Label-free optical biosensors have attracted much attention, and nanoporous metal-oxide membranes with uniform pore structure and diameter are promising candidates for platforms of label-free optical biosensors. However, development of such sensors with high sensitivity still remains challenging. In this paper, we report on the remarkably enhanced sensitivity of a label-free nanoporous optical waveguide (NPWG) sensor composed of a porous anodic alumina (PAA) waveguiding film and an aluminum cladding film. The enhanced sensitivity was achieved by engineering nanostructures and tuning optical properties of the PAA film. Careful tuning of the porosity, pore density, thickness, and refractive index of the PAA film could significantly improve the sensitivity of the NPWG sensor toward adsorption of bovine serum albumin (BSA) onto the PAA surface, and the optimized sensor responded to the adsorption of BSA with an extraordinarily large red shift (>300 nm) of a waveguide mode due to the large adsorption capacity of the PAA film and the inherently high sensitivity of the waveguide mode. The Fresnel calculations suggested that the potential sensitivity of the NPWG sensor was much higher than that of the conventional surface plasmon resonance (SPR) sensors.Keywords: biosensing; label-free; nanopores; optical waveguide sensor; porous anodic alumina

One-dimensional (1D) nanomaterials have unique applications due to their inherent physical properties. In this study, hexagonally ordered mesoporous silica hybrid anodic alumina membranes (AAM) were synthesized using template-guided synthesis with a number of nonionic n-alkyl-oligo(ethylene oxide), Brij-type (CxEOy), which are surfactants that have different molecular sizes and characteristics. The hexagonal mesoporous silicas are vertically aligned in the AAM channels with a predominantly columnar orientation. The hollow mesostructured silicas had tunable pore diameters varying from 3.7 to 5.1 nm. In this synthesis protocol, the surfactant molecular natures (corona/core features) are important for the controlled generation of ordered structures throughout AAM channels. The development of ultrafiltration membranes composed of silica mesostructures could be used effectively in separating silver nanoparticles (Ag NPs) in both aqueous and organic solution phases. This would be relevant to the production of well-defined Ag NPs with unique properties. To create a size-exclusive separation system of Ag NPs, we grafted hydrophobic trimethylsilyl (TMS) groups onto the inner pores of the mesoporous silica hybrid AAM. The immobilization of the TMS groups allowed the columnar mesoporous silica inside AAM to retain this inner pore order without distortion during the separation of solution-phase Ag NPs in organic solvents that may cause tortuous-pore membranes. Mesoporous TMS-silicas inside 1D AAM channels were applicable as a size-exclusive separation system to isolate organic solution-phase Ag NPs of uniform morphology and size.Graphical abstractSize-exclusion separation of Ag NPs using hybrid membranes.Research highlights► Highly ordered mesoporous silicas hybrid AAM membranes were successfully fabricated through a template-guided method with a number of Brij-type surfactants. ► The mesoporous silicas are vertically aligned in the AAM channels, with a predominantly columnar orientation. ► The membranes show promise as a size-exclusive separation system in isolating Ag NPs in both aqueous and organic solution phases. ► The development of size-exclusive membranes composed of mesostructures would be indispensible for the production of uniform Ag NPs with unique properties.

We report a protocol for the direct synthesis of hexagonal silica nanostrands inside anodic alumina membranes using cationic surfactants as templates. When coated with layers of trimethylsilyl moieties, the nanostrands were a powerful tool for the ultrafine filtration of noble metal and semiconductor nanoparticles.

