This award is for developing an integrated microsystem platform that can incorporate an array of bio-interfaces into an accurate continuous-use electrochemical characterization system suitable for a wide range of proteins. This system would provide capabilities for protein characterization, including: (1) simultaneous activity measurement for many soluble and membrane proteins, (2) rapid, automated interrogation using multiple electrochemical techniques, (3) microthermoregulation of individual protein sites, and (4) reduced costs per assay. A prototype device will be tested by studying novel proteins of Galdieria sulphuraria, including a phosphate transporter, ion channels, and dehydrogenase enzymes. The genome of Galdieria sulphuraria has recently been sequenced at the PI's university. This unicellular eukaryote is able to grow at high temperatures, extremely low pH, and high concentrations of toxic metals. The numerous, highly stable, enzymes and membrane transporters that impart these capabilities are of great interest from the standpoints of structural biology and biotechnology. However, research progress on Galdieria proteomics is hampered by the lack of cost-effective, reliable, and high-throughput methods to rapidly characterize the proteins' functional properties.
Development of this array microsystem would significantly advance progress in functional proteomics, with potential benefit to biological research and biotechnology development. This multidisciplinary project will be integrated into education by direct involvement of graduate and undergraduate student researchers, the development of a new course, and participation in a novel project-based Multidisciplinary Bioprocessing Laboratory course. In addition, an educational module for K-12 students will be developed and incorporated into programs for students from underrepresented groups, in coordination with the university's Diversity Programs Office.
This NSF project involved four primary objectives: 1) Develop biomimetic interfaces for on-chip implementation, 2) Develop microfabrication techniques for a miniaturized, multi-protein, array-on-chip platform that incorporates microfluidics and temperature control of individual electrodes, 3) Develop an integrated circuit with range/sensitivity adaptive multi-mode electrochemical readout and thermal feedback control, 4) Integrate the components into a model microsystem to study proteins. The major outcomes from these research objectives are summarized individually below. Several types of bio-interfaces were developed to measure protein functions electrochemically. Some interfaces measured electrical currents generated by chemical reactions, while others measured ion leakage across cell membranes through channel proteins. Fig. 1 shows how a Neisseria meningitidis PorB (II) channel protein embedded in an artificial cell membrane closes in three steps in response to an applied voltage. This channel protein is thought to be involved in some types of bacterial meningitis. Temperature plays an important role in protein characterization and sensing due to its strong effect on reaction rate. A thermal control microsystem for protein array sensing was developed and tested in a liquid environment. The on-chip microhotplates were implemented in standard CMOS without post-CMOS fabrication for thermal isolation. With only a 5V supply, up to 45°C can be achieved in liquid, which is well suited for protein-based interfaces. Results show that heating is contained sufficiently within each site to permit setting each array sites to a different temperature. Using feedback control, temperature can be held to within 0.7?C of the set point, enabling precise control of on-chip protein interfaces during characterization or sensing. Fig. 2 shows the 3x3 array on a CMOS chip along with a schematic of the layers within an individual microhotplate. An integrated CMOS amperometric instrument with on-chip electrodes was designed and implemented. The mixed-signal integrated circuit supports a variety of electrochemical measurement techniques including linear sweep, constant potential, cyclic and pulse voltammetry. Implemented in 0.5µm CMOS, the 3×3mm2 chip dissipates 22.5mW for a 200kHz clock. The highly programmable chip provides a wide range of user-controlled stimulus rate and amplitude settings with a maximum scan range of 2V and scan rates between 1mV/sec and 400V/sec. The amperometric readout circuit provides ±500fA linear resolution and supports inputs up to ±47µA. This chip is shown in Fig. 3a. In addition to amperometric techniques, impedance spectroscopy is a powerful tool for characterizing biomaterials that exhibit a frequency dependent behavior to an applied electric field. Thus, a fully integrated multi-channel impedance extraction circuit that can both generate AC stimulus signals over a broad frequency range and also measure and digitize the real and imaginary components of the impedance response was developed. The circuit was fabricated in 0.5μm CMOS and consumes 355μW at 3.3V. Tailored for protein and lipid bilayer characterization, the signal generator produces sinusoidal waves from 1Hz to 10kHz. To suit a variety of applications, the impedance extraction circuit provides a programmable current measurement range from 100pA to 100nA with a measured resolution of ~100fA. Occupying only 0.045mm2 per measurement channel, the circuit is compact enough to include nearly 100 channels and the signal generator on 3 ´ 3mm die. A prototype with five readout channels on a 1.5x1.5mm CMOS chip is shown in Fig. 3b. To combine the biointerfaces and instrumentation electronics developed within this project into a fully integrated microsystem array, a wide variety of electrode arrays schemes and chip packaging techniques were explored. Planar microfabricated electrode arrays were implemented and optimized for both enzyme and tethered lipid bilayer interfaces. Fig. 4a shows the most highly integrated system achieved in this project. A 2×2 gold electrode array was fabricated on the surface of the CMOS amperometric instrumentation chip. An all-parylene packaging scheme was developed for compatibility with liquid test environments as well as harsh electrode cleaning processes. The chip was tested using cyclic voltammetry of different concentrations of potassium ferricyanide and, as shown in Fig. 4b, results were nearly identical to measurements using commercial instruments.
