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A Chamber-Based Digital PCR Based on a Microfluidic Chip for the Absolute Quantification and Analysis of KRAS
Jie Ren1, Gangwei Xu2,3, Hongna Liu1
1Hunan Key Laboratory of Biomedical Nanomaterials and Devices, Hunan University of Technology, Zhuzhou 412007, China.
Abstract:
The Kirsten rat sarcoma virus gene (KRAS) is the most common tumor in human cancer, and KRAS plays an important role in the growth of tumor cells. Normal KRAS inhibits tumor cell growth. When mutated, it will continuously stimulate cell growth, resulting in tumor development. There are currently few drugs that target the KRAS gene. Here, we developed a microfluidic chip. The chip design uses parallel fluid channels combined with cylindrical chamber arrays to generate 20,000 cylindrical microchambers. The microfluidic chip designed by us can be used for the microsegmentation of KRAS gene samples. The thermal cycling required for the PCR stage is performed on a flat-panel instrument and detected using a four-color fluorescence system. "Glass-PDMS-glass" sandwich structure effectively reduces reagent volatilization; in addition, a valve is installed at the sample inlet and outlet on the upper layer of the chip to facilitate automatic control. The liquid separation performance of the chip was verified by an automated platform. Finally, using the constructed KRAS gene mutation detection system, it is verified that the chip has good application potential for digital polymerase chain reaction (dPCR). The experimental results show that the chip has a stable performance and can achieve a dynamic detection range of four orders of magnitude and a gene mutation detection of 0.2%. In addition, the four-color fluorescence detection system developed based on the chip can distinguish three different KRAS gene mutation types simultaneously on a single chip.
Insights
A novel microfluidic chip enables precise detection of Kirsten rat sarcoma virus (KRAS) gene mutations, crucial for cancer development. This technology offers a new avenue for targeted cancer therapies by improving diagnostic capabilities.
Area of Science:
- Biotechnology
- Molecular Biology
- Oncology
Background:
- The Kirsten rat sarcoma virus (KRAS) gene is frequently mutated in human cancers, driving tumor cell proliferation.
- Targeting KRAS mutations is challenging due to limited effective drugs.
- Accurate detection of KRAS mutations is essential for personalized cancer treatment.
Purpose of the Study:
- To develop an innovative microfluidic chip for efficient KRAS gene sample microsegmentation.
- To create a sensitive and specific system for KRAS gene mutation detection.
- To evaluate the chip's performance in digital polymerase chain reaction (dPCR) applications.
Main Methods:
- A microfluidic chip with parallel fluid channels and cylindrical microchambers was designed and fabricated using a "Glass-PDMS-glass" sandwich structure.
- Automated platforms were used to verify the chip's liquid separation performance.
- A four-color fluorescence detection system integrated with the chip and a flat-panel instrument for thermal cycling was employed for KRAS mutation analysis.
Main Results:
- The microfluidic chip demonstrated stable performance with a dynamic detection range of four orders of magnitude.
- The system achieved a gene mutation detection limit of 0.2%.
- The four-color fluorescence system successfully distinguished three different KRAS gene mutation types simultaneously on a single chip.
Conclusions:
- The developed microfluidic chip shows significant potential for digital polymerase chain reaction (dPCR) applications in KRAS mutation detection.
- This technology offers a promising platform for sensitive and multiplexed cancer mutation analysis.
- The system facilitates improved diagnostic capabilities for KRAS-driven cancers.
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