Colorimetric paper bioassay for the detection of phenolic compounds.

A new type of paper based bioassay for the colorimetric detection of phenolic compounds including phenol, bisphenol A, catechol and cresols is reported. The sensor is based on a layer-by-layer (LbL) assembly approach formed by alternatively depositing layers of chitosan and alginate polyelectrolytes onto filter paper and physically entrapping the tyrosinase enzyme in between these layers. The sensor response is quantified as a color change resulting from the specific binding of the enzymatically generated quinone to the multilayers of immobilized chitosan on the paper. The color change can be quantified with the naked eye but a digitalized picture can also be used to provide more sensitive comparison to a calibrated color scheme. The sensor was optimized with respect to the number of layers, pH, enzyme, chitosan and alginate amounts. The colorimetric response was concentration dependent, with a detection limit of 0.86 (±0.1) µg/L for each of the phenolic compounds tested. The response time required for the sensor to reach steady-state color varied between 6 and 17 min depending on the phenolic substrate. The sensor showed excellent storage stability at room temperature for several months (92% residual activity after 260 days storage) and demonstrated good functionality in real environmental samples. A procedure to mass-produce the bioactive sensors by inkjet printing the LbL layers of polyelectrolyte and enzyme on paper is demonstrated.

Paper bioassay based on ceria nanoparticles as colorimetric probes.

We report the first use of redox nanoparticles of cerium oxide as colorimetric probes in bioanalysis. The method is based on changes in the physicochemical properties of ceria nanoparticles, used here as chromogenic indicators, in response to the analyte. We show that these particles can be fully integrated in a paper-based bioassay. To construct the sensor, ceria nanoparticles and glucose oxidase were coimmobilized onto filter paper using a silanization procedure. In the presence of glucose, the enzymatically generated hydrogen peroxide induces a visual color change of the ceria nanoparticles immobilized onto the bioactive sensing paper, from white-yellowish to dark orange, in a concentration-dependent manner. A detection limit of 0.5 mM glucose with a linear range up to 100 mM and a reproducibility of 4.3% for n = 11 ceria paper strips were obtained. The assay is fully reversible and can be reused for at least 10 consecutive measurement cycles, without significant loss of activity. Another unique feature is that it does not require external reagents, as all the sensing components are fixed onto the paper platform. The bioassay can be stored for at least 79 days at room temperature while maintaining the same analytical performance. An example of analytical application was demonstrated for the detection of glucose in human serum. The results demonstrate the potential of this type of nanoparticles as novel components in the development of robust colorimetric bioassays.

Interaction of lipid membrane with nanostructured surfaces.

Tiny details of the phospholipid (DMPC) membrane morphology in close vicinity to nanostructured silica surfaces have been discovered in the atomic force microscopy experiments. The structural features of the silica surface were varied in the experiments by the deposition of silica nanoparticles of different diameter on plane and smooth silica substrates. It was found that, due to the barrier function of the lipid membrane, only particles larger than 22 nm in diameter with a smooth surface were completely enveloped by the lipid membrane. However, nanoparticles with bumpy surfaces (curvature diameter of bumps as that of particles <22 nm) were only partially enveloped by the lipid bilayer. For the range of nanostructure dimensions between 1.2 and 22 nm, the lipid membrane underwent structural rearrangements by forming pores (holes). The nanoparticles were accommodated into the pores but not enveloped by the lipid bilayer. The study also found that the lipid membrane conformed to the substrate with surface structures of dimensions less than 1.2 nm without losing the membrane integrity. The experimental results are in accord with the analytical free energy model, which describes the membrane coverage, and numerical simulations which evaluate adhesion of the membrane and dynamics as a function of surface topology. The results obtained in this study are useful for the selection of dimensions and shapes for drug-delivery cargo and for the substrate for supported lipid bilayers. They also help in qualitative understanding the role of length scales involved in the mechanisms of endocytosis and cytotoxicity of nanoparticles. These findings provide a new approach for patterning supported lipid membranes with well-defined features in the 1.2-22 nm range.

Biofuel cell controlled by enzyme logic network--approaching physiologically regulated devices.

