The development of Point-of-Use (PoU) sensors is driven to provide simple, rapid and reliable test results for applications that require quantitative analysis, in particular in resource-limited settings. To bring a more comprehensive range of tests into the PoU, there is a need to evaluate and improve upon the sensing technologies in terms of the ease of fabrication, sensing capabilities and practicality. This thesis describes the design, fabrication and characterization of PoU sensors on various materials, including polydimethylsiloxane (PDMS), paper and cotton threads. The choice of substrate materials hassled to functionalities that are suitable for a wide spectrum of applications, which has been previously limited by the use of conventional substrates such as glass and silicon. There are technical challenges in terms of device fabrication onto relatively unexplored materials compared to silicon. In the case of PDMS, known techniques for metal patterning are restricted in terms of resolution, yield and scalability. The primary constraint is the incompatibility of its physical properties with conventional cleanroom techniques. A sacrificial layer assisted pattern transfer is reported in this thesis as a fabrication strategy for the reliable patterning of metallic structures of varying shape and dimension (down to the nm range) onto the elastomer. The fabrication process enabled the bottom-up design of a resistive based sensor for the real-time and reversible detection of volatile organic compounds (VOCs). The VOC sensor displayed sensitivity to chloroform and toluene vapour with a rapid response (~ 30 s) and recovery (~ 200 s) at room temperature. The use of paper as a substrate material was also investigated for the electrochemical detection of bilirubin in blood serum. The proposed sensor utilized only 15 μL of sample for the screening of hyperbilirubinaemia in < 2 min. Due to some of the disadvantages of using paper such as the need of sensor calibration prior to potentiometric measurements, cotton threads were explored as an alternative substrate to develop cost-effective PoU tests. Thread-based sensors were characterized for the multiplexing of Na+ and K+ in blood serum, which showed an accuracy of > 98.5%. The sensors were further applied for choline (Ch+) analysis in infant formula with a sensitivity down to µM range. Finally, the use of thread-based sensors for real time monitoring of bioconjugation reactions was investigated. The sensors described here have implications to deliver analysis at the PoU.