Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix.

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time is developed. As a proof of concept, real-time imaging of inter-cellular cyclic adenosine 3',5'-monophosphate (cAMP) signals in Dictyostelium discoideum amoeba cells is performed. A set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals is engineered. By combining multiple switches with different dynamic ranges, measure cAMP concentrations spanning three orders of magnitude in a single experiment can be measured. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements are achieved. Using different aptamer switches, this approach can be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

Real-time monitoring of drug pharmacokinetics within tumor tissue in live animals.

The efficacy and safety of a chemotherapy regimen fundamentally depends on its pharmacokinetics. This is currently measured based on blood samples, but the abnormal vasculature and physiological heterogeneity of the tumor microenvironment can produce radically different drug pharmacokinetics relative to the systemic circulation. We have developed an implantable microelectrode array sensor that can collect such tissue-based pharmacokinetic data by simultaneously measuring intratumoral pharmacokinetics from multiple sites. We use gold nanoporous microelectrodes that maintain robust sensor performance even after repeated tissue implantation and extended exposure to the tumor microenvironment. We demonstrate continuous in vivo monitoring of concentrations of the chemotherapy drug doxorubicin at multiple tumor sites in a rodent model and demonstrate clear differences in pharmacokinetics relative to the circulation that could meaningfully affect drug efficacy and safety. This platform could prove valuable for preclinical in vivo characterization of cancer therapeutics and may offer a foundation for future clinical applications.

Improvement of the Bag-Mediated Filtration System for Sampling Wastewater and Wastewater-Impacted Waters.

Environmental surveillance of poliovirus (PV) plays an important role in the global program for eradication of wild PV. The bag-mediated filtration system (BMFS) was first developed in 2014 and enhances PV surveillance when compared to the two-phase grab method currently recommended by the World Health Organization (WHO). In this study, the BMFS design was improved and tested for its usability in wastewater and wastewater-impacted surface waters in Nairobi, Kenya. Modifications made to the BMFS included the size, color, and shape of the collection bags, the filter housing used, and the device used to elute the samples from the filters. The modified BMFS concentrated 3-10 L down to 10 mL, which resulted in an effective volume assayed (900-3000 mL) that was 6-20 times greater than the effective volume assayed for samples processed by the WHO algorithm (150 mL). The system developed allows for sampling and in-field virus concentration, followed by transportation of the filter for further analysis with simpler logistics than the current methods. This may ultimately reduce the likelihood of false-negative samples by increasing the effective volume assayed compared to samples processed by the WHO algorithm, making the BMFS a valuable sampling system for wastewater and wastewater-impacted surface waters.