RESUMO
We established an efficient method for single-cell miRNA analysis by droplet microfluidics, which has high sensitivity of single molecule detection and high throughput. Single-cell analysis of multiple miRNAs in various cells shows that miRNA expression is closely related to cancer type. CTC analysis shows that the method is applicable for rare cell analysis.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Neoplasias/genética , Análise de Célula Única , Linhagem Celular Tumoral , Fluorescência , Humanos , Limite de DetecçãoRESUMO
Single-cell phenotypic profiling of circulating tumor cells (CTCs) in the blood of cancer patients can reveal vital tumor biology information. Even though various approaches have been provided to enrich and detect CTCs, it remains challenging for consecutive CTC sorting, enumeration, and single-cell characterizations. Here, we report an integrated microfluidic device (IMD) for single-cell phenotypic profiling of CTCs that enables automated CTCs sorting from whole blood following continuous single-cell phenotypic analysis while satisfying the requirements of both high purity (92 ± 3%) of cell sorting and high-throughput processing capacity (5 mL whole blood/3 h). Using this new technique we test the phenotypes of individual CTCs collected from xenograft tumor-bearing mice and colorectal (CRC) patients at different tumor stages. We obtained a correlation between CTC characterization and clinical tumor stage and treatment response. The developed IMD offers a high-throughput, convenient, and rapid strategy to study individual CTCs toward minimally invasive cancer therapy prediction and disease monitoring and has the potential to be translated to clinic for liquid biopsy.
Assuntos
Antígenos CD/sangue , Biomarcadores Tumorais/sangue , Caderinas/sangue , Neoplasias Colorretais/sangue , Molécula de Adesão da Célula Epitelial/sangue , Dispositivos Lab-On-A-Chip , Células Neoplásicas Circulantes/metabolismo , Análise de Célula Única/métodos , Animais , Antígenos CD/genética , Biomarcadores Tumorais/genética , Caderinas/genética , Contagem de Células , Linhagem Celular Tumoral , Separação Celular/instrumentação , Separação Celular/métodos , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Molécula de Adesão da Célula Epitelial/genética , Feminino , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Glicoconjugados/química , Xenoenxertos , Humanos , Biópsia Líquida , Camundongos , Camundongos Endogâmicos BALB C , Estadiamento de Neoplasias , Células Neoplásicas Circulantes/patologiaRESUMO
Intracellular trace Zn2+ and Cu2+ play important roles in the regulation of cell function. Considering the limitations of existing metal ion detection methods regarding sensitivity and applicability to living cells, an amplification strategy based on functional DNA self-assembly under DNAzyme catalysis to improve the sensitivity of intracellular Zn2+ and Cu2+ imaging is reported. In this process, metal ions as cofactor can activate the catalysis of DNAzyme to shear substrate chains, and each broken substrate chain can initiate consecutive hybridizations of hairpin probes (Hx) labeled with fluorophore, which can reflect the information on a single metal ion with multiple fluorophores. The detection limit can reach nearly 80 pM and high-sensitivity fluorescence imaging of intracellular Zn2+ and Cu2+ can be achieved. The results are important for research on cell function regulation associated with trace Zn2+ and Cu2+. This approach is also a new way to improve the sensitivity of other trace metal ion imaging.
Assuntos
Técnicas Biossensoriais/métodos , Cobre/análise , DNA Catalítico/química , Corantes Fluorescentes/química , Imagem Óptica/métodos , Zinco/análise , Cátions Bivalentes/análise , Sondas de DNA/química , Humanos , Limite de Detecção , Células MCF-7RESUMO
Various intracellular metal ions have closely related functional roles in the nervous system. An excess or deficiency of essential metal ions can contribute to neurodegenerative diseases. Thus, the detection of various metal ions in neurons is important for diagnosing and monitoring these diseases. In particular, single-cell analysis of multiple metal ions allows us to not only understand the cellular heterogeneity and differentiation but also determine the actual relationships among multiple metal ions in each individual cell. Aiming at the low efficient single-cell manipulation and interference of complex biological matrices within cells in the existing method for single-cell metal ion detection, in this manuscript, we present a convenient, sensitive, and reliable method to simultaneously identify and quantify multiple metal ions at the single-cell level using a microfluidic system. Using the combination of on-chip electrophoresis separation and multicolor fluorescence detection, we achieved the simultaneous analysis of Na+, K+, Ca2+, and Mg2+ in single PC-12 cells and studied changes in these four metal ions in Aß25-35-treated PC-12 cells, which is a model of Alzheimer's disease (AD). The data showed that metal ions imbalances in neuron-like cells may be associated with AD induced by Aß25-35. This method paves the way for multiple metal ion detection in single neuron-like cells, and the results provide insights regarding synergistic function of multiple metal ions in regulation of neurological diseases at the single-cell level.
