Since their discovery almost three decades ago, DNAzymes have been used extensively in biosensing. Depending on the type of DNAzyme being used, these functional oligonucleotides can act as molecular recognition elements within biosensors, offering high specificity to their target analyte, or as reporters capable of transducing a detectable signal. Several parameters need to be considered when designing a DNAzyme-based biosensor. In particular, given that many of these biosensors immobilize DNAzymes onto a sensing surface, selecting an appropriate immobilization strategy is vital. Suboptimal immobilization can result in both DNAzyme detachment and poor accessibility toward the target, leading to low sensing accuracy and sensitivity. Various approaches have been employed for DNAzyme immobilization within biosensors, ranging from amine and thiol-based covalent attachment to non-covalent strategies involving biotin–streptavidin interactions, DNA hybridization, electrostatic interactions, and physical entrapment. While the properties of each strategy inform its applicability within a proposed sensor, the selection of an appropriate strategy is largely dependent on the desired application. This is especially true given the diverse use of DNAzyme-based biosensors for the detection of pathogens, metal ions, and clinical biomarkers. In an effort to make the development of such sensors easier to navigate, this paper provides a comprehensive review of existing immobilization strategies, with a focus on their respective advantages, drawbacks, and optimal conditions for use. Next, common applications of existing DNAzyme-based biosensors are discussed. Last, emerging and future trends in the development of DNAzyme-based biosensors are discussed, and gaps in existing research worthy of exploration are identified.

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Horseshoe crab (HSC) hemolymph is the source of Limulus amebocyte lysate (LAL), a critical component in sterility testing that ensures drug and medical device safety for millions of patients every year. Wild HSC populations have been declining as a result of its use as whelk and eel bait, environmental changes, and its capture and bleeding for hemolymph by the biomedical industry, thus posing significant risks to species viability and the LAL raw material supply chain. We designed a controlled aquaculture habitat to husband HSCs and evaluated the effects of captivity on health markers (e.g., amebocyte density, hemocyanin levels, and LAL activity). We found HSC aquaculture to be practicable, with routine hemolymph harvesting resulting in high LAL quality, while safeguarding animal well-being with 100% HSC survival. Further, lowimpact hemolymph harvesting via an indwelling catheter revealed rapid amebocyte rebound kinetics after consecutive 10% hemolymph extractions. Sustainable supplies of LAL could also be adapted to address daunting trends in septicemia and antimicrobial resistance. LAL is uniquely sensitive and specific for gram-negative bacteria, which represent 70-80% of pathogens that typically lead to sepsis. However, erratic results associated with interfering substances plagued efforts to adapt LAL for clinical use in the past. We report the development of a new LAL-based assay that can detect gram-negative bacteria and endotoxins in human blood without interference using aquaculture-derived LAL. Based on this research, sustainable LAL production from aquaculture could potentially satisfy industry needs with a fraction of one year's current capture via year-round harvesting from a finite cohort of HSCs and expand raw materials supplies for potential future clinical applications.

The diversity of autologous cells being used and investigated for cancer therapy continues to increase. Mast cells (MCs) are tissue cells that contain a unique set of anti-cancer mediators and are found in and around tumors. We sought to exploit the anti-tumor mediators in MC granules to selectively target them to tumor cells using tumor specific immunoglobin E (IgE) and controllably trigger release of anti-tumor mediators upon tumor cell engagement. We used a human HER2/neu-specific IgE to arm human MCs through the high affinity IgE receptor (FcεRI). The ability of MCs to bind to and induce apoptosis of HER2/neu-positive cancer cells in vitro and in vivo was assessed. The interactions between MCs and cancer cells were investigated in real time using confocal microscopy. The mechanism of action using cytotoxic MCs was examined using gene array profiling. Genetically manipulating autologous MC to assess the effects of MC-specific mediators have on apoptosis of tumor cells was developed using siRNA. We found that HER2/neu tumor-specific IgE-sensitized MCs bound, penetrated, and killed HER2/neu-positive tumor masses in vitro. Tunneling nanotubes formed between MCs and tumor cells are described that parallel tumor cell apoptosis. In solid tumor, human breast cancer (BC) xenograft mouse models, infusion of HER2/neu IgE-sensitized human MCs co-localized to BC cells, decreased tumor burden, and prolonged overall survival without indications of toxicity. Gene microarray of tumor cells suggests a dependence on TNF and TGFβ signaling pathways leading to apoptosis. Knocking down MC-released tryptase did not affect apoptosis of cancer cells. These studies suggest MCs can be polarized from Type I hypersensitivity-mediating cells to cytotoxic cells that selectively target tumor cells and specifically triggered to release anti-tumor mediators. A strategy to investigate which MC mediators are responsible for the observed tumor killing is described so that rational decisions can be made in the future when selecting which mediators to target for deletion or those that could further polarize them to cytotoxic MC by adding other known anti-tumor agents. Using autologous human MC may provide further options for cancer therapeutics that offers a unique anti-cancer mechanism of action using tumor targeted IgE’s.

The emergence of cancer immunotherapies utilizing adoptive cell transfer (ACT) continues to be one of the most promising strategies for cancer treatment. Mast cells (MCs) which occur throughout vascularized tissues, are most commonly associated with Type I hypersensitivity, bind immunoglobin E (IgE) with high affinity, produce anti-cancer mediators such as tumor necrosis factor alpha (TNF-α) and granulocyte macrophage colony-stimulating factor (GM-CSF), and generally populate the tumor microenvironments. Yet, the role of MCs in cancer pathologies remains controversial with evidence for both anti-tumor and pro-tumor effects. Here, we review the studies examining the role of MCs in multiple forms of cancer, provide an alternative, MC-based hypothesis underlying the mechanism of therapeutic tumor IgE efficacy in clinical trials, and propose a novel strategy for using tumor-targeted, IgE-sensitized MCs as a platform for developing new cellular cancer immunotherapies. This autologous MC cancer immunotherapy could have several advantages over current cell-based cancer immunotherapies and provide new mechanistic strategies for cancer therapeutics alone or in combination with current approaches.