Microsc. Microanal. 23, 336–339, 2017 doi:10.1017/S1431927616012551
© MICROSCOPY SOCIETY OF AMERICA 2016
Toward the Atomic-Level Mass Analysis of Biomolecules by the Scanning Atom Probe
Osamu Nishikawa,1,* and Masahiro Taniguchi2
1Kanazawa Institute of Technology, Office of Industry-University Collaboration, 7-1 Nonoichi, Ishikawa 921-8501, Japan 2Department of Applied Chemistry, Kanazawa Institute of Technology, 7-1 Nonoichi, Ishikawa 921-8501, Japan
Abstract: In 1994, a new type of atom probe instrument, named the scanning atom probe (SAP), was proposed. The unique feature of the SAP is the introduction of a small extraction electrode, which scans over a specimen surface and confines the high field, required for field evaporation of surface atoms in a small space, between the specimen and the electrode. Thus, the SAP does not require a sharp specimen tip. This indicates that the SAP can mass analyze the specimens which are difficult to form in a sharp tip, such as organic materials and biomolecules. Clean single wall carbon nanotubes (CNT), made by high-pressure carbon monoxide process are found to be the best substrates for biomolecules. Various amino acids and dipeptide biomolecules were successfully mass analyzed, revealing characteristic clusters formed by strongly bound atoms in the specimens. The mass analysis indicates that SAP analysis of biomolecules is not only qualitative, but also quantitative.
Key words: Scanning atom probe (SAP), biomolecule, amino acid, dipeptide, carbon nano tube (CNT), binding state, quantitative-qualitative analysis
INTRODUCTION
Atom probe (AP) (Müller et al., 1968; Nishikawa et al., 1981) is a combination of an atomic resolution field ion micro- scope and amass analyzerwith single ion sensitivity. Various metallic and semiconductor specimens have been mass analyzed at the atomic level (Cerezo & Smith, 2001; Nishikawa et al., 1984; Nishikawa et al., 2003). However, organic molecules and polymers are not typically analyzed because the preparation of sharp needles of these soft materials is extremely difficult. In order to remove this barrier, a small funnel-shaped extraction electrode was introduced in the conventional AP (Nishikawa & Kimoto,
of a soft polythiophene film (Nishikawa & Taniguchi, 2005) and tetra-N-butylammonium hydroxide detecting char- acteristic clusters such as NC4H9,NC8H18, and NC12H27 (Nishikawa et al., 2006). These clusters are formed by the strongly bound atoms of the molecules. Conversely, non-directionally and uniformly bound metal atoms are field evaporated atom by atom. Thus the clusters reflect the binding states of the atoms composing molecules.
*Corresponding author.
oynishikawa@gmail.com Received June 28, 2016; accepted November 11, 2016
1994;Nishikawa et al., 2000). The electrode confines the high field required for the field evaporation of surface atoms in a small space between the apex of a minute protrusion on a flat specimen and an open hole at the sharp end of the electrode. The electrode can scan over a specimen surface as a scanning tunneling microscope (Binnig et al., 1983) and stay above the apex of a protrusion. This instrument is termed, the scanning atom probe (SAP). The extraction electrode has allowed the mass analysis
find a suitable support material. An extensive search lead to clean single walled carbon nanotubes (SWCNT) as the most suitable substrates for the biomolecules, because the mass spectra of SWCNT exhibit only single atom ions, which can be easily separated from the cluster ions of the molecules (Nishikawa et al., 2008).
For the mass analysis of biomolecules, it is necessary to EXPERIMENTAL
The design and structure of the SAP is described elsewhere (Nishikawa et al., 2008). The SAP mass analysis proceeds by applying a positive pulsed voltage, VP, of 5 ns duration to a specimen and superposing a positive DC voltage VDC. The
ratio of VP to (VDC+VP) is set at 20%. Flight times of the field evaporated ions are measured with 1 ns accuracy. Accordingly, the mass resolution m/Δm of the SAP is better than 1,000. The estimated field strength above the carbon ball is of the order of 10–20 V/nm. As specimen atoms are field evaporated by nanosecond
voltage pulses, conductive specimens are desirable for the SAP analysis. However, biomolecules are not conductive. Thus, the molecules are deposited on a conductive substrate. In order to avoid the catalytic reaction of metals and semi- conductors, carbon is assumed to be a suitable substrate. For the present study, SWCNTs produced by a high-pressure carbon monoxide process are employed because of their cleanliness. Solutions of the amino acids to be analyzed, were made by
dissolving 1g of the amino acid in 10–30mLof ultra-pure water. Solutionsofpeptidesweremadebydissolving76mgof Glu-Glu (a-L-glutamyl-L-glutamicacid) in 50mL of 0.1N
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