Controlling carbon nanodot fluorescence for optical biosensing.

In this work we report on the optical properties of specific synthetic carbon nano-dots (CDs) and their suitability for the development of optical biosensors. We examine the photoluminescence behavior of these CDs under different conditions, in their native form, as well as when conjugated to the catalytic protein glucose oxidase (GOx) for the construction of optical glucose biosensors. The effect of pH and hydrogen peroxide on the observed spectra is examined as the basis for the biosensor development. The CDs examined here have inherent surface amino functional groups which allow for easy conjugation to biomolecules via EDC-NHS, providing a well defined platform for biosensing applications. We conclude that the well controlled, stable, and highly efficient fluorescence behavior of the CDs in solution or in conjugate, provides the grounds for this class of materials to be used in a variety of arrangements for the development of optical and optoelectrochemical detection systems.

Dendrimers as tunable vectors of drug delivery systems and biomedical and ocular applications.

Dendrimers are large polymeric structures with nanosize dimensions (1-10 nm) and unique physicochemical properties. The major advantage of dendrimers compared with linear polymers is their spherical-shaped structure. During synthesis, the size and shape of the dendrimer can be customized and controlled, so the finished macromolecule will have a specific "architecture" and terminal groups. These characteristics will determine its suitability for drug delivery, diagnostic imaging, and as a genetic material carrier. This review will focus initially on the unique properties of dendrimers and their use in biomedical applications, as antibacterial, antitumor, and diagnostic agents. Subsequently, emphasis will be given to their use in drug delivery for ocular diseases.

The stabilization of Au NP-AChE nanocomposites by biosilica encapsulation for the development of a thiocholine biosensor.

We report on the construction of an amperometric biosensor based on the immobilization of the enzyme acetylcholinesterase (AChE) onto gold nanoparticles (Au NPs). The active enzyme is covalently bound directly onto the surface of the Au NPs via a thiol bond. This immobilization provides increased stability and high electron-transfer between the colloidal Au NPs, the catalyst and the transducer surface. To further increase the biosensor stability by protecting the enzyme from denaturation and protease attack, a layer of biosilica was grown around the Au NP enzyme nanocomposite. All steps, i.e., the conjugation of the enzyme to the gold nanoparticles and the encapsulation into biosilica, are monitored and confirmed by ATR-FT-IR spectroscopy. The stabilizing effect of the entrapment was evaluated amperometrically, while the operation of the biosensor was monitored over a period of 4 months. The initial sensitivity of the biosensor was calculated to be 27.58 nA mM(-1) with a linear response to the concentration of the substrate in the range from 0.04 to 0.4 mM. It is thus shown that the biosilica nanocomposites doped with Au NPs-AChE conjugates create a system that provides both signal mediation and significant enzyme stabilization over the existing AChE biosensor. The biosensor had retained all its activity at the end of the 4 months, compared with the normal AChE biosensor whose activity reached 50% after only 42 days of operation.

Carbon-nanofiber-based nanocomposite membrane as a highly stable solid-state junction for reference electrodes.

There is currently a need for a reliable solid-state reference electrode, especially in applications such as autonomous sensing or long-term environmental monitoring. We present here for the first time a novel solid-state nanofiber junction reference electrode (NFJRE) incorporating a junction consisting of poly(methyl methacrylate) and carbon graphene stacked nanofibers. The NFJRE operates by using the membrane polymer junction, which has a very high glass transition temperature (T(g)) and small diffusion coefficient, to control the diffusion of ions, and the carbon nanofibers lower the junction resistance and act as ion-to-electron transducers. The fabrication of the NFJRE is detailed, and its behavior is characterized in terms of its impedance, stability, and behavior in comparison with traditional reference electrodes. The NFJRE showed a response of <5-13 mV toward a variety of electrolyte solutions from 10(-5) to 10(-2) M, <10 mV over a pH range of 2-12, and excellent behavior when used with voltammetric methods.

