Fully Automated Microfluidic Chip-Based Embryo Sexing Technology

Academic Background

In the egg-laying poultry industry, newly hatched male chicks are often culled immediately after hatching, as they neither lay eggs nor produce high-quality meat. Each year, approximately 372 million male chicks are culled in the European Union. This practice has sparked ethical and animal welfare concerns, prompting several European countries (such as Germany, France, and Italy) to legislate bans on this behavior. To address the issue, in ovo sexing has emerged as the most promising alternative. However, existing in ovo sexing technologies fail to simultaneously meet the requirements of high accuracy (>98%), low cost, minimal disturbance to embryos, compatibility with all eggshell colors, and processing speeds exceeding 20,000 eggs per hour.

To overcome these challenges, researchers have developed a microfluidic chip technology based on recombinase polymerase amplification (RPA), aiming to achieve automated, high-sensitivity embryo sexing. This technology can detect the female-specific HINTW gene in just 30 minutes at 37.7°C within an incubator, providing an efficient and cost-effective solution for the egg-laying poultry industry.

Paper Source

This paper was authored by Simão Monteiro Belo dos Santos, Celine Wegsteen, Dries Vloemans, Matthias Corion, Bart De Ketelaere, Dragana Spasic, and Jeroen Lammertyn, researchers from the Department of Biosystems at KU Leuven (Belgium). The team has extensive research experience in biosensing and microfluidic technologies. The paper was published in 2024 in the journal npj Biosensing, titled Fully automated sample-to-result simple-RPA microfluidic chip towards in ovo sexing application.

Research Workflow and Results

1. Research Objectives and Background

The primary objective of this study was to develop a microfluidic chip (simple-RPA chip) capable of automatically performing embryo sexing within an incubator. The chip leverages RPA technology to rapidly detect the female-specific HINTW gene at 37.7°C, enabling early-stage embryo sexing (days 6 to 9 of incubation). This technology minimizes disruption to embryos while meeting the high-throughput demands of industrial processing.

2. Experimental Design and Workflow

The study was divided into the following key steps:

a) Off-Chip Optimization of the RPA Bioassay

First, the researchers optimized the RPA bioassay under off-chip conditions. By testing different primer concentrations (0.5, 2.5, 5, and 10 µM) and incubation times (5, 10, 15, and 20 minutes), they determined the optimal primer concentration (2.5 µM) and incubation time (15 minutes). Experimental results showed that the primer concentration of 2.5 µM provided the highest signal-to-noise ratio (SNR=0.92), while the incubation time of 15 minutes enabled rapid detection without sacrificing sensitivity.

b) Sensitivity Testing

After optimizing the RPA bioassay, the research team tested its sensitivity by performing a serial dilution of synthetic DNA (ranging from 0.05 to 1.6 × 10⁻⁵ ng/µL). They found that RPA detection sensitivity was 8 × 10⁻⁵ ng/µL and 1.6 × 10⁻⁵ ng/µL when using a PCR thermal cycler and an incubator as the heat source, respectively. This result demonstrated that an incubator as a heat source could provide higher sensitivity, making it suitable for practical applications.

c) Development of the Simple-RPA Chip

The researchers developed a microfluidic chip called simple-RPA based on the off-chip-optimized RPA bioassay. This chip uses SIMPLE (Self-powered Imbibing Microfluidic Pump by Liquid Encapsulation) technology, allowing liquid operations to be fully automated with a single press. Key components of the chip design include: - Sample Processing Unit: Precisely measures 1 µL of the sample and mixes it with RPA reagents. - 3D Mixing Chambers: Enables efficient mixing through an expansion effect. - Detection Unit: Integrates a lateral flow strip (LFS) for colorimetric detection of RPA-amplified products.

d) Performance Testing of the Chip

The research team tested the optimized RPA bioassay on the chip. Results showed that the simple-RPA chip could detect the synthetic HINTW DNA within 30 minutes, with a limit of detection (LOD) of 8 × 10⁻⁵ ng/µL. These results were consistent with those in the off-chip experiments, demonstrating the chip’s reliability and practicality.

3. Key Findings and Conclusions

The key findings of the study include: - Successfully optimized the RPA bioassay, determining the best primer concentration and incubation time. - Developed a fully automated microfluidic chip (simple-RPA chip) capable of embryo sexing within an incubator. - The chip’s detection limit of 8 × 10⁻⁵ ng/µL meets the requirements for practical applications.

4. Significance and Value of the Research

This study provides an efficient and cost-effective embryo sexing technology for the egg-laying poultry industry. By combining RPA technology with a microfluidic chip, the researchers achieved automated, high-sensitivity detection, avoiding the complexity and high costs of traditional PCR techniques. Moreover, this technology has broad application potential in other fields, such as pathogen detection in medicine, veterinary science, and agriculture.

5. Highlights of the Research

• High Sensitivity: The simple-RPA chip can detect the HINTW gene at concentrations as low as 8 × 10⁻⁵ ng/µL.
• Full Automation: The chip is activated with a single press, reducing the risk of human error and contamination.
• Low Cost: The chip manufacturing cost is less than €1, making it suitable for large-scale production.

Summary

This study developed a microfluidic chip based on RPA technology, providing an efficient and low-cost solution for embryo sexing. The technology not only meets the needs of the egg-laying poultry industry but also has extensive application potential, offering new avenues for rapid detection in various other fields.