Protein aggregation is associated with many neurodegenerative diseases. We applied mid-infrared photothermal (MIP) to dissect the secondary structure of protein aggregates in living yeast cells modeling Huntington’s disease. We validated that MIP spectroscopy could reveal β-sheet enrichment by significant spectral changes and confirmed the β-sheet enrichment of aggregates in high throughput by wide-field fluorescence-detected MIP. By label-free identification of protein aggregates, we observed a further red shift in spectra for aggregates without GFP tag. Finally, by performing MIP spectroscopy with fine spatial steps, we discovered a partition of secondary structures between β-rich core and ɑ-rich shell of the aggregates.
The standard laboratory procedure for determining the antibiotic susceptibility of a pathogen (an antimicrobial susceptibility test, AST) measures the inhibition of growth, and requires several days. This can delay effective therapy and lead to antibiotic overuse and misuse. Recent work (Wei Hou et al, Lab on a Chip 2015) has shown that resistant and susceptible pathogens will have very different gene expression profiles shortly following antibiotic exposure, and that these expression biomarkers may be used to accurately identify the pathogen species, strain and antibiotic susceptibility without growth. We therefore developed an ultrasensitive ‘digital microarray’ for performing rapid & quantitative gene expression analysis as part of a rapid AST. The digital microarray uses plasmonic gold nanorods (GNRs) functionalized with DNA to specifically label each target RNA that binds to the microarray. Each GNR on the array is then individually detected based on its light scattering, with an interferometric microscopy technique called SP- IRIS. Our optimized high-throughput version of SP-IRIS is able to scan a typical array of 500 spots in less than 10 minutes. Due to its single molecule readout, the assay has a limit of detection of less than 1 femtomolar following just 2 hours of incubation. Altogether, digital microarrays are about 10,000-fold more sensitive than fluorescence microarrays, yet maintain all of the strengths of the platform including low cost and high multiplexing. The reproducibility and robustness of the multiplexed assay will next be evaluated with clinically relevant pathogenic strains of E. coli as part of a functional rapid AST.
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