Biosilicated CdSe/ZnS quantum dots as photoluminescent transducers for acetylcholinesterase-based biosensors.

CdSe/ZnS core/shell quantum dots (QDs) are functionalized with mercaptoundecanoic acid (MUA) and subsequently covered with poly-L-lysine (PLL) as the template for the formation of the silica outer shell. This nanocomposite is used as a transduction and stabilization system for optical biosensor development. The covalent immobilization of the enzyme acetylcholinesterase from Drosophila melanogaster (AChE) during the formation of the biomimetically synthesized silica is used here as a model, relatively unstable enzyme, as a proof of principle. The enzyme is successfully immobilized onto the QDs and then stabilized by the PLL capping and the subsequent formation of the outer nanoporous silica thin shell, giving rise to the QD/AChE/PLL/silica biosensor. It is shown that the poly-L-lysine templated silica outer shell does not modify the optical properties of the quantum dots, while it protects the enzyme from unfolding and denaturation. The small pores of the silica shell allow for the free diffusion of the analyte to the active center of the enzyme, while it does not allow for the proteases to reach the enzyme. The response of the QD/AChE/PLL/silica nano-biosensor to its substrate, acetylcholine chloride, is evaluated by monitoring the changes in the QDs' photoluminescence which are related to the changes in pH. These pH changes of the surrounding environment of the QDs are induced by the enzymatic reaction, and are associated with the analyte concentration in the solution. The biodetection system proposed is shown to be stable with a storage lifetime of more than 2 months. The data presented provides the grounds for the application of this nanostructured biosensor for the detection of AChE inhibitors.

Pesticide detection with a liposome-based nano-biosensor.

Monitoring of the organophosphorus pesticides dichlorvos and paraoxon at very low levels has been achieved with liposome-based nano-biosensors. The enzyme acetylcholinesterase was effectively stabilized within the internal nano-environment of the liposomes. Within the liposomes, the pH sensitive fluorescent indicator pyranine was also immobilized for the optical transduction of the enzymatic activity. Increasing amounts of pesticides lead to the decrease of the enzymatic activity for the hydrolysis of the acetylcholine and thus to a decrease in the fluorescent signal of the pH indicator. The decrease of the liposome biosensors signal is relative to the concentration of dichlorvos and paraoxon down to 10(-10)M levels. This biosensor system has been applied successfully to the detection of total toxicity in drinking water samples. Also a colorimetric screening device for pesticide analysis has been evaluated.

Immobilization of enzymes into nanocavities for the improvement of biosensor stability.

Nanoporous materials with different pore sizes are evaluated as immobilization and stabilization matrices of proteins for the development of highly stable biosensors. It has been proven experimentally that confinement of proteins in cages with a diameter that is 2-6 times larger than their size increases considerably the stability of the biomolecules, as has been shown earlier by theoretical calculations. Porous silica beads with pore sizes of 10nm were utilized for the immobilization of the enzymes HRP and GOx with diameters in the order of 5 and 7 nm, respectively. The sensitivity of the corresponding biosensor systems was monitored for 70 h under continuous operation conditions (+600 mV) and it was found that the stabilization factor of GOx is 1.7 times higher compared to HRP. Also the stabilization efficiency of enzymes against leaching and inactivation in porous polymer beads with pore diameters of 10 and 30 nm was examined. The leaching rate of the enzyme AChE from the 30 nm polymer beads was found to be 1.1 times higher than that from the 10nm beads. At the same time the remaining activity of GOx biosensors after 5 days of continuous operation conditions (+600 mV) was found to be 2.1 times higher when the enzyme had been immobilized in the 10nm beads compared to the 30 nm beads. It is thus evident that the matching between the pore size of nanoporous materials and the molecular size of enzymes is essential for the development of biosensors with extended shelf and operational lifetimes.

Fluorescence detection of enzymatic activity within a liposome based nano-biosensor.

