Year: 2024
Type: conference-paper
Abstract: Several European countries have implemented new legislation to stop the culling of day-old male chicks, killed due to the lack of use in the laying hen industry, and hence improve animal welfare.However, the industry is still seeking low-cost solutions that can handle all egg colors with >98.5 % accuracy and minimal embryo disturbance, while allowing sexing before day 7 of incubation at a throughput of >20 000 eggs per hour [1].In our recent work, we have shown that real-time polymerase chain reaction (qPCR) offers 100 % accuracy for detecting female-specific genes (e.g., HINTW) in allantoic fluid (AF) samples (Figure 1A) [2], although, it still lacked sufficient throughput, next to being costly, and relying on handling the embryos outside the incubators for a long period (1.5 hours).In this context, recombinase polymerase amplification (RPA; Figure 1B) has been reported as a good alternative to qPCR, with similar accuracy and sensitivity, while taking only 10 -30 minutes at isothermal temperatures [3], e.g., 37.7 °C in egg incubators.Moreover, RPA can be combined with lateral flow strips (LFS; Figure 1C) for colorimetric read-out and integrated with microfluidic platforms [3].Therefore, in this work, we first established off-chip RPA of synthetic HINTW gene, which showed significant differences between male and female samples (Figure 1D).Following this, we developed a SIMPLE*-RPA platform, achieving on-chip RPA of HINTW gene without needing external devices or complex handling (Figure 1E).The SIMPLE-RPA consists of three distinct fluidic layers (detection, microfluidic channel and SIMPLE pump layers) and is assembled through lamination of low-cost plastic materials.Upon fingerpress activation, working liquid was absorbed into a porous material (i.e., filter paper) to produce the necessary pressure gradients in the microfluidic channels.The generated pressure and combinations of hydrophobic burst valves and stop valves in the microfluidic channels allowed for driving the fluid flow for the necessary RPA reaction steps (i.e., sample metering (1 μL), merging and mixing with RPA reagents (9 μL), RPA incubation, mixing with LFS buffer, and carrying the RPA amplicon to the LFS; Figure 1F).The use of 3D expansion chambers allowed for efficient mixing and enhanced DNA amplification [6].The whole on-chip bioassay was less than 20 minutes inside an incubator at 37.7 °C.The SIMPLE-RPA shows full compatibility for chick embryo sexing, requiring only a few microliters of sample and a single finger press activation step, whereas the results are observed by the naked eye and require no external pumping device or fluorescence excitation of probes.Moreover, the platform allows use in other applications (e.g., virus detection) since different steps are easily tuned and combined with others (e.g., DNA extraction).Lastly, this platform is assembled using inexpensive materials (PET, PMMA, and paper), has a small footprint, and is compatible with large-scale manufacturing.Future work will focus on validating the SIMPLE-RPA with AF samples and additional reduction of costs for RPA and LFS.*SIMPLE: Self-powered Imbibing Microfluidic Pump by Liquid Encapsulation (in-house developed microfluidic chip [5,6]) Word count: 492
DOI: 10.70477/hlyb7957
Institution: KU Leuven
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: gold
OpenAlex ID: W4408828182