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Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay
Published on: February 23, 2020
Elongated oligonucleotide-linked immunosorbent assay for sensitive detection of a biomarker in a microwell
Ki-Cheol Han1, Eun Gyeong Yang, Dae-Ro Ahn
1Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea.
Insights
A new elongated oligonucleotide-linked immunoassay (EOLISA) offers a 10-fold increase in sensitivity for detecting disease biomarkers like fatty acid binding protein (FABP). This enhanced sensitivity aids in the early diagnosis of critical illnesses.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunotechnology
Background:
- Immunoassays are crucial for detecting disease biomarkers and pathogens.
- Conventional enzyme-linked immunosorbent assay (ELISA) lacks the sensitivity needed for early disease diagnosis.
- Higher sensitivity assays are required for detecting low-level biomarkers.
Purpose of the Study:
- To develop a highly sensitive immunoassay for early disease diagnosis.
- To introduce the elongated oligonucleotide-linked immunoassay (EOLISA) for enhanced biomarker detection.
- To evaluate EOLISA's performance compared to ELISA.
Main Methods:
- Development of EOLISA utilizing 80-mer DNA oligonucleotides.
- Incorporation of a fluorogenic RNA probe and RNase H for signal amplification.
- Signal amplification achieved through iterative DNA-RNA duplexation and RNA degradation.
Main Results:
- EOLISA demonstrated significantly improved detection sensitivity.
- The assay achieved approximately 10-fold greater sensitivity than ELISA.
- Successful detection of fatty acid binding protein (FABP) in the 0-1 ng mL(-1) range.
Conclusions:
- EOLISA offers a simple and reliable method for sensitive biomarker detection.
- The assay shows potential for clinical diagnostics, particularly for early disease detection.
- EOLISA provides a valuable advancement over conventional immunoassay techniques.
Abstract:
The immunoassay is a representative method for detecting disease biomarkers and pathogenic biological agents. While the conventional enzyme-linked immunosorbent assay (ELISA) has been routinely used for the analysis of biological samples, methods with higher sensitivity are still in demand because the detection of low-level biomarkers is important for early diagnosis of lethal diseases. In this study, we developed a sensitive immunoassay called elongated oligonucleotide-linked immunoassay (EOLISA), employing long DNA oligonucleotides (80-mer), a fluorogenic RNA probe and RNase H for signal amplification. The elongated DNA oligonucleotides led to a highly amplified fluorescence signal via iterative cycles of DNA-RNA duplexation and subsequent degradation of the RNA in the duplex by RNase H. The immunoassay was evaluated for sensitive detection of fatty acid binding protein (FABP) in the 0-1 ng mL(-1) range. When compared with ELISA, EOLISA showed about 10-fold improved detection sensitivity. With its simple procedure and reliable detection performance in the conventional platform, the proposed immunoassay is expected to have potential applications in clinical diagnostics.
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