Customized Instrumentation for Small-Volume Electrochemistry and Microfluidics used in Bioanalytical Applications

Abstract

Disease biomarker detection allows diagnosis of both chronic and acute conditions. Although current detection methods are adequate for several targets (e.g. enzymatic biosensors), the stability and limited number of targets leave room for new biosensor development. In this dissertation, new tools are developed in both microfluidic flow control and electrochemical instrumentation to further advance biosensor applicability. Surface-confined electrochemical biosensors require uniform electrode surfaces free from fouling onto which self-assembled monolayers are formed. Researchers are required to laboriously hand polish multiple electrodes to carry out experiments. In Chapter 2, an electrode polishing robot (EPBot) is created and investigated. The EPBot is a 3D-printed articulating arm robot, drawing a figure “8” continuously over 10 min to mimic hand polishing. The EPBot is shown to be similar to hand polishing by both electrochemical experiments and atomic force microscopy. Bioanalytical detection methods are also influenced by temperature. Chapter 3 describes the construction of two thermal controllers, applied to either optical or electrochemical detection method development. The first controller was based on the LabVIEW virtual instrument environment capable of generating precise temperature steps with a Peltier device. The controller was used to interrogate thermal response of branched DNA detection systems. These studies reiterate the importance of thermal conditions in DNA detection systems and present data showing shifts in detection sensitivity as temperature varies. The second thermal controller, driven by the Arduino programming environment was designed to incorporate portability and a second Peltier element capable of a 70 °C gradient generation. Finally, a single micro-Peltier version was successfully applied to quantitative microfluidic thermofluorimetric DNA melt detection of both insulin and thrombin. The surface-confined nucleic acid based electrochemical detection methods used in the aforementioned chapter exhibit a trade-off between signal intensity and charging current when using pulse voltammetry techniques. We show that differential measurements between two identical working electrodes, where one electrode is exposed to Faradaic current, allow significant reduction of charging current. For this reason, a real-time analog differential potentiostat was designed, constructed, and characterized in Chapter 4. The differential potentiostat (DiffStat) was validated by comparison to a conventional potentiostat with common electrochemical experiments: chronoamperometry, cyclic voltammetry, and square wave voltammetry. A drastic removal of charging current was shown. The DiffStat was then applied to surface-confined DNA systems at duplicate gold electrodes with increasing surface area. A reduction in both noise and baseline current were shown with the DiffStat when compared to the conventional potentiostat. Various assay modes of operation were then demonstrated using the DiffStat, and matrix effects were shown to be negligible. Chapter 5 details the construction of customized support equipment utilized in other chapters within this dissertation. Equipment included in this chapter are: CO2 laser drill, UV LED photolithography exposure unit, microfluidic solenoid valve controller, electronic pressure meters, UV laser controller, PID laboratory oven controller, microscope LED light source, and a reflow soldering oven. The lab-on-a-chip concept incorporates laboratory operations into a microfluidic device such as sample clean-up, separation, and detection, but these often require many actively-controlled valves on-chip. Moving fluidic valve control into the chip by creating microfluidic logic circuits can increase the number of operations on a chip and reduce external drive component requirements. Fluidic logic circuits can leverage fluidic analogs of electronic components, e.g. resistors, diodes, and transistors. Like electronic devices, such fluidic components would ideally be tunable, but few examples exist. Novel, tunable fluidic resistors, diodes, and transistors were designed and investigated in Chapter 6. Finally, in Chapter 7 this dissertation is summarized, and future work is presented.