No abstract

Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps onto a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in under 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings.

The 2009 Influenza A (H1N1) pandemic disproportionately affected the developing world and high-lighted the key inadequacies of traditional diagnostic methods that make them unsuitable for use in resource-limited settings, from expensive equipment and infrastructure requirements to unacceptably long turnaround times. While rapid immunoassay diagnostic tests were much less costly and more context-appropriate, they suffered from drastically low sensitivities and high false negative rates. An accurate, sensitive, and specific molecular diagnostic that is also rapid, low-cost, and independent of laboratory infrastructure is needed for effective point-of-care detection and epidemiological control in these developing regions. We developed a paper-based assay that allows for the extraction and purification of RNA directly from human clinical nasopharyngeal specimens through a poly(ether sulfone) paper matrix, H1N1-specific in situ isothermal amplification directly within the same paper matrix, and immediate visual detection on lateral flow strips. The complete sample-to-answer assay can be performed at the point-of-care in just 45 min, without the need for expensive equipment or laboratory infrastructure, and it has a clinically relevant viral load detection limit of 106 copies/mL, offering a 10-fold improvement over current rapid immunoassays.

Layer-by-layer (LbL) films have multiple features which make them attractive for drug delivery including the possibility of sequential delivery of growth factors, however, to date, proof of concept has been lacking for protein delivery from such films. Here, LbL polyelectrolyte films constructed with lysozyme (a model protein) and a hydrolytically degradable and biocompatible synthetic polycation are shown to be capable of release attractive for the localized delivery of therapeutic proteins from implanted medical devices. Milligram/cm 2 scale release with power law or linear profile can be achieved over 3 weeks to 3 months at room temperature. The release rate at 37°C increases in a way that is compatible with a surface erosion mechanism of release from 100 days to 5 days. This time scale of release can be tuned by changing the degradability of the synthetic polycation, and an increase to 34 days of release at 37°C is seen by increasing the hydrophobicity of this degradable polyester. The enzyme released from these films retains 80-100% functionality, underscoring the mild processing conditions that are apt to preserve fragile protein function. These results uncover many possibilities for incorporation of therapeutic proteins to modulate the interaction between implanted surfaces and the cells they contact.

Fibroblast growth factor 2 (FGF-2) is a potent mediator of stem cell differentiation and proliferation. Although FGF-2 has a well-established role in promoting bone tissue formation, flaws in its delivery have limited its clinical utility. Polyelectrolyte multilayer films represent a novel system for FGF-2 delivery that has promise for local, precisely controlled and sustained release of FGF-2 from surfaces of interest including medical implants and tissue engineering scaffolds. In this work, the loading and release of FGF-2 from synthetic hydrolytically degradable multilayer thin films of various architectures is explored; drug loading was tunable using at least three parameters (number of nanolayers, counterpolyanion, and type of degradable polycation) and yielded values of 7-45 ng/cm2 of FGF-2. Release time varied between 24 hours and approximately five days. FGF-2 released from these films retained in vitro activity, promoting the proliferation of MC3T3 pre-osteoblast cells. The use of biologically derived counterpolyanions heparin sulfate and chondroitin sulfate in the multilayer structures enhanced FGF-2 activity. The control over drug loading and release kinetics inform future in vivo bone and tissue regeneration models for the exploration of clinical relevance of LbL growth factor delivery films.