Cancer-ID: Toward Identification of Cancer by Tumor-Derived Extracellular Vesicles in Blood.

Extracellular vesicles (EVs) have great potential as biomarkers since their composition and concentration in biofluids are disease state dependent and their cargo can contain disease-related information. Large tumor-derived EVs (tdEVs, >1 µm) in blood from cancer patients are associated with poor outcome, and changes in their number can be used to monitor therapy effectiveness. Whereas, small tumor-derived EVs (<1 µm) are likely to outnumber their larger counterparts, thereby offering better statistical significance, identification and quantification of small tdEVs are more challenging. In the blood of cancer patients, a subpopulation of EVs originate from tumor cells, but these EVs are outnumbered by non-EV particles and EVs from other origin. In the Dutch NWO Perspectief Cancer-ID program, we developed and evaluated detection and characterization techniques to distinguish EVs from non-EV particles and other EVs. Despite low signal amplitudes, we identified characteristics of these small tdEVs that may enable the enumeration of small tdEVs and extract relevant information. The insights obtained from Cancer-ID can help to explore the full potential of tdEVs in the clinic.

Isolation of single cells for protein therapeutics using microwell selection and Surface Plasmon Resonance imaging.

Here the feasibility is demonstrated that by combining Surface Plasmon Resonance Imaging (SPRi) and self-sorting microwell technology product secretion of individual cells can be monitored. Additionally isolation of the selected cells can be performed by punching the cells from the microwells using coordinates of the positions of microwells obtained with SPRi. Cells of interest can be retrieved sterile from the microwell array for further cultivation.

Modification of ISFETs with a monolayer of latex beads for specific detection of proteins.

The so-called ion-step method is a novel potentiometric approach that can detect protein adsorbed onto the gate area of modified ion-sensitive field-effect transistors (ISFETs). In this report, a generic technology is described for immobilization of peptides and proteins to the ISFET gate in order to confer specific binding properties to the ISFET. For this, the surface of the ISFET was covered with a monolayer of Amino beads (diameter, 0.9 microm) followed by immobilization of protein ligands onto these beads. Amino beads are latex spheres that contain primary amino groups at the outer surface. Preactivation of the latex-bound amino groups with glutaraldehyde, and consecutive incubation with polylysine resulted in covalent immobilization of this polyamine, as revealed by ion stepping measurements. For ImmunoFET applications, human serum albumin (HSA) was immobilized onto the Amino bead-covered ISFETs, by passive adsorption but also by covalent coupling. Resulting devices were used for qualitative detection of alpha-HSA antibodies by means of the ion step method. The binding of antibody was very specific and fast (most of the binding was accomplished in 15 min) with signal yields up to 17 mV. Efforts to increase the antibody-binding capacity of the solid phase on the ISFET exploiting amino group activation (with glutaraldehyde or other homobifunctional cross linkers) before HSA coupling, did not improve signal yield. The bead technology described in this report is an easy, generic method for coating the ISFET with a solid phase that, using the ion-step method, can be applied to immunosensing.