EGFR point mutation detection of single circulating tumor cells for lung cancer using a micro-well array

Gao, Wanlei; Zhang, Xiaofen; Yuan, Haojun; Wang, Yanmin; Zhou, Hongbo; jinsoo, Han; Jia, Chunping; Jin, Qinghui; Cong, Hui; Zhao, Jianlong

doi:10.1016/j.bios.2019.111326

Cited by 20 publications

(15 citation statements)

References 35 publications

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“…124 The cells are patterned into single-cell arrays on the microwell chip with the assistance of external forces, such as centrifugal force, magnetism, gravity, and vacuum. [125][126][127][128][129][130] Since the scale of microwells is easily expanded by chip design and fabrication, the microwell array-based chips can meet the requirements of high throughput readily. Gao et al proposed a microwell array containing more than 20,000 units to separate CTCs into independent units by centrifugation (Figure 5C).…”

Section: Microwell Arraysmentioning

confidence: 99%

“…Gao et al proposed a microwell array containing more than 20,000 units to separate CTCs into independent units by centrifugation (Figure 5C). 125 After on-chip cell lysis and PCR amplification, duplex EGFR mutation genes were parallelly read out from single CTCs.…”

Section: Microwell Arraysmentioning

confidence: 99%

mentioning

confidence: 99%

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Advanced techniques for gene heterogeneity research: Single‐cell sequencing and on‐chip gene analysis systems

Dong

Wang

Yin

et al. 2022

VIEW

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Gene heterogeneity leads to the differences in cellular behaviors in a wide range, such as tumor drug‐resistant mutation, epithelial‐mesenchymal transition, and migration, posing significant challenges to the development of biomedicine. Traditional gene analysis methods, such as polymerase chain reaction, employ a mass of cells as the gene source, resulting in that the gene properties from a specific single cell are hidden in massive gene information. Recent decades have seen the emerging single‐cell gene analysis techniques with their unprecedented opportunities to study gene heterogeneity with high precision and high throughput. In this review, we summarized the state‐of‐the‐art techniques for single‐cell sequencing and on‐chip gene analysis systems. The principles of each technique are introduced in detail, with the focus on the application scenarios in gene heterogeneity research. Looking forward, we also introduced the challenges in current technologies and point out the future direction for facilitating the technical improvement and clinical applications of single‐cell gene analysis techniques.

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Section: Microwell Arraysmentioning

confidence: 99%

Section: Microwell Arraysmentioning

confidence: 99%

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Advanced techniques for gene heterogeneity research: Single‐cell sequencing and on‐chip gene analysis systems

Dong

Wang

Yin

et al. 2022

VIEW

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“…By analyzing and identify the speci c biomarkers, it can provide precise and individualized treatment to cancer patients. The expression of several proteins has been recognized as a suitable predictive biomarker for monitoring tumor response to ICI or target therapy, such as PD-L1, HER2 and VEGF [31][32][33] . These biomarkers are traditionally assessed via tissue-based technique of immunohistochemistry (IHC).…”

Section: Downstream Analysis Of the Enriched Ctcsmentioning

confidence: 99%

Development and clinical validation of a microfluidic-based platform for CTC enrichment and downstream molecular analysis

Cai

Deng

Wang

et al. 2022

Preprint

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Circulating tumor cells (CTCs) are shed from primary or metastatic tumors into the peripheral blood circulation which carry a wealth of information for cancer diagnosis, treatment and prognosis. However, most of current CTCs isolation and detection methods provide only cancer cell counting information which is far from meeting clinical needs. In addition to the numbers of CTCs, the target proteins and gene mutations carried by CTCs can also be used for clinical diagnosis, disease monitoring and therapeutic selection. In this work, we develop a novel microfluidic-based CTCs separation and enrichment platform that enables the extraction of CTCs information, including cell number, epithelial-mesenchymal transition (EMT) subtypes, protein expression levels, and target gene mutations. The platform offers a high CTCs recovery rate (> 85%), high CTCs purification (∼104 enrichment) and intact viable CTCs for downstream analysis. This platform can successfully enrich tumor cells from a 4 mL blood sample within 15 minutes. CTCs were detected in clinical samples from cancer patients with a detection rate of 95.8%. Furthermore, the CTCs subtypes (epithelial, mesenchymal or mix type), the expression levels of selected proteins (PD-L1, HER2, VEGF), and the target mutations in selected genes (EGFR, KRAS, BRAF) could also be directly analyzed by immunofluorescence and digital PCR for clinical utility. PD-L1 expression detected in the CTCs was consistent with the immunohistochemical results. This microfluidic-based CTCs enrichment platform and downstream molecular analysis provide a possible alternative to tissue biopsy for precision cancer management, especially for patients whose tissue biopsies are unavailable.

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“…Typically, PCR thermal cycling includes three stages: denaturation (~ 95 °C), annealing (~ 55 °C), and extension (~ 72 °C). With microfluidics-assisted PCR, the performance of mutation point detection (Gao et al 2019 ; Rowlands et al 2019 ) and gene-expression (Sarma et al 2019 ) in single-cell level has been significantly improved with high sensitivity. Compared with WGA methods, PCR is able to more efficiently amplify and identify target genes in single cell level, which is beneficial to cancer diagnosis (Dalerba et al 2011 ).…”

Section: Single-cell Nucleic Acid Analysis Based On Microfluidicsmentioning

confidence: 99%

Methods and platforms for analysis of nucleic acids from single-cell based on microfluidics

Liu

Dong

et al. 2021

Microfluid Nanofluid

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Single-cell nucleic acid analysis aims at discovering the genetic differences between individual cells which is well known as the cellular heterogeneity. This technology facilitates cancer diagnosis, stem cell research, immune system analysis, and other life science applications. The conventional platforms for single-cell nucleic acid analysis more rely on manual operation or bulky devices. Recently, the emerging microfluidic technology has provided a perfect platform for single-cell nucleic acid analysis with the characteristic of accurate and automatic single-cell manipulation. In this review, we briefly summarized the procedure of single-cell nucleic acid analysis including single-cell isolation, single-cell lysis, nucleic acid amplification, and genetic analysis. And then, three representative microfluidic platforms for single-cell nucleic acid analysis are concluded as valve-, microwell-, and droplet-based platforms. Furthermore, we described the state-of-the-art integrated single-cell nucleic acid analysis systems based on the three platforms. Finally, the future development and challenges of microfluidics-based single-cell nucleic acid analysis are discussed as well.

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EGFR point mutation detection of single circulating tumor cells for lung cancer using a micro-well array

Cited by 20 publications

References 35 publications

Advanced techniques for gene heterogeneity research: Single‐cell sequencing and on‐chip gene analysis systems

Advanced techniques for gene heterogeneity research: Single‐cell sequencing and on‐chip gene analysis systems

Development and clinical validation of a microfluidic-based platform for CTC enrichment and downstream molecular analysis

Methods and platforms for analysis of nucleic acids from single-cell based on microfluidics

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