A "smart" biofuel cell switchable ON and OFF upon application of several chemical signals processed by an enzyme logic network was designed. The biocomputing system performing logic operations on the input signals was composed of four enzymes: alcohol dehydrogenase (ADH), amyloglucosidase (AGS), invertase (INV) and glucose dehydrogenase (GDH). These enzymes were activated by different combinations of chemical input signals: NADH, acetaldehyde, maltose and sucrose. The sequence of biochemical reactions catalyzed by the enzymes models a logic network composed of concatenated AND/OR gates. Upon application of specific "successful" patterns of the chemical input signals, the cascade of biochemical reactions resulted in the formation of gluconic acid, thus producing acidic pH in the solution. This resulted in the activation of a pH-sensitive redox-polymer-modified cathode in the biofuel cell, thus, switching ON the entire cell and dramatically increasing its power output. Application of another chemical signal (urea in the presence of urease) resulted in the return to the initial neutral pH value, when the O(2)-reducing cathode and the entire cell are in the mute state. The reversible activation-inactivation of the biofuel cell was controlled by the enzymatic reactions logically processing a number of chemical input signals applied in different combinations. The studied biofuel cell exemplifies a new kind of bioelectronic device where the bioelectronic function is controlled by a biocomputing system. Such devices will provide a new dimension in bioelectronics and biocomputing benefiting from the integration of both concepts.

Biomolecular oxidative damage activated by enzymatic logic systems: biologically inspired approach.

Systems that perform oxidative damage to biomolecules through catalytic cascades in the presence of iron-redox labile species were activated by enzymatic logic gates that process chemical input signals according to built-in logic operations. AND/OR enzymatic logic gates were composed of glucose oxidase (GOx) and GOx/esterase, respectively. The AND/OR logic functions of the enzyme gates were activated by application of glucose-oxygen and glucose-ethyl acetate input signals, respectively. The enzymatic logic gates, upon activation by specific patterns of the chemical input signals, produced acidic solutions and triggered release of redox labile iron species from a complex that is unstable under acidic conditions. This resulted in the activation of a catalytic cascade, which produced reactive oxygen species (ROS) and subsequently yielded oxidative damage in biomolecules. Functional integration of the enzyme-based logic systems with the catalytic redox cascade that performs damage in biomolecules on demand is a first step towards "smart" systems capable of programmed detection, identification, and neutralization of potential biohazards.

Bioelectrocatalytic system coupled with enzyme-based biocomputing ensembles performing boolean logic operations: approaching "smart" physiologically controlled biointerfaces.

The modified electrode for electrocatalytic oxidation of NADH was developed using a pH-switchable redox interface. The operation of the modified electrode was controlled by logic operations performed by enzyme systems processing biochemical input signals. The electrocatalytic oxidation of NADH was activated upon appropriate combinations of the signals processed by the AND/OR logic operations performed by the enzymes. The modified interface was reset in a mute nonactive state by another enzyme reaction. The coupling between the enzyme logic systems and the bioelectrocatalytic interface was achieved by pH changes produced in situ by the enzyme reactions, resulting in different protonation states of the polymeric matrix associated with the electrode surface. The bioelectrocatalytic system integrated with biochemical computing systems opens the way to novel "smart" interfaces for multisignal biosensors and signal-controlled biofuel cells. In a long perspective, this approach will allow physiological control of implantable bioelectronic devices.

Biochemically controlled bioelectrocatalytic interface.

A switchable bioelectrocatalytic system for glucose oxidation controlled by external biochemical signals exemplifies interfacing between bioelectronic and biochemical ensembles.

Boolean logic gates that use enzymes as input signals.

Biochemical systems that demonstrate the Boolean logic operations AND, OR, XOR, and InhibA were developed by using soluble compounds, which represent the chemical "devices", and the enzymes glucose oxidase (GOx), glucose dehydrogenase (GDH), alcohol dehydrogenase (AlcDH), and microperoxidase-11 (MP-11), which operated as the input signals that activated the logic gates. The enzymes were used as soluble materials and as immobilized biocatalysts. The studied systems are proposed to be a step towards the construction of "smart" signal-responsive materials with built-in Boolean logic.

Biocomputing security system: concatenated enzyme-based logic gates operating as a biomolecular keypad lock.

