Coupling stimuli-responsive magnetic nanoparticles with antibody-antigen detection in immunoassays.

Because current homogeneous immunoassays show some limitations, particularly low sensitivity, we developed a new immunoassay to overcome these limitations. The approach was based on magnetic nanoparticles with a thermoresponsive polymer layer, a negatively charged polymer, and streptavidin-biotin-based antibody-antigen detection and yielded higher sensitivity than commonly used heterogeneous immunoassays. Because no special equipment is needed, it can be applied to currently available absorbance-based systems for high-throughput assays.

Effects of conditions for preparing nanoparticles composed of aminoethylcarbamoyl-beta-cyclodextrin and ethylene glycol diglycidyl ether on trap efficiency of a guest molecule.

Nanoparticles comprising copolymers of aminoethylcarbamoyl-beta-cyclodextrin (AEC-beta-CD) and ethylene glycol diglycidyl ether (EGDGE) are prepared by an interfacial polyaddition reaction in a miniemulsion system. Polymers are formed in a W/O emulsion containing 0.25-10.0% (w/w) water and 5.0% (w/w) surfactant (MO-3S, tetraglycerin monoester, HLB 8.8), where simple particles are predominantly obtained when the water content is 1.0% and 5.0%. Notably, nano-size small particles (diameter: 0.3 microm) are formed under the condition of 5.0% water and 5.0% surfactant, which have the highest beta-CD contents (75.5 wt.%) and the most positive zeta-potential (53.6 mV). The zeta-potential measurement indicates that the obtained particles have positive charge due to protonation of their amino groups below around pH 10. Actually, uptake of 8-anilino-1-naphthalenesulfonic acid (ANS) bearing negative charge (SO(3)(-)) and moderate hydrophobicity depends on the magnitude of zeta-potential of the particles; viz., the particles with zeta-potential of 53.6 mV show the highest efficiency of uptake. The diameter and the beta-CD contents are closely related with the water/surfactant ratio, and the zeta-potentials are dependent on both the diameter and the beta-CD contents. Inclusion of ANS into the CD cavity of EGDGE/AEC-beta-CD particles can be controlled by electrostatic interaction between ANS (negatively charged) and the particle (positively charged). Namely, synergistic effect of cavity-inclusion and electrostatic interaction can dominate the uptake of guest molecules by the particles.

Motor protein nano-biomachine powered by self-supplying ATP.

A new nano-biomachine has been created from microtubules (MTs) and hetero-bifunctional polymer particles bearing pyruvate kinase, which is propelled on glass surfaces coated with kinesin by use of self-supplying ATP.

Rapid binding of concanavalin A and maltose-polyrotaxane conjugates due to mobile motion of alpha-cyclodextrins threaded onto a poly(ethylene glycol).

Maltose-polyrotaxane conjugates (Mal-PRXs), in which maltose-conjugated alpha-cyclodextrins (alpha-CDs) are threaded onto a poly(ethylene glycol) (PEG) chain capped with benzyloxycarbonyl-L-tyrosine, were characterized in terms of their molecular motion and the relation to multivalent interactions between the maltose moiety and concanavalin A from Canavalia ensiformis (Con A). Spin-lattice relaxation time (T1) and spin-spin relaxation time (T2) of alpha-CD C(1), maltosyl C(1), and PEG methylene protons in the Mal-PRXs revealed that the mobile motion of alpha-CDs in the polyrotaxane governed the molecular motion of maltosyl groups in alpha-CDs and threading PEG chain. The association constant (Ka) of the Mal-PRXs with 22, 38 and 53% of alpha-CD threading was 5.7 x 10(4), 1.1 x 10(6), and 5.3 x 10(5) (M(-1)-maltose), respectively. The largest Ka value of the Mal-PRX with 38% of alpha-CD threading was well correlated with the T1 and T2 values of maltosyl groups and alpha-CD, suggesting that the mobile motion of maltose-conjugated alpha-CDs in the Mal-PRX contributes to the highest affinity with Con A. Initial rate of binding with Con A was also governed by the mobile motion of maltose-conjugated alpha-CDs. Therefore, we concluded that both highly molecular motion due to the mobile motion of maltose-conjugated alpha-CDs and multivalency of the Mal-PRXs contributes to inducing rapid Con A binding.

