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Updated: May 27, 2026
Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy
Published on: September 11, 2015
Simultaneous detection of multiple mutations in epidermal growth factor receptor based on fluorescence quenching of
Taegyeong Kang1, Hosub Lee, Dongcheol Choe
1Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Republic of Korea.
Abstract:
We have developed a simultaneous detection method for two common mutations in the epidermal growth factor receptor gene based on the fluorescence quenching phenomenon caused by aggregation of CdSe quantum dots. For detection of the in-frame deletion in exon 19 and the L858R point mutation in exon 21, water-soluble CdSe quantum dots with two sizes were functionalized using four different types of probe oligonucleotides. Addition of target oligonucleotides with the deletion mutation in exon 19 into the suspensions caused crosslinking-induced aggregation of green-emitting quantum dots, followed by the fluorescence quenching while that with the L858R point mutation resulted in aggregation of yellow-emitting quantum dots. In addition, targets with both deletion and point mutations caused aggregation of both green- and yellow-emitting quantum dots. This method allows a simultaneous detection of mutations in exon 19 and 21 of EGFR gene in a single experiment. We found that minimum mutant concentration that could be detected by this method was as low as 2% for deletion mutation, and 5% for point mutation. PCR products of EGFR gene were also used to confirm that our method could be used to detect mutation in amplified DNA fragments.
Insights
This study introduces a novel method for simultaneously detecting two common epidermal growth factor receptor (EGFR) gene mutations using quantum dots. The technique offers sensitive detection of exon 19 deletions and L858R point mutations in a single experiment.
Area of Science:
- Biochemistry
- Molecular Biology
- Nanotechnology
Background:
- Epidermal Growth Factor Receptor (EGFR) gene mutations are crucial in non-small cell lung cancer pathogenesis.
- Accurate and simultaneous detection of common EGFR mutations (exon 19 deletion, L858R) is vital for targeted therapy selection.
- Existing detection methods may lack efficiency or simultaneous detection capabilities for multiple mutations.
Purpose of the Study:
- To develop a novel, simultaneous detection method for two common EGFR mutations: exon 19 deletion and L858R point mutation.
- To utilize the fluorescence quenching of Cadmium Selenide (CdSe) quantum dots for mutation detection.
- To establish a sensitive and efficient assay for clinical applications.
Main Methods:
- Functionalization of water-soluble CdSe quantum dots (green and yellow emitting) with specific probe oligonucleotides.
- Utilizing the crosslinking-induced aggregation and subsequent fluorescence quenching of quantum dots upon binding to target mutant oligonucleotides.
- Simultaneous detection of both mutations in a single reaction by observing distinct fluorescence changes.
Main Results:
- The method successfully differentiated and detected exon 19 deletion (green-emitting QDs) and L858R mutation (yellow-emitting QDs) through distinct fluorescence quenching.
- Simultaneous detection of both mutations was achieved, indicated by the aggregation of both green and yellow quantum dots.
- The assay demonstrated high sensitivity, detecting mutant concentrations as low as 2% for deletion and 5% for point mutation.
- Detection was confirmed using PCR-amplified EGFR gene fragments.
Conclusions:
- A novel, simultaneous detection method for common EGFR mutations (exon 19 deletion and L858R) was successfully developed using CdSe quantum dots.
- The fluorescence quenching-based assay offers high sensitivity and specificity for detecting these critical mutations.
- This method holds potential for efficient and simultaneous molecular diagnosis in clinical settings.
