February 2006. February 2006. "Elect5romagnetic 'Lab on A Chip'

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Received 1 December 2005; revised 11 January 2006; accepted 13 January 2006. Available online 28 February 2006.

Abstract
In this study we developed an innovative electrochemical ‘lab on a chip’ system that contains an array of nano-volume electrochemical cells on a silicon chip. Each of the electrochemical cells can be monitored simultaneously and independently, and each cell contains three embedded electrodes, which enable performance of all types of electrochemical measurements. The integration of living organisms on an electrochemical array chip that can emulates reactions of living organisms and senses essential biological functions have never been demonstrated before. In order to show the wide range of applications that can be benefited from this device, biological components including chemicals, enzymes, bacteria and bio-films were integrated within the nano-chambers for various applications. During the measurement period the bacteria remained active, enabling cellular gene expression and enzymatic activity to be monitored on line.

The miniaturized device was designed in two parts to enable multiple measurements: a disposable silicon chip containing an array of nano-volume electrochemical cells that are housing the biological material, and a reusable unit that includes a multiplexer and a potentiostat connected to a pocket PC for sensing and data analysis.

This electrochemical ‘lab on a chip’ was evaluated by measuring various biological reactions including the microbial current response to toxic chemicals. These bacteria were genetically engineered to respond to toxic chemicals by activating cascade of mechanisms, which leads to the generation of electrical current. A measurable current signal, well above the noise level, was produced within 5 min of exposure to phenol, a representative toxicant. Our work shows faster and more sensitive functional physiological detection due to the unique concept demonstrated here.

Keywords: Electrochemical detection; Nano-chip; Bio-chip; Biosensor; Bio-MEMS; Lab on a chip; MicroTAS

Article Outline
1. Introduction
2. Experimental
2.1. Bio-chip design
2.1.1. Chip process
2.1.1.1. Gold electrodes
2.1.1.2. Wall formation
2.1.1.3. Reference electrode
2.1.1.4. Chip packaging
2.1.1.5. Bonding
2.2. Experimental set-up
2.2.1. Electrochemical measurements
2.2.2. Enzymatic measurements
2.2.3. Bacterial based functional measurements
2.2.4. On-chip bacterial bio-film response to phenol
3. Results and discussion
3.1. Bio-chip fabrication
3.1.1. Electrochemical behavior of the device
3.1.2. Physiological applications of the electrochemical nano-bio-chip
3.1.3. Bacterial based functional measurements
3.1.4. Bacterial entrapped into Agar-gel measurements
4. Conclusions
Acknowledgements
References
Vitae




Fig. 1. Schematic diagram of the major fabrication steps (in transverse section). Key: The cleaned silicon wafer was isolated (SiO2), coated with W/Ti film. A thin film of gold (Au) was deposited by rf sputtering and than coated with positive S-18-18 photoresist (1). The photoresist was patterned and than developed, leaving three microelectrodes patterned (2). The sample was coated with a positive photoresist SU-8 (3). The SU-8 was patterned and developed, leaving three microelectrodes at bottom of a chamber (4).


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Fig. 2. Images of the electrochemical silicon chip wire bonded to the PCB platform. (a) Array of eight 100 nL electrochemical cells on a silicon chip. The chip is glued to the tailored PCB platform (4 cm × 4.8 cm), and the chip's gold pads (500 μm × 500 μm) are wire bonded to the gold PCB's electrodes. The PCB board enters directly to the socket of an external sensing circuit. (b) Electrochemical cells (r = 800 μm) on chip consist of three embedded electrodes: gold working electrode, gold counter electrode, and Ag/AgCl reference electrode.


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Fig. 3. Amperometric response to successive aliquots additions of redox compound K4Fe(CN)6 in 0.1 M KCl to 100 nL electrochemical final cell volume. Measurements were performed at fixed 350 mV working potential vs. Ag/AgCl. Inset: Calibration curve of Fe final concentrations (μM) vs. current signal (nA).


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Fig. 4. Amperometric response of the activity of Alkaline Phosphatase. Different concentrations of the enzyme ranging from 36 to 690 pg/cell were placed in the nano-bio-chip. The substrate PAPP was added to final concentration of 1 mg/ml. The enzymatic product, p-aminophenol (P-AP) was measured at 220 mV vs. Ag/AgCl. Inset: Calibration curve of alkaline phosphatase activity shown as Δcurrent/Δtime.


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Fig. 5. Recombinant bacterial current response on chip to increasing concentrations of phenol: (a) after 300 s and (b) after 600 s. The E. coli reporters are fabA, grpE and dnaK. Amperometric measurements were performed immediately after phenol addition (1 min) at 220 mV working potential vs. Ag/AgCl reference electrode. The dashed line represents a linear interpolation between the minimal detection concentration (1.6 ppm) and zero phenol concentration.


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Fig. 6. Amperometric response curves of biosensors agar film to phenol exposure. Agar containing bacteria and PAPG substrate was added to the electrochemical 100 nL volume chambers on the chip, and was exposed to 10 ppm of phenol. The enzymatic activity was monitored on line by amperometric technique (V = 220 mV).


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