A biomolecular security system mimicking a keypad lock device was developed using enzyme-based concatenated AND logic gates resulting in the implication logic network.

Interaction of nanoparticles with lipid membrane.

A nanoscale range of surface feature curvatures where lipid membranes lose integrity and form pores has been found experimentally. The pores were experimentally observed in the l-alpha-dimyristoyl phosphatidylcholine membrane around 1.2-22 nm polar nanoparticles deposited on mica surface. Lipid bilayer envelops or closely follows surface features with the curvatures outside of that region. This finding provides essential information for the understanding of nanoparticle-lipid membrane interaction, cytotoxicity, preparation of biomolecular templates and supported lipid membranes on rough and patterned surfaces.

Assembling of dense fluorescent supramolecular webs via self-propelled star-shaped aggregates.

We report a novel mechanism of assembly of dendronized rod molecules into a dense supramolecular fluorescencent web featuring self-propelled mechanistic inward motion of star-shaped aggregates within a solution droplet. We suggest that such a motion (observed in real time) is caused by the self-repulsion of the growing star-shaped nuclei from the liquid-solid-air interface in the course of one-dimensional growth of the anchored arms. An intriguing mechanism discovered here involves microscopic (hundred micrometers) directional motion of the microscopic aggregates driven by one-dimensional molecular assembly, which opens a new venue for guided assembly of dense mesoscopic supramolecular webs. Such assemblies can serve as interesting microfluidic networks, a web of optical switches, and model systems for studying intercellular communication.

Formation of silver nanoparticles at the air-water interface mediated by a monolayer of functionalized hyperbranched molecules.

Nanofibrillar micellar structures formed by the amphiphilic hyperbranched molecules within a Langmuir monolayer were utilized as matter for silver nanoparticle formation from the ion-containing water subphase. We observed that silver nanoparticles were formed within the multifunctional amphiphilic hyperbranched molecules. The diameter of nanoparticles varied from 2-4 nm and was controlled by the core dimensions and the interfibrillar free surface area. Furthermore, upon addition of potassium nitrate to the subphase, the Langmuir monolayer templated the nanoparticles' formation along the nanofibrillar structures. The suggested mechanism of nanoparticle formation involves the oxidation of primary amino groups by silver catalysis facilitated by "caging" of silver ions within surface areas dominated by multibranched cores. This system provides an example of a one-step process in which hyperbranched molecules with outer alkyl tails and compressed amine-hydroxyl cores mediated the formation of stable nanoparticles placed along/among/beneath the nanofibrillar micelles.

Assembling of amphiphilic highly branched molecules in supramolecular nanofibers.

We found that the amplification of weak multiple interactions between numerous peripheral branches of irregular, flexible, polydisperse, and highly branched molecules can facilitate their self-assembly into nanofibrillar micellar structures at solid surfaces and the formation of perfect long microfibers in the course of crystallization from solution. The core-shell architecture of the amphiphilic dendritic molecules provides exceptional stability of one-dimensional nanofibrillar structures. The critical condition for the formation of the nanofibrillar structures is the presence of both alkyl tails in the outer shell and amine groups in the core/inner shell. The multiple intermolecular hydrogen bonding and polar interactions between flexible cores stabilize these nanofibers and make them robust albeit flexible. This example demonstrates that one-dimensional supramolecular assembling at different spatial scales (both nanofibers and microfibers) can be achieved without a tedious, multistep synthesis of shape-persistent molecules.

Biomolecular stress-sensitive gauges: surface-mediated immobilization of mechanosensitive membrane protein.

We report the observation of structural reorganizations associated with unique, stress-assisted gating of mechanosensitive (MscL) membrane protein on a silicon surface modified with alkyl-terminated monolayers. We observed that the shape of MscL membrane proteins changed dramatically depending upon the packing density of alkyl tails and the surface tension of the supporting organic layer. High-resolution atomic force microscopy confirmed a transition from an elongated, prolate shape of MscL molecules within a monolayer with low surface tension to a flattened, oblate shape with a wide central opening within a monolayer with high surface tension. These observations are consistent with the conformation reorganizations associated with the two-stage, "iris"-like expansion proposed for the gating of the MscL molecules.