Solid Phase PCR on 3D Microstructure ArrayChip for Pathogen Detection Application.

Advanced free angle photolithography (FAPL) is presented for making 3D supercritical angle fluorescence (SAF) microstructures and transfer them on to polymeric chips using injection molding technique for low-cost microfluidic devices embedded with optical sensing structures. A solid phase polymerase chain reaction (SP-PCR) is used as model technique, which allows rapid and sensitive detection of pathogen DNA on-chip. This article presents the detailed fabrication of SAF structure and SP-PCR application on SAF structure for pathogen detection. This protocol of developing SAF structures using the FAPL process, increases the number of SAF per mm. FAPL was performed via a motorized stage to control the angle of incidence and to achieve the desired bucket-shapes (dimensions of 50 μm to 150 μm with a slope) required for the 3D optical sensing. Due to the unique properties of SAF structures, it enhances the fluorescent signal by 46 times. Increasing the number of SAF structures and reducing the size resulted in reduction of sample volume required per test along with improvement in the limit of detection (LOD) due to a smaller size. This article also presents the experimental details of SP-PCR using DNA oligos bound to the SAF structures for on-chip pathogen detection and a comparison between different sizes of SAF structures. The direct on-chip SP-PCR paves the path for the application of this technique in point-of-care devices.