A metal-clad waveguide (MCWG) sensor comprised of a nanoporous waveguiding layer on a metal cladding layer is advantageous in sensing of biomolecules because of a high surface area of nanopores and a sharp dip in the reflection spectrum due to characteristics of the MCWG mode. Here, a porous anodic alumina (PAA)/aluminum (Al) film was fabricated on a glass substrate as a MCWG sensor with the Kretschmann geometry, and the sensor response was examined for both colorless bovine serum albumin (BSA) and colored metal complexes by measurements of reflection spectra and Fresnel calculations. The BSA adsorption on the PAA layer induced a parallel redshift of the waveguide coupling dip in the reflection spectrum. The experimental results were well simulated by the five-phase Fresnel calculations which indicated that the redshift of the dip was linearly dependent on the adsorbed amount of BSA. When the response of a MCWG sensor with a PAA layer was compared with that of a MCWG sensor with a nonporous alumina layer, the former showed larger redshift than the latter, due to a large adsorbed amount of BSA in the PAA layer with high surface area. For the adsorption of colored Ru[Bphen3]2+ and Fe[Phen3]2+, the effect of both the real and imaginary parts of the complex refractive index on the sensor response was examined. As a result, a redshift of the waveguide coupling dip was observed for both metal complexes irrespective of the wavelength region examined; this could be ascribed to the changes in the real part of the refractive index due to the adsorption of metal complexes on the PAA layer. Meanwhile, an increase in the reflectivity was observed when the coupling wavelength was close to that of the absorption bands of the metal complexes; this could be ascribed to the changes in the imaginary part of the refractive index of the PAA layer. Using the sensor response caused by the changes in the imaginary part, absorption spectral profiles of metal complexes could be reproduced.

By using iodide (I−) as a quencher, we successfully improve the fluorescence response of amiloride when binding to thymine opposite an AP site in a 21-meric DNA duplex. From fluorescence measurements, as compared to the NaCl solutions, the addition of NaI as a quencher as well as salt to adjust the ionic strength effectively suppresses the background fluorescence from unbound amiloride in a solution. The Stern–Volmer analysis shows that the bound amiloride to the nucleobase at the AP site is unexposed to NaI quencher. Therefore the high signal-to-background fluorescence response of amiloride is obtained. Such enhancement in fluorescence response of amiloride by using the quencher can provide the significant improvement of the detection limit for DNA duplexes carrying T target base. The method presented in this study is simple and effective. The present method could be applicable to other detection system where microenvironment of fluorophores changes at a recognition event.

A label-free adenosine sensor with emissive response is designed based on an AP site-containing aptamer/DNA duplex and a small fluorescent molecule 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND).

An optical waveguide sensor was fabricated by forming a multilayer film made by porous anodic alumina (PAA) and Al layers on a glass substrate. The fabricated sensor system was based on the monitoring of a waveguide coupling mode, which is sensitive to the change in the refractive index of the PAA layer caused by collection of target molecules into the pores of the PAA layer. The PAA/Al multilayer film was formed by partial anodization of an Al film deposited on the glass substrate, and the waveguide coupling mode was examined by measuring angular spectra (reflectivity dependence on the incident angle of monitoring light; green He−Ne laser, 534.5 nm). A deep and sharp waveguide coupling dip was obtained for the PAA/Al multilayer system where the thicknesses of the PAA and Al layers were 200 and 17 nm, respectively. The optical sensor response of the PAA/Al multilayer system was compared to the responses of a surface plasmon resonance (SPR) sensor made by a Au thin film on a SF10 glass substrate. It was inferred that the optical waveguide sensor made by the PAA/Al multilayer could detect a smaller change in the refractive index of a solution, and it provided higher resolution than the SPR sensor. The sensor response for a change in the complex refractive index of the PAA layer was examined next, and it was found that the optical waveguide sensor was sensitive to the change in the imaginary part of the complex refractive index rather than the change in the real part. This result indicated that the sensitivity of the optical waveguide sensor could be improved by using the light absorption of a target compound.

Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX. This leads to the known ligands 3-methyl isoxanthopterin (3-MIX) and 8-methyl isoxanthopterin (8-MIX), and the binding affinity for C or T is expected to be tuned and improved by methyl substitution. Indeed, 3-MIX selectively binds to T more strongly than IX with a binding constant of 1.5 × 106 M−1 and it loses its binding affinity for C. In contrast, 8-MIX selectively binds to C over T with a binding constant of 1.0 × 106 M−1 and the binding affinity is greatly improved compared to the parent ligand IX. The thermodynamics of the ligand–nucleotide interaction is analyzed by isothermal calorimetric titrations, and the results show that the interaction follows a 1:1 stoichiometry and is enthalpy-driven. The introduction of methyl groups at both N-3 and N-8 positions results in an increase in enthalpy of the ligand–nucleotide interaction, which leads to the improved binding affinity.Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX. This leads to the known ligands 3-methyl isoxanthopterin (3-MIX) and 8-methyl isoxanthopterin (8-MIX), and the binding affinity for C or T is expected to be tuned and improved by methyl substitution. Indeed, 3-MIX selectively binds to T more strongly than IX with a binding constant of 1.5 × 106 M−1 and it loses its binding affinity for C. In contrast, 8-MIX selectively binds to C over T with a binding constant of 1.0 × 106 M−1 and the binding affinity is greatly improved compared to the parent ligand IX. The thermodynamics of the ligand–nucleotide interaction is analyzed by isothermal calorimetric titrations, and the results show that the interaction follows a 1:1 stoichiometry and is enthalpy-driven. The introduction of methyl groups at both N-3 and N-8 positions results in an increase in enthalpy of the ligand–nucleotide interaction, which leads to the improved binding affinity.

The binding behavior of lumiflavin, a biologically vital ligand, with DNA duplexes containing an abasic (AP) site and various target nucleobases opposite the AP site is studied. Lumiflavin binds selectively to thymine (T) opposite the AP site in a DNA duplex over other nucleobases. Using 1H NMR spectroscopy and fluorescence measurements, we show that ligand−DNA complexation takes place by hydrogen-bond formation between the ligand and the target nucleobases and by stacking interactions between the ligand and the nucleobases flanking the AP site. From isothermal titration calorimetric experiments, we find that ligand incorporation into the AP sites is primarily enthalpy-driven. Examination of ionic strength dependency of ligand binding with DNA reveals that ligand−DNA complexation is a manifestation of both electrostatic and nonelectrostatic interactions and that the contribution from the nonelectrolyte effect is fundamental for the stabilization of the ligand−DNA complex. In comparison to riboflavin, reported previously as a T-selective ligand, lumiflavin binds to the DNA much more strongly and is a more promising ligand for efficient detection of T-related single nucleotide polymorphisms.

6,7-Dimethyllumazine more selectively binds to adenine (A) base opposite the abasic site in DNA duplexes (5′-TCC AG GCA AC-3′/3′-AGG TC CGT TG-5′, = AP site (Spacer C3), = A, T, C and G) than the other three nucleobases with a dissociation constant Kd of ca. 1.0 μM; substituted methyl groups enhance the binding affinity to A and the selectivity for A over T, compared to the parent molecule, lumazine.

Immobilization of glucose oxidase (GOD) within a hybrid mesoporous membrane with 12 nm pore diameter was successfully achieved, resulting in catalytically high efficiency during flow of a glucose solution across the membrane.

Alloxazine can bind to adenine selectively over other nucleobases opposite an abasic site in DNA duplexes (5′-TCC AG GCA AC-3′/3′-AGG TC CGT TG-5′, = AP site, = A, T, C, G) with a dissociation constant of 0.82 µM (pH 7.0, I = 0.11 M, at 5 °C), and it is applicable to SNPs typing of PCR amplification products based on the binding-induced fluorescence response.

