Biomimetic Stress Sensitive Hydrogel Controlled by DNA Nanoswitches.

One of the most intriguing and important aspects of biological supramolecular materials is its ability to adapt macroscopic properties in response to environmental cues for controlling cellular processes. Recently, bulk matrix stiffness, in particular, stress sensitivity, has been established as a key mechanical cue in cellular function and development. However, stress-stiffening capacity and the ability to control and exploit this key characteristic is relatively new to the field of biomimetic materials. In this work, DNA-responsive hydrogels, composed of semiflexible PIC polymers equipped with DNA cross-linkers, were engineered to create mimics of natural biopolymer networks that capture these essential elastic properties and can be controlled by external stimuli. We show that the elastic properties are governed by the molecular structure of the cross-linker, which can be readily varied providing access to a broad range of highly tunable soft hydrogels with diverse stress-stiffening regimes. By using cross-linkers based on DNA nanoswitches, responsive to pH or ligands, internal control elements of mechanical properties are implemented that allow for dynamic control of elastic properties with high specificity. The work broadens the current knowledge necessary for the development of user defined biomimetic materials with stress stiffening capacity.

On/off-switchable LSPR nano-immunoassay for troponin-T.

Regeneration of immunosensors is a longstanding challenge. We have developed a re-usable troponin-T (TnT) immunoassay based on localised surface plasmon resonance (LSPR) at gold nanorods (GNR). Thermosensitive poly(N-isopropylacrylamide) (PNIPAAM) was functionalised with anti-TnT to control the affinity interaction with TnT. The LSPR was extremely sensitive to the dielectric constant of the surrounding medium as modulated by antigen binding after 20 min incubation at 37 °C. Computational modelling incorporating molecular docking, molecular dynamics and free energy calculations was used to elucidate the interactions between the various subsystems namely, IgG-antibody (c.f., anti-TnT), PNIPAAM and/or TnT. This study demonstrates a remarkable temperature dependent immuno-interaction due to changes in the PNIPAAM secondary structures, i.e., globular and coil, at above or below the lower critical solution temperature (LCST). A series of concentrations of TnT were measured by correlating the λLSPR shift with relative changes in extinction intensity at the distinct plasmonic maximum (i.e., 832 nm). The magnitude of the red shift in λLSPR was nearly linear with increasing concentration of TnT, over the range 7.6 × 10-15 to 9.1 × 10-4 g/mL. The LSPR based nano-immunoassay could be simply regenerated by switching the polymer conformation and creating a gradient of microenvironments between the two states with a modest change in temperature.

Studies on an on/off-switchable immunosensor for troponin T.

Regeneration is a key goal in the design of immunosensors. In this study, we report the temperature-regulated interaction of N-isopropylacrylamide (PNIPAAm) functionalised cardiac troponin T (cTnT) with anti-cTnT. Covalently bonded PNIPAAm on an anti-cTnT bioelectrode showed on/off-switchability, regeneration capacity and temperature triggered sensitivity for cTnT. Above the lower critical solution temperature (LCST), PNIPAAm provides a liphophilic microenvironment with specific volume reduction at the bioelectrode surface, making available binding space for cTnT, and facilitating analyte recognition. Computational studies provide details about the structural changes occurring at the electrode above and below the LCST. Furthermore, free energies associated with the binding of cTnT with PNIPAAm at 25 (ΔGcoil=-6.0 Kcal/mole) and 37 °C (ΔGglobular=-41.0 kcal/mole) were calculated to elucidate the interaction and stability of the antigen-antibody complex. The responsiveness of such assemblies opens the way for miniaturised, smart immuno-technologies with 'built-in' programmable interactions of antigen-antibody upon receiving stimuli.

Fabrication of a tunable glucose biosensor based on zinc oxide/chitosan-graft-poly(vinyl alcohol) core-shell nanocomposite.

A potentiometrically tuned-glucose biosensor was fabricated using core-shell nanocomposite based on zinc oxide encapsulated chitosan-graft-poly(vinyl alcohol) (ZnO/CHIT-g-PVAL). In a typical experiment, ZnO/CHIT-g-PVAL core-shell nanocomposite containing <20 nm ZnO nanoparticles was synthesized using wet-chemical method. The glucose responsive bio-electrode, i.e., glucose oxidase/ZnO/chitosan-graft-poly(vinyl alcohol) (GOD/ZnO/CHIT-g-PVAL/ITO) was obtained by immobilization of glucose oxidase (GOD) onto the electrode made of resulting ZnO core-shell nanocomposite coated on the indium-tin oxide (ITO) glass substrate. The ZnO/CHIT-g-PVAL/ITO and GOD/ZnO/CHIT-g-PVAL electrodes were characterized with Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM), whereas ZnO/CHIT-g-PVAL size of core-shell nanoparticles were measured using transmission electron microscopy (TEM). The electrostatic interaction between GOD and ZnO/CHIT-g-PVAL provided the resulting tuned enzyme electrode with a high degree of enzyme immobilization and excellent lifetime stability. The response studies were carried out as a function of glucose concentration with potentiometric measurement. The GOD/ZnO/CHIT-g-PVAL/ITO bioelectrode has showed a linear potential response to the glucose concentration ranging from 2 µM to 1.2mM. The glucose biosensor exhibited a fast surface-controlled redox biochemistry with a detection limit of 0.2 µM, a sensitivity of >0.04 V/µM and a response time of three sec. ZnO/CHIT-g-PVAL core-shell nanocomposite could be a promising nanomaterials for a range of enzymic biosensors.

Detection of p53 gene point mutation using sequence-specific molecularly imprinted PoPD electrode.

An amperometric sequence-specific molecularly imprinted single-stranded oligodeoxyribonucleotide (ss-ODN) biosensor was fabricated and characterised in this study. Using ss-ODN as the template and o-phenylenediamine as the functional monomer, the ODN biosensor was fabricated by an electropolymerisation process on an indium-tin oxide (ITO) coated glass substrate. The template ss-ODN was washed out of the ss-ODN/poly(o-phenylenediamine)(PoPD)/ITO electrode using sterilised basic ethanol-water. The resulting ss-ODN imprinted PoPD/ITO electrode was characterised using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and cyclic voltammetry (CV). The amperometric responses, i.e., Δi as a function of the target ss-ODN concentration was studied. The biosensor using ss-ODN imprinted PoPD/ITO as the working electrode showed a linear Δ current response to the target ss-ODN concentration within the range of 0.01-300 fM. The biosensor showed a sensitivity of 0.62 µA/fM, with a response time of 14s. The present novel molecularly imprinted ss-ODN biosensor could greatly benefit in terms of cost-effectiveness, storage stability, ultra sensitivity and selectivity together with the potential for improved commercial genetic sensors.