Microcalorimetric Biosensors for DNA Melting Curve Analysis.

摘要

Microcalorimetry enables the study of the thermodynamic properties of solid materials such as polymers and metal thin films. Recent advancements in microfabrication techniques and material science has extended the use of microcalorimetry to investigate thermodynamically driven reactions in biological macromolecules suspended in a fluid medium. Microcalorimetry enables direct measurement of small temperature or energy changes associated with the reaction within minuscule sample volumes.;In this work we exploit the fundamental principles of calorimetry and amalgamate novel silicon microfabrication techniques with custom designed capture electronics to create highly sensitive portable biosensors with sub-microliter resolution. The thesis lays the foundation for strategic enhancement of the design features through a guided evolution process. The performance metrics and limitations of each device generation were used as the key determinants for progressive renovation. We illustrate the design methodology and characterization process of developing microcalorimeters for measuring the heat of the reaction of biological liquid samples while the device is freely suspended in air without external insulation. The microcalorimeters thus developed successfully detected enthalpy changes in the order of 2 microJ with a sample consumption of 0.4 microL and an average response time of 3.5 min.;Melting curve analysis (MCA) was selected as the pertinent application to demonstrate the performance of all device generations. MCA is a popular method for studying thermal characteristics of nucleic acids. DNA melting curve analysis primarily involves heating a fluorescently tagged mixture of double-stranded DNA (dsDNA) for detecting the melting temperature of dsDNA when it dissociates into its component single-stranded DNA (ssDNA). The shape and position of melting curves thus obtained suggest information about the length, specific heat capacity and relative base pair content of the DNA sample. Commercial MCA systems require sample volumes of about 150 microL with typical sample concentrations of 0.2 mg/ml along with a measurement time on the order of 15 -- 90 min.;All generations of our microcalorimetric devices consisted of a thin film heater and thermometer integrated on a common substrate to perform heating and sensing functions. Finite element modeling of the thin film microelectrodes and bioreactor chamber enabled optimized designs for uniform temperature distribution. The first generation glass-based devices had the capability to circumvent disadvantages such as evaporation and bubble-formation during conventional heating processes on microfluidic chips through a novel step-wise pressurization technique. The subsequent designs incorporated droplet samples encapsulated by oil to control evaporation. Wax, egg-white protein lysozyme, salmon testes DNA and purified genomic DNA with known melting temperatures were used as melting models to successfully demonstrate melting curve analysis.