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Introduction

The major goal of biochips is their use for rapid and economical characterization of biological samples. We have devoted considerable effort to the development of novel bio-chip systems, which are urgently required in proteome research. We have proposed a novel biochip-concept for a protein detection system involving labeled structured peptides as capture molecules and the ‘protein-fingerprint’ method, which affords a barcode-like visualization, while the protein-protein interaction can be mimicked by a protein-peptide interaction [Reviewed in 1, 2, 3]. The novel chip, designated PepTenChip&reg, satisfies all these requirements for protein detection, specificity, reproducibility, sensitivity, easy handling, stability (storage/transport) and production economics. The novel chip material made from amorphous carbon has been developed, which has significant advantages over conventional glass slides. In practice our system has been shown to discriminate the structures of a number of different proteins. Important factors for peptide micro-arrays are the construction of peptide libraries, the use of chip materials with a suitable surface chemistry, the deposition of peptide solutions by an arrayer, detection and data mining. We have achieved several applications of the PepTenChip&reg [4, 5]. Although PepTenChip® and related products are get into the market since 2010, optimization and further improvement are continuously carrying out.

1. Nokihara, K. et.al., Kobunsi Ronbunshu, 61, pp 523, 2004.(in Japanese)
2. Nokihara, K. et.al.,Solid-Phase Synthesis & Combinatorial Chemical Libraries 2004, Epton, R. ed.; Mayflower Scientific, UK, pp 83, 2004
3. Nokihara, K., Future Materials, 6, 42, 2006, (Review in Japanese)
4. Nokihara, K et. al., Peptide Science 2007, Aimoto, S., Ono, S.eds.; Japanese Peptide Society, pp106, 2008.
5. Kawasaki, T., Ohyama, T., Hirata, A. and Nokihara, K., Bull. Chem. Soc. Jpn., 83, 799-801. 2010.

Capture molecules

Arrayed molecules of PepTenChip&reg are designed peptides, which had been synthesized, purified and characterized one by one by the highly efficient manner in improved SPPS. These are several hundreds of labeled structured peptides of which secondary structures are confirmed by CD spectra. Hence the SPOT syntheses can not be employed, since the quality assurance is very important for quantitative analyses and reproducibility in biodetection. In fact one of the most important issues in bio-chips for protein detection is quantitative analysis.

Novel material for PepTenChip®

The novel chip material made from amorphous carbon has been developed, which has significant advantages over conventional glass slides, those are:

  1. mechanically more stable
  2. chemically inert
  3. shows no self-fluorescence
  4. easy manufactured by laser drawing, thus micro-channel effects can be easily maximized
  5. environmentally friendly (regeneration is easier)
  6. higher thermal conductivity and higher electro-conductivity (heating & cooling can be easily performed, and can be used for electrochemical reactions)

The grinding flatness is < 10 micron that has been performed by hard-disk technology. The surface chemistry which we have developed (patents pending) allows extremely low back ground and non-specific adsorption, uniform distribution of functional groups on the plate surface and their concentration on the surface is much higher than conventional materials, thus immobilization is much easier. Ion analysis using a detector of electric conductivity has been developed to confirm the amounts of functional group on the surface (patents applied for). This is indispensable for quality control of each bio-chip and is important as conventional X-ray photoelectron spectroscopy data do not correlate directly with reaction stoichiometry. The results revealed that the loading amounts on the present amino-carbon plate were ca. 40 pmol/㎟. Commercial slide glass has significant nonspecific absorption and not allowed to determine precisely. Together with present improved surface technology the arraying conditions have also been successfully optimized to give at least 10-20 amounts of immobilized peptides than previously. To improve the spot formation tedious protocols have been proposed (metal ions or detergent was used as additives), since microarray spots often exhibit ring-like structures, which produce difficulties in quantitative characterization. The PepTenChipR gives uniformed spot-distribution using the improved surface technology. The basic chemical modification of our surface is through an amino group and further derivatization to carboxyl group, bromoacetyl group, succinimide ester, maleimide or biotin-streptavidin is easily achieved. Thus, the present material with special surface processing technologies provides excellent possible applications for bio-chips, sample trays for micro analyzers such as MALDI-TOF, ultra micro/nano-plates (assay), and micro reactors.

PepTenChip®Applications

The present paper describes the construction of structured peptide and glycopeptide libraries and the development of the novel chip-material using amorphous carbon with a special surface chemistry. Fluorescently labeled a-helical, b-loop and b-strand peptides (total number of ca. 2500 and ca. 100 O-glycosylated peptides) have been successfully prepared by improved solid-phase syntheses and characterization. The peptide solution (350 pico Liter), which contained Cys-residues for immobilization on the chip-surface, was deposited to form a ca. 100 micron spot on a novel material described below by a Piezorray?. Amorphous carbon plates were manufactured which are mechanically stable, chemically inert, none self-fluorescent, easily manufactured for laser drawing, and of high thermal and electro-conductivity. Our surface chemistry allows extremely low background and non-specific adsorption, uniform distribution of functional groups on the plate surface and a much higher concentration on the surface than conventional materials, thereby making immobilization much easier. The results revealed that the loading amounts on the present amino-carbon plate were ca. 40 pmol/㎟.
Bio-detection of several toxicant proteins has been achieved as an application of O-glycopeptides libraries. We demonstrate here that toxicant proteins show different responses to different glycopeptides using the present chip in a dose dependent manner and finger prints can be obtained against such toxicant proteins in solution using titer plates to give a similar detection range to that of conventional ELISA. In fact, solution assay requires larger amounts of arrayed peptides and analytes. We have constructed a bio-chip using the above plates. At the present time the optimized amounts of arrayed peptides on a chip was 9 femto mole and toxicant protein as an analyte was ca. 20ng. The assay has also been performed in the presence of 2% milk to simulate practical conditions. These results suggested that glycopeptide arrays show promising applications as a toxin detection tool.

1. Nokihara, K. et.al., Kobunsi Ronbunshu, 61, pp 523, 2004. (in Japanese)
2. Nokihara, K. et.al.,Solid-Phase Synthesis & Combinatorial Chemical Libraries 2004, Epton, R. ed.; Mayflower Scientific, UK, pp 83, 2004
3. Nokihara, K., Future Materials, 6, 42, 2006, (Review in Japanese)
4. Nokihara, K et. al., Peptide Science 2007, Aimoto, S., Ono, S. eds.; Japanese Peptide Society, pp106, 2008.
5. Kawasaki, T., Ohyama, T., Hirata, A. and Nokihara, K., Bull. Chem. Soc. Jpn., 83, 799-801. 2010.
6. Nokihara, K., et. al., Peptide Science 2008, ed. Nomizu, M., Japanese Peptide Society, pp95, 2009.
7. Nokihara, K., et. al., Peptide Science 2009, Okamoto, K., ed.; Japanese Peptide Society, pp337, 2010.

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