Research Program
Nanoscale Cell Biology

Objectives
Quantitative measurements of neurotransmitters are of enormous significance for neuroscience research as well as medical treatments. The aim of this project is the design, fabrication, and characterization of active miniature electrochemical pixel array devices to assay quantal exocytosis from thousands of cells in a day. Using appropriate chemical modification with charged polymer brushes on silica insulator surface we aim to guide rat hippocampal neurons to grow over microelectrodes, which are modified with glutamate oxidase to detect also glutamate release from cultured neurons in response to stimulation.

Methods
Low-level circuit design techniques and deep-submicrometer CMOS foundry processes are employed in developing a pixel array that measures amperometric currents with on-chip amplifiers and read-out. A low noise regulated cascade amplifier (RCA) potentiostat was designed and fabricated at MOSIS with 0.5 µm CMOS technology [1]. In post-processing, the aluminum pads are platinized by electron beam deposition using the newly acquired FEI Strata 400 STEM Dual Beam Focused Ion Beam.Chromaffin cells are placed directly on the e-beam fabricated platinum electrodes on the CMOS die and amperometric spikes indicating quantal release events are recorded with the potentistat circuit maintaining the electrode voltage at 0.7 V. Surface initiated atom transfer radical polymerization (SI-ATRP) was used to grow polymer brushes on both, gold electrode surfaces and silica insulator surfaces. Photolithography was used to fabricate microelectrode arrays and pattern positively charged brushes to guide neurons to grow across electrodes. Confocal fluorescence microscope was used to characterize neurons cultured on polymer brush modified surfaces. Amperometry was used to detect glutamate on glutamate oxidase modified microelectrodes and will be used to detect glutamate release from neurons cultured on the electrode array.

Summary

A U.S. patent application was filed for the electrochemical CMOS pixel array [2]. The electrochemical detectors of the chip are platinized aluminum pads. Electron beam deposition of Pt on the aluminum pads was markedly improved (Fig. 1) using the newly acquired FEI Strata 400 STEM Dual Beam Focused Ion Beamwhich is equipped with both ion beam and electron beam. Electron beam deposition is less damaging to the sample and more defined deposition is possible. For the first time, single vesicle release events from chromaffin cells were recorded On-Chip. Fig. 2 shows the amperometric currents recorded at 700 mV electrode voltage. The noise was at sub pico-ampere level. The trace includes a total of 59 events A typical exocytotic event (at 140s into the recording) has a peak of 40pA, a half width of 16ms and a total charge of 586fC, parameters that are very similar to those obtained with carbon fiber electrodes and conventional amplifiers. A first prototype for a 2-dimensional 100 electrode potentiostat array with (10mm)² electrodes and 45µm x 25mm pitch was fabricated in 0.5mm AMIS technology. Glutamate detection was achieved on glutamate oxidase modified microelectrodes showing a linear concentration dependence (Fig. 3). A positively charged polymer brush modified surface was shown for the first time to be a very good substrate for neuron culture and growth guidance. Photolithography was used to pattern this cationic polymer brush. In areas between the polymer brushes, the surface was backfilled with a polyethylene glycol (PEG) monolayer. Such polymer brush/PEG hybrid surfaces showed good spatial control of neuron outgrowth. The combination with glutamate detection by surface electrodes from patterned neurons appears feasible.

Accomplishments

• First on-chip measurements of single vesicle release events using a fully integrated CMOS chip enabled by improved post-fabrication of Pt electrodes using e-beam deposition
• Precise calibration curves of glutamate detection were achieved on glutamate oxidase modified microelectrodes
• An antibacterial polymer brush modified surface was used as neuron culture substrate for the first time and patterned to guide neuron outgrowth