Cell tracking is an emergent area in nanobiotechnology, promising the study of individual cells or the identification of populations of cultured cells. In our approach, microtools designed for extracellular tagging are prepared, because using biofunctionalized polysilicon barcodes to tag cell membranes externally avoids the inconveniences of cell internalization. The crucial covalent biofunctionalization process determining the ultimate functionality was studied in order to find the optimum conditions to link a biomolecule to a polysilicon barcode surface using a self-assembled monolayer (SAM) as the connector. Specifically, a lectin (wheat germ agglutinin, WGA) was used because of its capacity to recognize some specific carbohydrates present on the surface of most mammalian cells. Self-assembled monolayers were prepared on polysilicon surfaces including aldehyde groups as terminal functions to study the suitability of their covalent chemical bonding to WGA. Some parameters, such as the polysilicon surface roughness or the concentration of WGA, proved to be crucial for successful biofunctionalization and bioactivity. The SAMs were characterized by contact angle measurements, time-of-flight secondary ion mass spectrometry (TOF-SIMS), laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS), and atomic force microscopy (AFM). The biofunctionalization step was also characterized by fluorescence microscopy and, in the case of barcodes, by adhesion experiments to the zona pellucida of mouse embryos. These experiments showed high barcode retention rates after 96 h of culture as well as high embryo viability to the blastocyst stage, indicating the robustness of the biofunctionalization and, therefore, the potential of these new microtools to be used for cell tagging.
study question: Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of cultured embryos?summary answer: The results achieved provide a proof of concept for a direct embryo tagging system using biofunctionalized polysilicon barcodes, which could help to minimize the risk of mismatching errors (mix-ups) in human assisted reproduction technologies.what is known already: Even though the occurrence of mix-ups is rare, several cases have been reported in fertility clinics around the world. Measures to prevent the risk of mix-ups in human assisted reproduction technologies are therefore required. study design, size, duration: Mouse embryos were tagged with 10 barcodes and the effectiveness of the tagging system was tested during fresh in vitro culture (n ¼140) and after embryo cryopreservation (n ¼ 84). Finally, the full-term development of tagged embryos was evaluated (n ¼105).participants/materials, setting, methods: Mouse pronuclear embryos were individually rolled over wheat germ agglutinin-biofunctionalized polysilicon barcodes to distribute them uniformly around the ZONA PELLUCIDA surface. Embryo viability and retention of barcodes were determined during 96 h of culture. The identification of tagged embryos was performed every 24 h in an inverted microscope and without embryo manipulation to simulate an automatic reading procedure. Full-term development of the tagged embryos was assessed after their transfer to pseudo-pregnant females. To test the validity of the embryo tagging system after a cryopreservation process, tagged embryos were frozen at the 2-cell stage using a slow freezing protocol, and followed in culture for 72 h after thawing.main results and the role of chance: Neither the in vitro or in vivo development of tagged embryos was adversely affected. The tagging system also proved effective during an embryo cryopreservation process. Global identification rates higher than 96 and 92% in fresh and frozen-thawed tagged embryos, respectively, were obtained when simulating an automatic barcode reading system, although these rates could be increased to 100% by simply rotating the embryos during the reading process.limitations, reasons for caution: The direct embryo tagging developed here has exclusively been tested in mouse embryos. Its effectiveness in other species, such as the human, is currently being tested.
Three nanostructured vehicles are proposed as potential carriers for photosensitizers to be used in photodynamic therapy: spherical nanoparticles, hexahedral microparticles and cylindrical magnetic nanorods. A comparative study of their photodynamic properties was performed, and the influence of their size and the amount of loaded porphyrin was considered to discuss their effects in the observed photodynamic activity. All the vehicles have a gold surface, allowing functionalization with a disulfide-containing porphyrin as the photosensitizer, as well as with a PEG-containing thiol to improve their biocompatibility and water solubility. The activity of the porphyrin loaded in each vehicle was assessed through in vitro photocytotoxicity studies using HeLa cells. A synergic effect for the porphyrin toxicity was observed in all of the vehicles. The zinc-containing porphyrin showed better production of singlet oxygen, and proved more photocytotoxic both in solution and loaded in any of the vehicles. The magnetism of the nanorods allows targeting with a magnetic field, but causes their aggregation, hampering the porphyrin's activity. Microparticles showed lower cell internalization but their bigger size allowed a high porphyrin loading, which translated into high photocytotoxicity. The highest cell internalization and photocytotoxicity was observed for the porphyrin-loaded nanoparticles, suggesting that a smaller size is favored in cell uptake
Increasingly, advances in microtechnologies are focused on obtaining new chips intended for applications in fields such as nanomedicine and cell biology, taking advantage of the ability of microelectronics to manufacture devices with cell dimensions and a large variety of features. Here, we report a technology for the fabrication of multi-material chips, using polysilicon and gold as device layers, to be used as bi-functional cellinternalizable devices. In our case, one of the main technological challenges is to overcome the low adherence between these two materials, especially because of their small contact-area, only 9 µm 2. Thus, in order to circumvent this difficulty a chromium adherent-layer was deposited in between. After fabrication, the devices following this design can be successfully internalized inside living macrophages without affecting their viability. The advantage of having multiple material layers in one device is the potential to render multi-tasking chips, as once they are appropriately functionalized, we can provide the chip the ability of being bi-functional. Hence, and as a proof of concept, two different proteins, Wheat Germ Agglutinin (WGA) and Concanavalin (ConA), were 3 immobilized on the chip surface through self-assembled monolayers using orthogonal chemistry. The results of this work show a well-controlled fabrication, the bi-functional capabilities and no cell-toxicity of intracellular polysilicon-chromium-gold chips. These devices have a promising future as intracellular functional platforms for biosensing, drug delivery and diagnosis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.