Abstract
Nanopores are a versatile technique for the detection and characterization of single molecules in solution. An ongoing challenge in the field is to find methods to selectively detect specific biomolecules. In this work we describe a new technique for sensing specific proteins using unmodified solid-state nanopores. We engineered a double strand of DNA by hybridizing nearly two hundred oligonucleotides to a linearized version of the m13mp18 virus genome. This engineered double strand, which we call a DNA carrier, allows positioning of protein binding sites at nanometer accurate intervals along its contour via DNA conjugation chemistry. We measure the ionic current signal of translocating DNA carriers as a function of the number of binding sites and show detection down to the single protein level. Furthermore, we use DNA carriers to develop an assay for identifying a single protein species within a protein mixture.
Highlights
Nanopores have emerged to become an important tool in biophysics and single molecule sensing
Stochastic binding of a protein to a ligand attached to the entrance of a biological nanopore has been used extensively as a method for detection of folded proteins.[3−6] Alternatively several strategies based on stochastic blocking of α-hemolysin by single stranded DNA−protein complexes have been reported[7] and some proteins can be detected by their effect on current−voltage curves due to binding to biological pores.[8]
We refer to the double stranded DNA formed in this way as a “DNA carrier” since we use it to selectively drive protein molecules through a solid-state nanopore
Summary
Nanopores have emerged to become an important tool in biophysics and single molecule sensing. Biological pores used for nanopore sensing have typical diameters on the order 1−2 nm, which limits the range of analytes that will freely translocate. Stochastic binding of a protein to a ligand attached to the entrance of a biological nanopore has been used extensively as a method for detection of folded proteins.[3−6] Alternatively several strategies based on stochastic blocking of α-hemolysin by single stranded DNA−protein complexes have been reported[7] and some proteins can be detected by their effect on current−voltage curves due to binding to biological pores.[8] Denaturation by chemical[9] or thermal[10] means has been used to unfold globular proteins and permit studies of their translocations through α-hemolysin. Techniques of unfolding using high mechanical force with oligonucleotide tethers[11,12] or unfoldase[13,14] enzymes have been developed which provide new avenues for biological nanopore based protein detection
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