Controlling the mechanism of trypsin inhibition by the numbers of alpha-cyclodextrins and carboxyl groups in carboxyethylester-polyrotaxanes.

Carboxyethylester-polyrotaxanes (CEE-polyrotaxanes) with the various number of CEE-modified alpha-cyclodextrins (CEE-alpha-CDs) were synthesized, and the effects of the number of CEE-alpha-CDs on calcium binding and trypsin inhibition were investigated. Calcium binding affinity was dependent on the density of the CEE groups accompanied with the number of alpha-CD threading in the CEE-polyrotaxanes. The high number of CEE-alpha-CDs leads to greater inhibition of trypsin activity than poly(acrylic acid), which is mainly due to the good calcium binding affinity. The CEE-polyrotaxane with the smallest number of CEE-alpha-CDs temporally interacted with trypsin, which was well correlated with the inhibition and recovery of trypsin activity. Therefore, the number of CEE-alpha-CDs in the CEE-polyrotaxanes can control the inhibition mechanism of trypsin activity.

Effect of the mobility of ligands in polyrotaxanes on order structure of water clusters.

The effect of the mobility of ligands (maltose groups) in the polyrotaxanes (pRXs) on the structure of the surrounding water molecules was investigated. Raman spectra of collective OH stretching vibration of water molecules in aqueous solutions of maltose-pRX conjugates with different alpha-cyclodextrin (alpha-CD) threading on a poly(ethylene glycol) (PEG) chain was measured. The mobility of maltose groups was estimated by measuring the relaxation time T2 of the C1 protons in maltose groups bound on alpha-CD by NMR experiment. A positive correlation between the Raman intensity of the collective band and the relaxation time T2 was obtained. This result indicates that the degree of order of the water clusters is higher as the mobility of maltose groups increases in these conjugate solutions. It is suggested that rapid motion of maltose groups in the pRX conjugate can contribute to preserving ordered structure of the bulk water clusters.

Supramolecular design for multivalent interaction: maltose mobility along polyrotaxane enhanced binding with concanavalin A.

High molecular mobility of maltose-conjugated alpha-cyclodextrins (alpha-CDs) along a poly(ethylene glycol) (PEG) chain due to the mechanically locked structure of polyrotaxanes enhanced multivalent interactions between maltose and concanavalin A (Con A). When maltose groups are conjugated with alpha-CDs that were threaded onto a PEG capped with benzyloxycarbonyl l-tyrosine (polyrotaxane), Con A-induced hemagglutination was greatly inhibited by polyrotaxanes with a certain threading % of alpha-CDs. Such an inhibitory effect was significantly superior to the other type of conjugates, in which poly(acrylic acid) was used as a backbone for maltose conjugation. The spin-spin relaxation time (T2) of the maltose C(1) proton in the polyrotaxane at a typical alpha-CD threading % was significantly larger than that of any other conjugate, which was well related to the inhibitory effect. Therefore, we concluded that the high mobility of maltose groups along the polyrotaxane structure contributes to enhanced Con A recognition.

Carboxyethylester-polyrotaxanes as a new calcium chelating polymer: synthesis, calcium binding and mechanism of trypsin inhibition.

A carboxyethylester-polyrotaxane was synthesized as a novel calcium chelating polymer in the field of oral drug delivery and characterized in terms of mechanism of trypsin inhibition. Here, carboxyethylester (CEE) groups are introduced to all the primary hydroxyl groups in alpha-cyclodextrins (alpha-CDs), which are threaded onto a poly(ethylene glycol) chain capped with bulky end-groups (polyrotaxane). The solubility of the CEE-polyrotaxane in physiological conditions increased with pH, indicating ionization-related solubility similar to conventional polyacrylates. The ability of calcium (Ca2+) chelation was found to increase in the order of poly(acrylic acid) (PAA)>CEE-polyrotaxanez.Gt;CEE-alpha-CD, suggesting that the increased density of carboxyl groups enhances the Ca2+ chelating ability. The activity of trypsin was inhibited by these compounds in the same order of the calcium chelation. However, the inhibitory effect of CEE-polyrotaxane was reduced by adding excess Ca2+ without precipitation that was observed in the presence of PAA. Such the reduced inhibition and precipitation by CEE-alpha-CD was not observed. Therefore, the inhibitory effect of CEE-polyrotaxane is due to Ca2+ chelation from trypsin without non-specific interaction.