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We present a simple, facile method to micropattern planar metal electrodes defined by the geometry of a microfluidic channel network template. By introducing aqueous solutions of metal into reversibly adhered PDMS devices by desiccation instead of flow, we are able to produce difficult to pattern “dead end” or discontinuous features with ease. We characterize electrodes fabricated using this method and perform electrical lysis of mammalian cancer cells and demonstrate their use as part of an antibody capture assay for GFP. Cell lysis in microwell arrays is achieved using the electrodes and the protein released is detected using an antibody microarray. We show how the template channels used as part of the workflow for patterning the electrodes may be produced using photolithography-free methods, such as laser micromachining and PDMS master moulding, and demonstrate how the use of an immiscible phase may be employed to create electrode spacings on the order of 25–50 μm, that overcome the current resolution limits of such methods. This work demonstrates how the rapid prototyping of electrodes for use in total analysis systems can be achieved on the bench with little or no need for centralized facilities.

Probing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy. Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of more than 1 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01–1 µm. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells.

Computational models predicting cell damage responses to transient temperature rises generated by exposure to lasers have implemented the damage integral (Ω), which time integrates the chemical reaction rate constant described by Arrhenius. However, few published reports of empirical temperature histories (thermal profiles) correlated with damage outcomes at the cellular level are available to validate the breadth of applicability of the damage integral. In our study, an analysis of photothermal damage rate processes in cultured retinal pigment epithelium cells indicated good agreement between temperature rise, exposure duration (τ), and threshold cellular damage. Full-frame thermograms recorded at high magnification during laser exposures were overlaid with fluorescence damage images taken 1 h postexposure. From the image overlays, pixels of the thermogram correlated with the boundary of cell death were used to extract threshold thermal profiles. Assessing photothermal responses at these boundaries standardized all data points, irrespective of laser irradiance, damage size, or optical and thermal properties of the cells. These results support the hypothesis that data from boundaries of cell death were equivalent to a minimum visible lesion, where the damage integral approached unity (Ω=1) at the end of the exposure duration. Empirically resolved Arrhenius coefficients for use in the damage integral determined from exposures at wavelengths of 2 μm and 532 nm and durations of 0.05–20 s were consistent with literature values. Varying ambient temperature (Tamb) between 20°C and 40°C during laser exposure did not change the τ-dependent threshold peak temperature (Tp). We also show that, although threshold laser irradiance varied due to pigmentation differences, threshold temperatures were irradiance independent.

Paper microfluidics has attracted much attention since its first introduction around one decade ago due to the merits such as low cost, ease of fabrication and operation, portability, and facile integration with other devices. The dominant application for paper microfluidics still lies in point-of-care testing (POCT), which holds great promise to provide diagnostic tools to meet the ASSURED criteria. With micro/nanostructures inside, paper substrates provide a natural 3D scaffold to mimic native cellular microenvironments and create excellent biointerfaces for cell analysis applications, such as long-term 3D cell culture, cell capture/phenotyping, and cell-related biochemical analysis (small molecules, protein DNA, etc.). This review summarizes cell-related applications based on various engineered paper microdevices and provides some perspectives for paper microfluidics-based cell analysis.

Bacteriophage (phage), which are viruses that infect bacteria only, have shown promise as vehicles for targeted cancer gene therapy, albeit with poor efficiency. Recently, we generated an improved version of phage vectors by incorporating cis genetic elements of adeno‐associated virus (AAV). This novel AAV/phage hybrid (AAVP) efficiently delivered systemically administered therapeutic genes to various tumor targets by displaying an integrin tumor‐targeting ligand on the phage capsid. However, inherent limitations in bacteriophage mean that these AAVP vectors still need to be improved. One of the limitations of AAVP in mammalian cells may be its susceptibility to proteasomal degradation. The proteasome is upregulated in cancer and it is known that it constitutes a barrier to gene delivery by certain eukaryotic viruses. We report here that inhibition of proteasome improved targeted reporter gene delivery by AAVP in cancer cells in vitro and in tumors in vivo after intravenous vector administration to tumor‐bearing mice. We also show enhanced targeted tumor cell killing by AAVP upon proteasome inhibition. The AAVP particles persisted significantly in cancer cells in vitro and in tumors in vivo after systemic administration, and accumulated polyubiquitinated coat proteins. Our results suggest that the proteasome is indeed a barrier to tumor targeting by AAVP and indicate that a combination of proteasome‐inhibiting drugs and AAVP should be considered for clinical anticancer therapy., Highlights ► Proteasome in cancer is a barrier to tumor targeting by RGD4C/AAVP.► Proteasome inhibiting drugs enhance targeted gene transfer by RGD4C/AAVP.► Proteasome inhibition increases targeted cancer gene therapy by RGD4C/AAVP.► RGD4C/AAVP persistence in cancer is improved by proteasome inhibition

International audience; An European Union (EU)-funded project QLK1-CT-2001-00930 (BIOSAFEPAPER) involves the development, validation and intercalibration of a short-term battery of toxicological tests for the safety assessment of food-contact paper and board. Dissemination of the results to industry, legislators (e.g. DG Consumer Protection, DG Enterprises, DG Research), standardization bodies such as CEN, and consumers will create an agreed risk evaluation procedure. The project involves pre-normative research in order to establish a set of in-vitro cytotoxicity and genotoxicity tests that will be easily adaptable to food-contact fibre-based materials and have endpoints relevant to consumer safety, including sub-lethal cellular events. These tests will be performed on samples representing actual migration conditions from food-contact paper and board with respect to different foodstuffs, and should form an experimental basis for scientifically sound recommendations for a harmonized system of risk evaluation and product testing.