A novel hydrogen bond-forming ligand for pyrimidine/purinetransversion, which contains both a fluorescent naphthyridine moiety and a ferrocenyl group as an electrochemical indicator, is described. Hydrogen bond-mediated recognition for a target nucleobase at an abasic site in a DNA duplex is confirmed by both fluorescence and electrochemical measurements. The analysis by fluorescence titration reveals that the ligand shows significant fluorescent quenching upon formation of a 1 : 1 complex with the target nucleobase opposite the abasic site, and the selectivity is in the order of cytosine > thymine > adenine, guanine, reflecting the stability of the hydrogen bond formation.

Solvation dynamics in alcohols confined in silica nanochannels was examined by time-resolved fluorescence spectroscopy using coumarin 153 (C153) as a fluorescent probe. Surfactant-templated mesoporous silica was fabricated inside the pores of an anodic alumina membrane. The surfactant was removed by calcination to give mesoporous silica (Cal-NAM) containing one-dimensional (1D) silica nanochannels (diameter, 3.1 nm) whose inner surface was covered with silanol groups. By treating Cal-NAM with trimethylchlorosilane, trimethylsilyl (TMS) groups were formed on the inner surface of the silica nanochannels (TMS-NAM). Fluorescence dynamic Stokes shifts of C153 were measured in alcohols (ethanol, butanol, hexanol, and decanol) confined in the silica nanochannels of Cal- and TMS-NAMs, and the time-dependent fluorescence decay profiles could be best fitted by a biexponential function. The estimated solvent relaxation times were much larger than those observed in bulk alcohols for both Cal- and TMS-NAMs when ethanol or butanol was used as a solvent, indicating that the mobility of these alcohol molecules was restricted within the silica nanochannels. However, hexanol or decanol in Cal- and TMS-NAMs did not cause a remarkable increase in the solvent relaxation time in contrast to ethanol or butanol. Therefore, it was concluded that a relatively rigid assembly of alcohols (an alcohol chain) was formed within the silica nanochannels by hydrogen bonding interaction and van der Waals force between the surface functional groups of the silica nanochannels and alcohol molecules and by the successive interaction between alcohol molecules when alcohol with a short alkyl chain (ethanol or butanol) was used as a solvent.

6,7-Dimethyllumazine more selectively binds to adenine (A) base opposite the abasic site in DNA duplexes (5′-TCC AG GCA AC-3′/3′-AGG TC CGT TG-5′, = AP site (Spacer C3), = A, T, C and G) than the other three nucleobases with a dissociation constant Kd of ca. 1.0 μM; substituted methyl groups enhance the binding affinity to A and the selectivity for A over T, compared to the parent molecule, lumazine.

Immobilization of glucose oxidase (GOD) within a hybrid mesoporous membrane with 12 nm pore diameter was successfully achieved, resulting in catalytically high efficiency during flow of a glucose solution across the membrane.

Alloxazine can bind to adenine selectively over other nucleobases opposite an abasic site in DNA duplexes (5′-TCC AG GCA AC-3′/3′-AGG TC CGT TG-5′, = AP site, = A, T, C, G) with a dissociation constant of 0.82 µM (pH 7.0, I = 0.11 M, at 5 °C), and it is applicable to SNPs typing of PCR amplification products based on the binding-induced fluorescence response.

A novel hydrogen bond-forming ligand for pyrimidine/purinetransversion, which contains both a fluorescent naphthyridine moiety and a ferrocenyl group as an electrochemical indicator, is described. Hydrogen bond-mediated recognition for a target nucleobase at an abasic site in a DNA duplex is confirmed by both fluorescence and electrochemical measurements. The analysis by fluorescence titration reveals that the ligand shows significant fluorescent quenching upon formation of a 1 : 1 complex with the target nucleobase opposite the abasic site, and the selectivity is in the order of cytosine > thymine > adenine, guanine, reflecting the stability of the hydrogen bond formation.

We report a protocol for the direct synthesis of hexagonal silica nanostrands inside anodic alumina membranes using cationic surfactants as templates. When coated with layers of trimethylsilyl moieties, the nanostrands were a powerful tool for the ultrafine filtration of noble metal and semiconductor nanoparticles.