M. Strano
Massachusetts Institute of Technology,
United States
Keywords: biosensor, chemical transfer
Summary:
Our laboratory at MIT has been interested over the past few years in new techniques to facilitate the transfer of chemical information from living organisms, specifically plants, animals and humans, for applications ranging from precision agriculture to precision medicine. This presentation will discuss recent advances on this topic. As tool towards this end, fluorescent nanosensors hold the potential to revolutionize life sciences and medicine. However, their adaptation and translation into the in vivo environment is fundamentally hampered by unfavourable tissue scattering and intrinsic autofluorescence. Here we develop wavelength-induced frequency filtering (WIFF) whereby the fluorescence excitation wavelength is modulated across the absorption peak of a nanosensor, allowing the emission signal to be separated from the autofluorescence background, increasing the desired signal relative to noise, and internally referencing it to protect against artefacts. Using highly scattering phantom tissues, an SKH1-E mouse model and other complex tissue types, we show that WIFF improves the nanosensor signal-to-noise ratio across the visible and near-infrared spectra up to 52-fold. This improvement enables the ability to track fluorescent carbon nanotube sensor responses to riboflavin, ascorbic acid, hydrogen peroxide and a chemotherapeutic drug metabolite for depths up to 5.5 ± 0.1 cm when excited at 730 nm and emitting between 1,100 and 1,300 nm, even allowing the monitoring of riboflavin diffusion in thick tissue. As an application, nanosensors aided by WIFF detect the chemotherapeutic activity of temozolomide transcranially at 2.4 ± 0.1 cm through the porcine brain without the use of fibre optic or cranial window insertion. The ability of nanosensors to monitor previously inaccessible in vivo environments will be important for life-sciences research, therapeutics and medical diagnostics. Also towards this overall objective, our laboratory at MIT has been interested in exploring the relatively new interface between living plants and non-biological nanostructures to impart the former with new and enhanced functions, which we call Plant Nanobionics. We have developed a theory of subcellular uptake and kinetic trapping of a wide range of nanoparticles, validated in-vivo in living plants. Confocal visible and near infrared fluorescent microscopy and single particle tracking of Gold-Cystein-AF405 (GNP-Cys-AF405), Streptavidin-Quantum Dot (SA-QD), Dextran and Poly(acrylic acid) nanoceria, and various polymer-wrapped SWCNT, including lipid-PEG-SWCNT, chitosan-SWCNT and (AT)15-SWCNT, were used to demonstrate that particle size and the magnitude, but not the sign, of the zeta potential are key in determining whether a particle is spontaneously and kinetically trapped within chloroplasts or the cytosol. We develop a mathematical model of this Lipid Exchange Envelope Penetration (LEEP) mechanism, which agrees well with observations of this size and zeta potential dependence. As an application, we rationally designed a chitosan-complexed single-walled carbon nanotube (SWNT) as nanocarriers to selectively deliver plasmid DNA (pDNA) to chloroplasts of different plant species without external biolistic or chemical aid. We demonstrate chloroplast-targeted transgene delivery and expression in living mature arugula (Eruca sativa) and watercress (Nasturitium officinale) plants in planta and in isolated Arabidopsis thaliana mesophyll protoplasts. Another application of nanoparticles and nanotechnology to plant sciences is in the form of biochemical sensors that operate in planta and across diverse species. Using non-destructive optical nanosensors, we find that the spatial and temporal H2O2 concentration immediately post-wounding follows a simple logistic waveform for six dicot plant species: lettuce (Lactuca sativa), arugula (Eruca sativa), spinach (Spinacia oleracea), strawberry blite (Blitum capitatum), sorrel (Rumex acetosa), and Arabidopsis thaliana, ranked in order of wave speed from0.44 to 3.10 cm/min. The H2O2 wave tracks the concomitant surface potential wave measured electrochemically for the series of plants. We show that the plant NADPH oxidase RbohD, glutamate receptor-like channels (GLR3.3 and GLR3.6) are all critical to the propagation of the H2O2 waveform upon wounding. Our findings highlight the utility of a new type of nanosensor probe that is species-independent and capable of real-time, spatial and temporal biochemical measurements in planta.