title: Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays.
abstract: A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins 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. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, California 94720, USA.|California Institute for Quantitative Biosciences (qb3), University of California-Berkeley, Berkeley, California 94720, USA.
authors.forename: Markita Patricia
authors.initials: MP
authors.lastname: Landry
authors.affiliation: Department of Electrical Engineering &Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.|MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ando
authors.affiliation: Department of Electrical Engineering &Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.|MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.|Biophysics Program, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: Allen Y
authors.initials: AY
authors.lastname: Chen
authors.affiliation: Department of Electrical Engineering &Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.|MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Jicong
authors.initials: J
authors.lastname: Cao
authors.affiliation: The Rowland Institute at Harvard University, Cambridge, Massachusetts 02142, USA.|Department of Biology, The Catholic University of America, Washington, District of Columbia 20064, USA.
authors.forename: Vishal Isaac
authors.initials: VI
authors.lastname: Kottadiel
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, California 94720, USA.
authors.forename: Linda
authors.initials: L
authors.lastname: Chio
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, California 94720, USA.
authors.forename: Darwin
authors.identifier: 0000-0002-6839-1955
authors.initials: D
authors.lastname: Yang
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02138, USA.
authors.forename: Juyao
authors.initials: J
authors.lastname: Dong
authors.affiliation: Department of Electrical Engineering &Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.|MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Timothy K
authors.initials: TK
authors.lastname: Lu
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02138, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Strano
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D057230:Limit of Detection; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D037742:Nanotubes, Carbon / Q000737:chemistry*; D010843:Pichia / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D037742:Nanotubes, Carbon; D011506:Proteins