+2H2PO4-+2H+ Co3(PO4)2+3H2O 2Co+O2 2CoO P Co Au EW O2

Ag/AgCl Au ER

Sensing chip

Model 215 bench-top pH/mV meter BalanceTalk SLTM software MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu Toronto, Biosensors 2006

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Fabrication Process
Sensing Chip
Ni COC Au Co S1818 SU-8 SU-8 patterning

Au/Co evaporation and patterning

Ni electroplating

Co etching and patterning

SU-8 removal

Au etching

Injection molding

Ag/AgCl electroplating

Standard microfabrication technology

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

Toronto, Biosensors 2006

University of Cincinnati

Fabrication Process
Microfluidic Chip
Ni COC Au Co S1818 SU-8 SU-8 patterning

Au/Co evaporation and patterning

Ni electroplating

Co etching and patterning

SU-8 removal

Au etching Injection Mold process Ag/AgCl electroplating

Injection molding

High throughput plastic micromachining

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

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University of Cincinnati

Fabrication Process
Chip Bonding
Ni COC Au Co S1818 SU-8 SU-8 patterning

Au/Co evaporation and patterning

Ni electroplating

Co etching and patterning

SU-8 removal

Au etching

Injection molding

Ag/AgCl electroplating

UV adhesive bonding

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

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Fabricated Device

WE

WE Co
Inlet Microchamber RE 500 m

RE Ag/AgCl

Electric contact

Chip size: 1.5 cm2 cm Chamber volume: 2 l Electrode: 200 m1.5 mm

Outlet

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

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Experiment Results
Output potential vs. various KH2PO4 concentrations
-500 -550

-660

Potential (mV)

-600 -650 -700 -750 -800

10 M 10 M 10 M 10 M
-2 -3 -4 -5

Potential (mV)

-680 -700 -720 -740 -760 -780

100

200

300

400

500

-800

-5

-4

-3

-2

Time (sec)

log [Concentration (M)]

KH2PO4 has been diluted to different concentrations using buffer solution. The buffer solution was made by potassium hydrogen phthalate (KHP) and KCl in de-ionized water at pH 5.0. MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu Toronto, Biosensors 2006

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Experiment Results
Time-dependent potential response in 10-5 M KH2PO4
0

Potential (mV)

-200

-400

-600

-800

10

15

20

25

30

35

Time (min)
The proposed on-chip sensor presents a steady-state response for more than 30 minutes in 10-5 M KH2PO4 solution , which is sufficient for disposable sensor applications. MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu Toronto, Biosensors 2006

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Experiment Results
Output potential vs. various organic phosphate concentrations
-450 -480

-450 -480

Potential (mV)

-540 -570 -600 -630 -660 -5 -4 -3 -2

Potential (mV)

-510

-510 -540 -570 -600 -630 -660 -5 -4 -3 -2

log[Concentration (M)]

log[Concentration (M)]

Adenosine 5'-triphosphate (ATP)

Adenosine 5'-diphosphate (ADP)

ATP and ADP have been diluted to different concentrations using buffer solution. The buffer solution was made by potassium hydrogen phthalate (KHP) and KCl in de-ionized water at pH 5.0. MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu Toronto, Biosensors 2006

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Experiment Results
Reproducibility of the fabricated sensor
-700 -600

RSD = 0.6%

-1000

RSD = 2.5% and 2.1%

KH2PO4 ATP

Potential (mV)

-500 -400 -300 -200 -100 0 0 2 4 6 8 10

Potential (mV)

-800 -600 -400 -200 0

Number of injection
Potential responses to ten times repeated injections of 10-3 M ADP to the same phosphate sensor

Sensor number
Chip-to-chip deviation of four different phosphate sensors in measuring 10-3 M KH2PO4 and 10-3 M ATP

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

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Conclusions
A new on-chip phosphate sensor using planar cobalt micro electrodes has been designed and fabricated. The feasibility of using the proposed sensor to monitor both inorganic and organic phosphate has been presented. Can be quickly fabricated with low cost and high yield compared to the bulk cobalt-wire based phosphate sensor, while still keeping the good performance. Fully integrated with polymer microfluidic system and can be easy developed as multi-analyte polymer lab-on-a-chips. Especially suitable for the large-scale field deployment for mass environmental data collections and disposable point-ofcare testing (POCT) in clinical diagnostics.

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

Toronto, Biosensors 2006

University of Cincinnati

Acknowledgements
Financial support

NIH-P021-L684

Technical assistance

Mr. Ron Flenniken Institute for Nanoscale Science and Technology (INST) at the University of Cincinnati

MicroSystems and BioMEMS Lab www.BioMEMS.uc.edu

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