Single-Molecule Detection of Protein Efflux from Microorganisms using Synthetic Fluorescent Nanosensors
Linda Chio1, Darwin Yang1, Markita P. Landry1, 2
1 Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, California 94720
2 California Institute for Quantitative Biosciences (qb3), University of California-Berkeley, Berkeley, CA 94720
Protein secretion can be detected with SWNT sensor-functionalized microfluidic device. a) A single E. coli cell engineered to secrete Rap1 protein is immobilized in an agarose matrix above a monolayer of sensors for Rap1 protein. Induction with aTcprompts secretion of Rap1, detected from b) dividing and c) non-dividing E. coli cells in real-time. Rap1-secreting engineered d) HEK293 and 3) yeast cells can be placed directly atop sensor surface, and Rap1 protein secretion can be tracked. In all images, red represents near-infrared signal of SWNT, indicative of Rap1 protein detection from cells for up to 75 minutes post-induction.
A distinct advantage of nanosensor arrays is their ability to achieve ultra-low detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins secreted from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabeled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show the process of E. coli induction, protein synthesis, and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.