Rapid Isolation and Multiplexed Detection of Exosome Tumor Markers Via Queued Beads Combined with Quantum Dots in a Microarray

Tumor-derived exosomes are actively involved in cancer progression and metastasis and have emerged as a promising marker for cancer diagnosis in liquid biopsy.Because of their nanoscale size,complex biogenesis,and methodological limitations related to exosome isolation and detection,advancements in their analysis remain slow.Microfluidic technology offers a better analytic approach compared with conventional methods.Here,we developed a bead-based microarray for exosome isolation and multiplexed tumor marker detection.Using this method,exosomes are isolated by binding to antibodies on the bead surface,and tumor markers on the exosomes are detected through quantum dot(QD)probes.The beads are then uniformly trapped and queued among micropillars in the chip.This design benefits fluorescence observation by dispersing the signals into every single bead,thereby avoiding optical interference and enabling more accurate test results.We analyzed exosomes in the cell culture supernatant of lung cancer and endothelial cell lines,and different lung cancer markers labeled with three QD probes were used to conduct multiplexed detection of exosome surface protein markers.Lung cancer-derived samples showed much higher(~sixfold-tenfold)fluorescence intensity than endothelial cell samples,and different types of lung cancer samples showed distinctive marker expression levels.Additionally,using the chip to detect clinical plasma samples from cancer patients showed good diagnostic power and revealed a well consistency with conventional tests for serological markers.These results provide insight into a promising method for exosome tumor marker detection and early-stage cancer diagnosis.

Multiple exosome RNA analysis methods for lung cancer diagnosis through integrated on-chip microfluidic system

Exosomes are now raising focus as a prospective biomarker for cancer diagnostics and prognosis owing to its unique bio-origin and composition.Exosomes take part in cellular communication and receptor mediation and transfer their cargos(e.g.,proteins,m RNA and DNA).Quantitative analysis of tumor-related nucleic acid mutations can be a potential method to cancer diagnosis and prognosis in early stages.Here we present an integrated microfluidic system for exosome on-chip isolation and lung cancer RNA analysis through droplet digital PCR(dd PCR).Gradient dilution experiments show great linearity over a large concentration range with R^(2)=0.9998.Utilizing the system,four cell lines and two mutation targets were parallelly detected for mutation analysis.The experiments demonstrated mutation heterogeneity and the results were agree with cell researches.These results proved our integrated microfluidic system as a promising means for early cancer diagnosis and prognosis in the era of liquid biopsy.

Single-cell level point mutation analysis of circulating tumor cells through droplet microfluidics

Tumor heterogeneity plays a critical role in the determination of appropriate anticancer therapy.As cir-culating tumor cells(CTCs)contain all tumor-related information,the genetic changes on CTCs could help us choose the appropriate treatments for different patients.Single-base mutations are very common in tumor genetic changes which may result in drug resistance.Here,we introduce a single-cell mutation de-tection platform based on droplet microfluidics.This platform integrates cell capsulation,cell lysis,poly-merase chain reaction(PCR)and the observation process.The droplets’generation speed is over 6000 per minute and more than 600 cells could be encapsulated in one second.To verify the performance of our platform in practical use,we performed the mutation analysis of 4 kinds of cells with our platform and noted that the genetic status of each single cell was clearly discriminated.Moreover,these results agreed with those from direct sequencing.Compared with other forms of single-cell mutation detection techniques,our platform has high throughput,short experimental time and less experimental operations.