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Digital Polymerase Chain Reaction (dPCR) has emerged to be a highly precise and sensitive tool for molecular diagnosis with absolute quantification. The precise control over sample partitioning in dPCR has enabled the development of chip-based dispersion approaches (cdPCR). However, conventional cdPCR often introduces the risks of sample loss (or “dead volume”), resulting in inaccurate detection and requiring complex and expensive fabrication processes. This paper introduces a simple, low-cost through-hole microwell sample dispersion method using sample sliding technique. The chip includes a through-hole microwell array with hydrophilic inner walls and hydrophobic chip surfaces. Under the effect of capillary force and capillary burst pressure, samples introduced to the chip to the chip are partitioned into uniform through-hole arrays with high partitioning efficacy and eliminate the effect of “dead volume”. The through-hole microwells are designed and fabricated using CNC micro drilling method with a total of 625 cylindrical microwells, each hole holds approximately 19.84 ± 3.04 nL reaction volume, with a total of 12.40 ± 1.90 µL sample volume required for each test. The effect of evaporation during PCR is minimized by the application of transparent silicon oil on both sides of the chip. The morphology investigating of the fabricated microwells shows a uniform array of microwell with a diameter of 202.26 ± 1.19 µm. The chip dispersion performance evaluation using dyed solutions in different surface plasma treatment conditions demonstrated a complete sample partitioning rate with a maximum of 98.56 ± 1.27% sample filling rate at an optimum surface energy level of 330J. The effect of surface hydrophobic recovery on the partitioning rate is also studied, indicating a long maintenance period of microwell inner wall surface wettability. Besides, the effect of sample loss due to evaporation is also tested, with the result showing that approximately 90.45 ± 4.81% of sample is maintained inside the microwell under one-hour heating condition at 100 °C.

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A Low-Cost Method for the Fabrication of Through-Hole Microwell Digital PCR Chip Using CNC Micro Drilling

  • 会议论文

  • 发表日期：

    2025年

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  • Phuc-Thinh Bui,
  • Trong-Hieu Duong,
  • Dang-Khoa Tran,
  • Ngoc-Viet Tran,
  • Thanh-Hoan Ngo,
  • Thanh-Qua Nguyen

摘要

Digital Polymerase Chain Reaction (dPCR) has emerged to be a highly precise and sensitive tool for molecular diagnosis with absolute quantification. The precise control over sample partitioning in dPCR has enabled the development of chip-based dispersion approaches (cdPCR). However, conventional cdPCR often introduces the risks of sample loss (or “dead volume”), resulting in inaccurate detection and requiring complex and expensive fabrication processes. This paper introduces a simple, low-cost through-hole microwell sample dispersion method using sample sliding technique. The chip includes a through-hole microwell array with hydrophilic inner walls and hydrophobic chip surfaces. Under the effect of capillary force and capillary burst pressure, samples introduced to the chip to the chip are partitioned into uniform through-hole arrays with high partitioning efficacy and eliminate the effect of “dead volume”. The through-hole microwells are designed and fabricated using CNC micro drilling method with a total of 625 cylindrical microwells, each hole holds approximately 19.84 ± 3.04 nL reaction volume, with a total of 12.40 ± 1.90 µL sample volume required for each test. The effect of evaporation during PCR is minimized by the application of transparent silicon oil on both sides of the chip. The morphology investigating of the fabricated microwells shows a uniform array of microwell with a diameter of 202.26 ± 1.19 µm. The chip dispersion performance evaluation using dyed solutions in different surface plasma treatment conditions demonstrated a complete sample partitioning rate with a maximum of 98.56 ± 1.27% sample filling rate at an optimum surface energy level of 330J. The effect of surface hydrophobic recovery on the partitioning rate is also studied, indicating a long maintenance period of microwell inner wall surface wettability. Besides, the effect of sample loss due to evaporation is also tested, with the result showing that approximately 90.45 ± 4.81% of sample is maintained inside the microwell under one-hour heating condition at 100 °C.