Micro-structured polymer fixed targets for serial crystallography at synchrotrons and XFELs.

Fixed targets are a popular form of sample-delivery system used in serial crystallography at synchrotron and X-ray free-electron laser sources. They offer a wide range of sample-preparation options and are generally easy to use. The supports are typically made from silicon, quartz or polymer. Of these, currently, only silicon offers the ability to perform an aperture-aligned data collection where crystals are loaded into cavities in precise locations and sequentially rastered through, in step with the X-ray pulses. The polymer-based fixed targets have lacked the precision fabrication to enable this data-collection strategy and have been limited to directed-raster scans with crystals randomly distributed across the polymer surface. Here, the fabrication and first results from a new polymer-based fixed target, the micro-structured polymer fixed targets (MISP chips), are presented. MISP chips, like those made from silicon, have a precise array of cavities and fiducial markers. They consist of a structured polymer membrane and a stabilization frame. Crystals can be loaded into the cavities and the excess crystallization solution removed through apertures at their base. The fiducial markers allow for a rapid calculation of the aperture locations. The chips have a low X-ray background and, since they are optically transparent, also allow for an a priori analysis of crystal locations. This location mapping could, ultimately, optimize hit rates towards 100%. A black version of the MISP chip was produced to reduce light contamination for optical-pump/X-ray probe experiments. A study of the loading properties of the chips reveals that these types of fixed targets are best optimized for crystals of the order of 25 µm, but quality data can be collected from crystals as small as 5 µm. With the development of these chips, it has been proved that polymer-based fixed targets can be made with the precision required for aperture-alignment-based data-collection strategies. Further work can now be directed towards more cost-effective mass fabrication to make their use more sustainable for serial crystallography facilities and users.

Thermoplastic Microfluidics.

Thermoplastic polymers are besides glass the material of choice for the industrialization of microfluidic and organ-on-chip applications. In most cases, however, such devices are developed on the basis standard lithographic clean room technologies and subsequent casting into PDMS. This results in comparably fast progress in the development of functional designs but important aspects with respect to later industrialization are thereby largely neglected. For that reason, it is advisable to switch at a rather early stage of development from PDMS to a thermoplastic polymer such as, for instance, cyclo-olefin (co)polymer (COC, COP). By making this step, additional challenges related to the anticipated manufacturing process can be identified, which is particularly important when aiming at industrialization. We present herein a standard process sequence for mastering of microfluidic devices by two-photon polymerization and final transfer into COC films by hot embossing. In addition, we describe the laser micromanufacturing of polymeric mold inserts and subsequent prototype injection molding of small series of COP samples.

Versatile microporous polymer-based supports for serial macromolecular crystallography.

Serial data collection has emerged as a major tool for data collection at state-of-the-art light sources, such as microfocus beamlines at synchrotrons and X-ray free-electron lasers. Challenging targets, characterized by small crystal sizes, weak diffraction and stringent dose limits, benefit most from these methods. Here, the use of a thin support made of a polymer-based membrane for performing serial data collection or screening experiments is demonstrated. It is shown that these supports are suitable for a wide range of protein crystals suspended in liquids. The supports have also proved to be applicable to challenging cases such as membrane proteins growing in the sponge phase. The sample-deposition method is simple and robust, as well as flexible and adaptable to a variety of cases. It results in an optimally thin specimen providing low background while maintaining minute amounts of mother liquor around the crystals. The 2 × 2 mm area enables the deposition of up to several microlitres of liquid. Imaging and visualization of the crystals are straightforward on the highly transparent membrane. Thanks to their affordable fabrication, these supports have the potential to become an attractive option for serial experiments at synchrotrons and free-electron lasers.

Surface topology and functionality of freeform microlens arrays.

Nonsymmetric (also known as freeform) optical components have attracted a great deal of academic and industrial attention due to the substantial benefits they have demonstrated in imaging and nonimaging optical systems. Additionally, freeform microlens arrays (FMLAs) are very promising with regard to the growing demand for device miniaturization and cost reduction. As a flip side, FMLAs entail specific challenges in design, manufacturing, and characterization. Here we report on the latter and present an innovative characterization strategy that makes it possible to assess the quality of FMLAs quickly and accurately. The precisely measured surface topology of FMLAs was accurately represented using nonuniform rational basis-spline (NURBS) and its optical response was predicted by means of ray-tracing simulations. We show that for reliably measured surface topology, the results are in excellent agreement with the experimental measurements. We also show that, compared to previous studies, illuminance levels displayed in a logarithmic scale are more adequate for low light levels and represent a closer match to nonlinear human visual perception. We believe that the method presented here will contribute to speeding up the FMLA manufacturing process, one of the current downsides of this promising technology.

Electrochemical aptamer-based biosensor developed to monitor PSA and VEGF released by prostate cancer cells.

Early prostate cancer (PCa) diagnostic is crucial to enhance patient survival rates; besides, non-invasive platforms have been developed worldwide in order to precisely detect PCa biomarkers. Therefore, the aim of the present study is to develop a new aptamer-based biosensor through the self-assembling of thiolated aptamers for PSA and VEGF on the top of gold electrodes. This biosensor was tested in three prostate cell lines (RWPE-1, LNCaP and PC3). The results evidenced a stable and sensitive sensor presenting wide linear detection ranges (0.08-100 ng/mL for PSA and 0.15 ng-100 ng/mL for VEGF). Therefore, the aptasensor was able to detect the patterns of PSA and VEGF released in vitro by PCa cells, which gave new insights about the prostate cancer protein dynamics. Thus, it could be used as a non-invasive PCa clinical diagnosis instrument in the near future. Graphical Abstract Overview of the experimental design applied to the aptamer-based electrochemical sensor self-assembled on the thiolated hairpin structure. A filter membrane was added on top of working electrode to provide the cell-attachment surface after aptamer incubation, without compromising the aptamer layer. The pore membrane allowed target proteins to pass to the aptamer surface; the MCH backfilling avoided unspecific protein binding to the gold electrode surface.