LI ET AL. VOL. 7 ’ NO. 5 ’ 4129–4134 ’ 2013 www.acsnano.org 4129 April 22, 2013 C 2013 American Chemical Society Single Protein Molecule Detection by Glass Nanopores Wenhong Li, †,§ Nicholas A. W. Bell, †,§ Silvia Herna ´ ndez-Ainsa, † Vivek V. Thacker, † Alana M. Thackray, ‡ Raymond Bujdoso, ‡ and Ulrich F. Keyser †, * † Cavendish Laboratory, University of Cambridge, J.J. Thomson Avenue, Cambridge CB3 0HE, United Kingdom, and ‡ Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, United Kingdom. § These authors contributed equally. N anopores are a promising tech- nique for single molecule detection in solution. The premise of using nanopores for sensing is founded on the resistive-pulse technique, 1 which offers label- free detection and identification of single analytes in aqueous environments. The first application of resistive-pulse sensing for particle analysis was the Coulter Counter for detection of cells. 2 Subsequently, the technique has been expanded to include detection of particles such as pollen, viruses, or colloids coated with antibodies. 3À5 With a decreasing size of the detection pore, nano- meter scale objects have been detected, such as organic polymers, peptides, proteins and DNA. 6,7 Single protein molecules have been de- tected and their properties have been inves- tigated using various types of nanopores. Biological nanopores, such as R-hemolysin or aerolysin, have been used collectively to detect small and unfolded proteins, 8À11 study the structure of peptides, 12 analyze peptideÀ antibody interactions, 13 and measure selfÀ self peptide aggregation. 14 Biological nano- pores have the advantage of a well-defined geometry but have a limitation in that the protein under analysis must be unfolded in order to translocate through the nanopore orifice, which is typically 1À2 nm in diameter. Solid state nanopores, which are more stable than biological nanopores, have also been used for the detection and analysis of single protein molecules. 14À19 Nanopores in a silicon nitride membrane have been used to enable detection of bovine serum albumin, 15,16 the folding states of β-lactoglobulin, 17 and to study proteinÀprotein interactions. 18 With a PET membrane coated with gold nanotubes, one can distinguish BSA, phosphorylase B, and β-galactosidase by amplitude-duration scatter plots. 19 A lipid layer coating on a solid state nanopore can slow down protein trans- portation and eliminate nonspecific binding to the nanopore. 20 Positioning of a binding site in the nanopore gives speci ficity to pro- tein measurements and enables determina- tion of binding kinetics. 21 Glass capillaries have emerged as an alternative to both bio- logical and silicon-based nanopores. They can be produced with orifice diameters ranging from micrometers to nanometers, with the advantages of ease of manufacture and rela- tively low cost. Glass nanopores have been successfully used to detect different biological molecules such as λ-DNA. 22À25 Here, we show the ability of glass nanopores to detect a range of proteins of di fferent mo- lecular masses, including lysozyme (14 kDa), avidin (66 kDa), bovine β-lactoglobulin (18 kDa), ovalbumin (42 kDa), BSA (66 kDa), rabbit im- munoglobulin G (IgG) (150 kDa), and β-galac- tosidase (465 kDa). In addition, we show for * Address correspondence to ufk20@ cam.ac.uk. Received for review January 28, 2013 and accepted April 10, 2013. Published online 10.1021/nn4004567 ABSTRACT Nanopores can be used to detect and analyze single molecules in solution. We have used glass nanopores made by laser-assisted capillary-pulling, as a high- throughput and low cost method, to detect a range of label-free proteins: lysozyme, avidin, IgG, β-lactoglobulin, ovalbumin, bovine serum albumin (BSA), and β-galactosidase in solution. Furthermore, we show for the first time solid state nanopore measurements of mammalian prion protein, which in its abnormal form is associated with transmissible spongiform encephalopathies. Our approach provides a basis for protein characterization and the study of protein conformational diseases by nanopore detection. KEYWORDS: nanopores . nanocapillary . protein translocation . PrP . prions . single molecule detection . diagnosis ARTICLE