RESUMO
In this paper, we describe a biosensing instrument based on our previously developed photonic resonator absorption microscope (PRAM) that incorporates autofocus, digital representation of the gold nanoparticle (AuNP) accumulation, and the ability to gather time-series image sequences of AuNP attachment and detachment from the photonic crystal (PC) surface. The combined capabilities are used to fully automate PRAM image collection during biomolecular assays to enable tiling of PRAM images to provide millimeter-scale field of view. The instrument can also gather PRAM "movies" that enables digital showcasing and dynamic counting AuNPs as they arrive and depart from the PC surface. We utilize the capabilities in the context of two biomolecular assays for detection of protein biomarkers in a conventional AuNP-tagged sandwich format. Utilizing dynamic counting of AuNP attachment and detachment events during the assay we present a detection for microRNA-375 (miRNA-375) down to 1 aM with a 10-min, room temperature, enzyme-free approach, while revealing characteristics of the binding-rate and unbinding-rate of the biomolecular interactions. Our instrument can potentially find broad applications in multiplexed point-of-care diagnostic testing, and as a general-purpose tool for quantitative characterization of biomolecular binding kinetics with single-molecule resolution.
RESUMO
Glandular cancers are amongst the most prevalent types of cancer, which can develop in many different organs, presenting challenges in their detection as well as high treatment variability and failure rates. For that purpose, anticancer drugs are commonly tested in cancer cell lines grown in 2D tissue culture on plastic dishesin vitro, or in animal modelsin vivo. However, 2D culture models diverge significantly from the 3D characteristics of living tissues and animal models require extensive animal use and time. Glandular cancers, such as prostate cancer-the second leading cause of male cancer death-typically exist in co-centrical architectures where a cell layer surrounds an acellular lumen. Herein, this spatial cellular position and 3D architecture, containing dual compartments with different hydrogel materials, is engineered using a simple co-axial nozzle setup, in a single step utilizing prostate as a model of glandular cancer. The resulting hydrogel soft structures support viable prostate cancer cells of different cell lines and enable over-time maturation into cancer-mimicking aggregates surrounding the acellular core. The biofabricated cancer mimicking structures are then used as a model to predict the inhibitory efficacy of the poly ADP ribose polymerase inhibitor, Talazoparib, and the antiandrogen drug, Enzalutamide, in the growth of the cancer cell layer. Our results show that the obtained hydrogel constructs can be adapted to quickly obtain 3D cancer models which combine 3D physiological architectures with high-throughput screening to detect and optimize anti-cancer drugs in prostate and potentially other glandular cancer types.