Liquid biopsy offers unique opportunities for low invasive diagnosis, real-time patient monitoring and treatment selection. The phenotypic and molecular profile of circulating tumor cells (CTCs) can provide key information about the biology of tumor cells, contributing to personalized therapy. CTC isolation is still challenging, mainly due to their heterogeneity and rarity. To overcome this limitation, a microfluidic chip for label-free isolation of CTCs from peripheral blood was developed. This device, the CROSS chip, captures CTCs based on their size and deformability with an efficiency of 70%. Using 2 chips, 7.5 ml of whole blood are processed in 47 minutes with high purity, as compared to similar technologies and assessed by
in situ
immunofluorescence. The CROSS chip performance was compared to the CellSearch system in a set of metastatic colorectal cancer patients, resulting in higher capture of DAPI+/CK+/CD45− CTCs in all individuals tested. Importantly, CTC enumeration by CROSS chip enabled stratification of patients with different prognosis. Lastly, cells isolated in the CROSS chip were lysed and further subjected to molecular characterization by droplet digital PCR, which revealed a mutation in the
APC
gene for most patient samples analyzed, confirming their colorectal origin and the versatility of the technology for downstream applications.
Carbon dots have demonstrated great potential as luminescent nanoparticles in bioapplications. Although such nanoparticles appear to exhibit low toxicity compared to other metal luminescent nanomaterials, today we know that the toxicity of carbon dots (C-dots) strongly depends on the protocol of fabrication. In this work, aqueous fluorescent C-dots have been synthesized from cinnamon, red chilli, turmeric and black pepper, by a one-pot green hydrothermal method. The synthesized C-dots were firstly characterized by means of UV–vis, fluorescence, Fourier transform infrared and Raman spectroscopy, dynamic light scattering and transmission electron microscopy. The optical performance showed an outstanding ability for imaging purposes, with quantum yields up to 43.6%. Thus, the cytotoxicity of the above mentioned spice-derived C-dots was evaluated in vitro in human glioblastoma cells (LN-229 cancer cell line) and in human kidney cells (HK-2 non-cancerous cell line). Bioimaging and viability studies were performed with different C-dot concentrations from 0.1 to 2 mg·mL−1, exhibiting a higher uptake of C-dots in the cancer cultures compared to the non-cancerous cells. Results showed that the spice-derived C-dots inhibited cell viability dose-dependently after a 24 h incubation period, displaying a higher toxicity in LN-229, than in HK-2 cells. As a control, C-dots synthesized from citric acid did not show any significant toxicity in either cancerous or non-cancerous cells, implying that the tumour cell growth inhibition properties observed in the spice-derived C-dots can be attributed to the starting material employed for their fabrication. These results evidence that functional groups in the surface of the C-dots might be responsible for the selective cytotoxicity, as suggested by the presence of piperine in the surface of black pepper C-dots analysed by ESI-QTOF-MS.
The deposition and subsequent electrochemical dissolution of (PLL/DNA) 6 films on ITO electrodes was studied by means of ecOWLS and in situ AFM. ecOWLS experiments showed that (PLL/DNA) 6 films fabricated at 0 V are stable in physiological buffer (pH 7.4) and that applying a potential above 1.8 V induces only a partial and slow dissolution. On the contrary, the dissolution is much more effective and quicker if a potential is applied already during the deposition of the film. AFM experiments showed that (PLL/DNA) 6 films are constituted of 30 nm high, 100 nm diameter nanodroplets. The film morphology was not affected by the application of a potential during the fabrication. A custom made flow-cell allowed in situ following of the electrochemical dissolution revealing the continuous shrinking of the nanodroplets. The results were interpreted in the light of a model describing the variation of pH induced by the water electrolysis in the proximity of the ITO electrode.
Efficient isolation strategies not based on epithelial biomarker expression are required to enable non-biased enrichment of circulating tumor cells (CTCs). CTCs undergoing epithelial-mesenchymal transition (EMT) may be prognostically relevant, and importantly are not detected with conventional epithelial based approaches such as CellSearch®. A method for the non-biased isolation of cancer cells within a peripheral blood sample utilizing microfluidic mixing PDMS devices functionalized with anti-CD45 is reported. The introduction of micro and nanoscale roughness using a single step treatment with sulfuric acid significantly increases the binding yield of white blood cells (WBCs) to the anti-CD45 conjugated surfaces. Up to 99.99% WBC depletion is achieved with a tumor cell recovery yield of 50%. This high level of CTC enrichment is expected to facilitate the detailed characterization of CTCs using for instance, imaging flow cytometry as demonstrated here.
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