Dielectrophoresis of Cells and Electrochemical Enzyme Assays
This dissertation follows a three essay format. In the first essay, an array of interdigitated microelectrodes was modeled, optimized, and tested for the contactless alignment of living biological cells by negative dielectrophoresis (nDEP). The goal is to advance the state of artificial tissues to more closely resemble natural tissues. The modeling focused on optimization of the x component, dE/dx, of potential gradient to maximize the aligning force at minimal electric field to protect fragile cells. The optimized array was tested with micron-sized, hard polymeric beads and soft C2C12 mouse myoblast cells. The array was effective at directing the orientation of confluent C2C12 cells during their differentiation into myotubes for the formation of muscle fibers. The aligned myotubes were alive and capable of growth.
In the second essay, the compound 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate (TAPTA) was analyzed as a substrate for leukocyte esterase (LE) activity. The goal is to produce an assay to allow doctors to more easily identify infection in bodily fluids. LE is an enzyme produced by leukocytes (WBC). In the presence of LE, TAPTA released a redox-active fragment whose oxidation at an electrode provided a direct numerical measure of LE activity. The assays showed that LE recognized TAPTA as its substrate with the Michaelis constant Km and Imax equal to 0.24 mM and 0.13 mA cm-1, respectively. The esterolytic activity of leukocyte suspensions was determined by using the internally calibrated electrochemical continuous enzyme assay (ICECEA). One activity unit (U) of LE catalyzed the hydrolysis of 1.0 micromole of TAPTA per minute in a pH 7.40 phosphate buffer saline solution containing 10 % DMSO at 21 °C. The measured units were directly proportional to the number of leukocytes in the range of 0.028--4.2 U L-1 (9--690 microg L-1 LE protein). One white blood cell displayed the average esterolytic activity of 0.86 and 1.4 nU when the ultrasonic and chemical cytolysis was used, respectively. The ICECEA is an electrochemical alternative to optical assays for the determination of LE activity as an inflammatory biomarker and proxy for the presence of leukocytes.
The third essay introduces a new class of pyridine-based redox substrates for LE. The new substrates (compounds I-III) dissolve in water and yield a current that is directly proportional to LE activity offering the prospect of infection assays not disturbed by the optical properties of solution. The kinetic constants Km and kcat for the reactions of LE with compounds I-III were determined by the ICECEA at a glassy carbon electrode. The binding affinity of compounds I-III for LE was two orders of magnitude better than that of optical substrates. The specificity constant kcat/Km was equal to 2.6, 3.7, and 6.1 x 105 M-1 s-1 for compounds containing the pyridine (I), methoxy-pyridine ( II), and (methoxy-carbonyl)-pyridine (III), respectively, showing the rise in the catalytic efficiency in this order. The compounds I-III were successfully used in the ICECEA to measure the esterolytic activity of white blood cell suspensions down to 20 WBC microL-1 . The new substrates signal the advent of high-resolution electrochemical LE assays as a proxy for the presence of active leukocytes in the diagnosis of infections.