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

Background

The role of master mold is crucial to generate defect-free polymer chips in an injection molding process (Klank et al., 2002; Hong et al., 2010). This work presents a strategy to maximize on-chip 3D detection sites per mm² with reduced defects and improved smoothness. Micro milling technique is conventionally used to make a rapid mold for injection molding. Due to the rough surface of milling bits it can cause defects in microstructures during molding. Significant efforts are being made towards improvement of molds by decreasing surface roughness and defects to achieve a better and smoother polymer chip (Ogilvie et al., 2010). Adding the novel FAPL (free angle photolithography) process we report here an improved fabrication of SAF structures and their transfer to the master mold and then to the polymeric chip.

The advantage of using SAF structures can be explained if we consider fluorophore molecule available on the front of a generic surface of microstructure: for a generic structure, most of the emitted light onto the material is refracted outside the structure, and only small amount of light passes through to give a signal (Ruckstuhl and Verdes, 2004; Winterflood et al., 2013). Usually, this refracted fluorescent light is lost or not been gathered when using a flat surface (microscopic polymer slide) for signal capturing. Collection of this remaining part of light by a structure that exhibits supercritical angle of reflection enhances the signal intensity up to 46 times (Hung et al., 2014). SAF structure offers a higher sensitivity and better view for efficient signal collection compared with the flat surface. Fabrication of SAF with photolithography improves the number of structures on a unit area of chip and reduces the volume of sample required. Incorporation of SAF structures on microfluidic chip in combination with SP-PCR provides the capability of multiplexed SP-PCR reactions on-chip for effective low-cost and compact optical detection platform. Our experimental results demonstrated that with SAF array it achieves ultra-sensitive limit of detection (LoD) for fluorophores. This sensitivity is comparable to that obtained by a conventional microarray with data acquisition from a high-end laser scanner. This SAF array platform is advantageous in terms of having a low cost microfluidic platform, whilst retaining high sensitivity and multiplexing capability.

In this paper, we introduce FAPL fabrication process and demonstrate the possibility of achieving lithography-made SAF (L-SAF) structures with lower surface roughness and higher optical efficiency with respect to our previously described micro-milled SAF (M-SAF) (Hung et al., 2015). In particular, we present fabrication procedure (Figure 1) for L-SAF structures of different sizes (50 μm, 100 μm, 150 μm). Multiplexed SP-PCR reaction using DNA probes on L-SAF array for on-chip pathogen detection application (Sun et al., 2011) and our experimental outcomes confirmed that by using these improved SAF arrays, the limit of detection (LOD) could be improved up to 0.05 nM around 6.62 x 10³ molecules. The LOD is calculated by data obtained from a high-end fluorescent scanner.


Figure 1. Schematic representation of fabrication steps of L-SAF array using FAPL process. Spin coating of photoresist and an edge bead removal is presented in (A), a free angle UV exposure over the rotatory chuck and development of the exposed Si is presented in (B), the transfer of fabricated 3D L-SAF array to polymeric chip using electroplating of the seed layer of NiV (80 nm) and thick layer (350 μm) of Nickel (Ni) for making a shim for injection molding is presented in (C).