The encapsulation of enzymes in microenvironments and especially in liposomes, has proven to greatly improve enzyme stabilization against unfolding, denaturation and dilution effects. Combining this stabilization effect, with the fact that liposomes are optically translucent, we have designed nano-sized spherical biosensors. In this work liposome-based biosensors are prepared by encapsulating the enzyme acetylcholinesterase (AChE) in L-a phosphatidylcholine liposomes resulting in spherical optical biosensors with an average diameter of 300+/-4 nm. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The response of the liposome biosensor to the substrate acetylthiocholine chloride is relatively fast and reproducible, while the system is stable as has been shown by immobilization within sol-gel.

Tuning the sol-gel microenvironment for acetylcholinesterase encapsulation.

The effect of the sol-gel microenvironment on the activity of acetylcholinesterase, an enzyme of high bio-analytical interest, is presented and is correlated to the overall analytical performance of corresponding biosensors. The sol-gel membranes are initially optimized with respect to the catalyst and the TEOS:H2O ratio (r), for mechanical stability, porosity, and hydrophobicity as well as in terms of enzymatic activity. FT-IR and electrochemical impedance spectroscopy (EIS) are used to probe the configuration and rotational mobility of the enzyme within the sol-gel matrices. Overall, it is observed that the rotational mobility of the protein can be correlated with the sensitivity of the biosensor. Optimum biosensor performance is obtained for base-catalyzed sol-gels with r values close to 2. The biosensor has sensitivity of 2.5 microA/mm, a linear range of response between 1 and 3mm, response time of about 30s, and sensor-to-sensor reproducibility (RSD) of 3%. These analytical characteristics are far superior to previously reported sol-gel biosensors.

Genetically engineered acetylcholinesterase-based biosensor for attomolar detection of dichlorvos.

The design of a biosensor for the detection of dichlorvos at attomolar levels is described based on a highly sensitive double mutant (E69Y Y71D) of the Drosophila melanogaster acetylcholinesterase (Dm. AChE). This enzyme has a k(i) for dichlorvos equal to 487 microM(-1)min(-1), which is 300 and 20,000 times higher than that of the wild type Dm. AChE and the Electrophorus electricus AChE (E.el. AChE), respectively. The enzyme is immobilized into microporous-activated conductive carbon, and is used as such for the development of an inhibitor electrochemical biosensor. This E69Y Y71D mutant enables the decrease in the detection limit of the biosensor down to 10(-17) M, which is five orders of magnitude lower compared to the Electropharus electricus-based biosensor and eight orders of magnitude lower than the biosensors described so far.

Stabilization of enzymes in nanoporous materials for biosensor applications.

In this study we present the results obtained from efforts to stabilize the inherently unstable m-AChE in nanoporous materials, for the development of biosensors with increased operational stability. Based on existing theoretical models, the entrapment of proteins into relatively small rigid cages drastically increases the stability of these proteins, as this is manifested by their decreased tendency to unfold. The use of two different meso/nanomaterials for the immobilization of the m-AChE shows that there is both a decrease in the leaching of the protein from the biosensor membrane to the test solution, as well as a drastic increase in the operational stability of the resulting biosensor.

Gallium nitride-based potentiometric anion sensor.

The gallium nitride (GaN) semiconductor is used as the sensing element for the development of a potentiometric anion sensor. The anion recognition mechanism is based on the selective interaction of anions in solution with the epitaxial Ga-face polarity GaN (0001) wurtzite crystal film grown on sapphire. The native GaN crystal is used for the development of an ion blocked sensor. The potential is based on the Volta potential, generated at the semiconductor/solution interface and within the Helmholtz layer, due to specifically adsorbed anions. The selectivity of the sensor is based on the direct interaction of the anionic ligand with the outer electron-defective gallium atoms; thus, it is not dependent on the lipophilicity of the adsorbed charged species. The chemical resistivity of the GaN crystal provides sensors with excellent lifetime, signal stability, and reproducibility.