Bioengineering Laboratory

Outline

The principal purpose of our laboratory is to explore a new frontier of research that fuses engineering and biological science. On the basis of polymer chemistry, analytical chemistry, surface chemistry, biochemistry and molecular biology, we are studying new materials comprising biological components, novel methodology for bioanalysis and medical diagnosis and artificial systems for regulation of biological processes. These new ideas and materials are being applied to the field of biomaterials science, environmental science, micro/nanoscience, life science and medical engineering. For example, we have prepared novel DNA-vinyl polymer conjugates which have been applied to DNA biosensors, affinity electrophoresis, SNP-responsive diagnostic nanoparticles, gene-responsive hydrogels, artificial gene regulation systems, etc. This research may be classified into a new category called DNA engineering.

Other ongoing research subjects include (1) unique colloidal properties of DNA-carrying nanoparticles, (2) biochemical assays and single-cell manipulations on power-free microfluidic devices, (3) adsorption mechanisms of biopolyester-degrading enzymes, (4) amyloid formation with molecular chaperones and (5) observation of protein-protein interactions using single-molecule imaging. Our general policy implicit in each research subject is the creation of innovative functionality from well-defined interfaces made of various biomolecules.

Recent Research Topic

Bioanalytical systems using DNA nanoparticles

Fig. 1 Schematic illustrations of double-stranded DNA-carrying nanoparticles

(left: perfect match, right: terminal single-base mismatch)

We have prepared DNA nanoparticles with a nanometer-sized vinyl polymer core or a colloidal gold core surrounded by a single-stranded DNA corona. The DNA nanoparticles disperse completely in an aqueous medium due to electrostatic repulsion between anionic phosphate groups in the DNA backbone. Interestingly, when complementary single-stranded DNA, whose base number is identical to that of the DNA on the surface, is added to the dispersion of DNA nanoparticles to form the fully matched double helix on the surface, the DNA nanoparticles become unstable and spontaneously form aggregates in a non-crosslinking manner. This aggregation is detectable by the naked eye as turbidity change when using DNA-carrying polymeric nanoparticles or as a color change from red to purple induced by surface plasmon resonance (SPR) shift when using DNA-carrying gold nanoparticles. Furthermore, we have found that the double-stranded DNA-carrying nanoparticles acquire high colloidal stability for dispersal in an aqueous medium when a terminal single-base mismatch exists at the interface between the DNA corona and the dispersal medium.

The underlying principle in the unique colloidal behaviors, which should be described by the physicochemical properties of the DNA corona on the surface of the nanoparticles, still remains unclear. We have been performing structural analyses of DNA on nanoparticle surfaces with NMR, zeta-potential measurements of the DNA nanoparticles, and the structural analysis of aggregates with small-angle X-ray scattering measurements at SPring-8. It has been suggested that change in the colloidal stability of the DNA nanoparticles may be at least partially attributed to DNA structure-dependent entropic repulsion (steric destabilization).

Exploiting the unique colloidal behavior of the DNA nanoparticles, we have devised a facile single-nucleotide polymorphism genotyping method. We applied the SPR imaging technique on our original, power-free microfluidic devices for the detection of the nanoparticles aggregation; the limit of detection was greatly improved down to 19 fmol. Through a combination of non-crosslinking aggregation of DNA nanoparticles and molecular recognition by aptamers or aptazymes, we have also developed analytical systems for detecting cGMP, ATP, FMN, 8-oxoG, theophyllin, Hg(II) and Ag(I) with the naked eye. In addition, using self-cleavage reactions catalyzed by various aptazymes, we have succeeded in constructing nanometer-sized logic gate elements (AND and OR gates), wherein the inputs are various small biomolecules as the cofactors of aptazymes and the outputs are colorimetric changes induced by the non-crosslinking aggregation of the DNA-carrying gold nanoparticles.

Fig. 2 Turbidimetric detection of single-base difference DNA using DNA-carrying polymeric nanoparticles

(left: single-base-substituted DNA, right: normal DNA)

Fig. 3 Colorimetric detection of Hg(II) ions using DNA-carrying gold nanoparticles