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Angular-ratiometric plasmon-resonance based light scattering for bioaffinity sensing.

We describe an exciting opportunity for affinity biosensing using a ratiometric approach to the angular-dependent light scattering from bioactivated and subsequently aggregated noble metal colloids. This new model sensing platform utilizes the changes in particle scattering from very small colloids, which scatter light according to traditional Rayleigh theory, as compared to the changes in scattering observed by much larger colloidal aggregates, formed due to a bioaffinity reaction. These larger aggregates no longer scatter incident light in a Cos(2) theta dependence, as is the case for Rayleigh scattering, but instead scatter light in an increased forward direction as compared to the incident geometry. By subsequently taking the ratio of the scattered intensity at two angles, namely 90 degrees and 140 degrees , relative to the incident light, we can follow the association of biotinylated bovine serum albumin-coated 20 nm gold colloids, cross-linked by additions of streptavidin. This new model system can be potentially applied to many other nanoparticle assays and has many advantages over traditional fluorescence sensing and indeed light-scattering approaches. For example, a single nanoparticle can have the equivalent scattered intensity as 10(5) fluorescing fluorescein molecules substantially increasing detection; the angular distribution of scattered light from noble metal colloids is substantially easier to predict as compared to fluorescence; the scattered light is not quenched by biospecies; the ratiometric measurements described here are not dependent on colloid concentration as are other scattering techniques; and finally, the noble metal colloids are not prone to photodestruction, as is the case with organic fluorophores.

Affinity Labels↗

Effect of silicon oxidation on long-term cell selectivity of cell-patterned Au/SiO2 platforms.

Cellular patterning on silicon platforms is the basis for development of integrated cell-based biosensing devices, for which long-term cell selectivity and biostability remain a major challenge. We report the development of a silicon-based platform in a metal-insulator format capable of producing uniform and biostable cell patterns with long-term cell selectivity. Substrates patterned with arrays of gold electrodes were surface-engineered such that the electrodes were activated with fibronectin to mediate cell attachment and the silicon oxide background was passivated with PEG to resist protein adsorption and cell adhesion. Three types of oxide surfaces, i.e., native oxide, dry thermally grown oxide, and wet thermally grown oxide, were produced to illustrate the effect of oxide state of the surface on long-term cell selectivity. Results indicated that the cell selectivity over time differed dramatically among three patterned platforms and the best cell selectivity was found on the dry oxide surface for up to 10 days. Surface analysis results suggested that this enhancement in cell selectivity may be related to the presence of additional, more active oxide states on the dry oxide surface supporting the stability of PEG films and effectively suppressing the cell adhesion. This research offers a new strategy for development of stable and uniform cell-patterned surfaces, which is versatile for immobilization of silane-based chemicals for preparation of biostable interfaces.

Animals↗

Water soluble cryptophanes showing unprecedented affinity for xenon: candidates as NMR-based biosensors.

Cryptophanes bearing OCH(2)COOH groups in place of the methoxy groups represent a new class of xenon-carrier molecules soluble in water at biological pH. By using (1)H and (129)Xe NMR (thermally- and laser-polarized dissolved gas), the structural and dynamical behaviors of these host molecules as well as their interaction with xenon are studied. They are shown to exist in aqueous solution under different conformations in very slow exchange. A saddle form present for one of these conformations could explain the (1)H NMR spectra. Whereas the cryptophanes in such a conformation are unable to complex xenon, unprecedented high binding constants are found for cryptophanes in the other canonical crown-crown conformation. These host molecules could therefore be valuable candidates for biosensing using (129)Xe MRI.

Biosensing Techniques↗

Investigating the molecular mechanisms of in-plane mechanochemistry on cantilever arrays.

Free-standing cantilevers, which directly translate specific biochemical reactions into micromechanical motion, have recently attracted much attention as label-free biosensors and micro/nano robotic devices. To exploit this mechanochemical sensing technology, it is essential to develop a fundamental understanding of the origins of surface stress. Here we report a detailed study into the molecular basis of stress generation in aqueous environments focusing on the pH titration of model mercaptohexadecanoic acid self-assembled monolayers (SAMs), using in situ reference cantilevers coated with nonionizable hexadecanethiol SAMs. Semiautomated data analysis and a statistical model were developed to quantify cyclic deprotonation/protonation reactions on multiple arrays. In-plane force titrations were found to have the sensitivity to detect ionic hydrogen bond formation between protonated and nonprotonated carboxylic acid groups in the proximity of the surface pK1/2, which generated a mean tensile differential surface stress of +1.2 +/- 0.3 mN/m at pH 6.0, corresponding to 1 pN attractive force between two adjacent MHA molecules. Conversely, the magnitude of compressive differential surface stress was found to increase progressively with pH >/= 7.0, reaching a maximum of -14.5 +/- 0.5 mN/m at pH 9.0, attributed to enhanced electrostatic repulsion between deprotonated carboxylic acid groups. However, striking differences were observed in the micromechanical responses to different ionic strength and ion species present in the aqueous environment, highlighting the critical role of counter- and co-ions on surface stress. Our findings provide fundamental insights into the molecular mechanisms of in-plane mechanochemistry, which may be exploited for biosensing and nanoactuation applications.

Biosensing Techniques↗

Detection of traces of tetracyclines from fish with a bioluminescent sensor strain incorporating bacterial luciferase reporter genes.

Bioluminescent Escherichia coli K-12 strain for the specific detection of the tetracycline family of antimicrobial agents was optimized to work with fish samples. The biosensing strain contains a plasmid incorporating the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive element from transposon Tn10 (Korpela et al. Anal. Chem. 1998, 70, 4457-4462). The extraction procedure of oxytetracycline from rainbow trout (Oncorhynchus mykiss) tissue was optimized. There was neither need for centrifugation of homogenized tissue nor use of organic solvents. The lowest levels of detection of tetracycline and oxytetracycline from spiked fish tissue were 20 and 50 microg/kg, respectively, in a 2-h assay. The optimized assay protocol was tested with fish that were given a single oral dose of high and low concentrations of oxytetracycline. The assay was able to detect oxytetracycline residues below the European Union maximum residue limits, and the results correlated well with those obtained by conventional HPLC (R = 0.81).

Animals↗

Optimized biorecognition of cytochrome c 551 and azurin immobilized on thiol-terminated monolayers assembled on Au(111) substrates.

Molecular recognition between two redox partners, azurin and cytochrome c 551, is studied at the single-molecule level by means of atomic force spectroscopy, after optimizing azurin adsorption on gold via sulfhydryl-terminated alkanethiol spacers. Our experiments provide evidence of specific interaction between the two partners, thereby demonstrating that azurin preserves biorecognition capability when assembled on gold via these spacers. Additionally, the measured single-molecule kinetic reaction rate results are consistent with a likely transient nature of the complex. Interestingly, the immobilization strategy adopted here, which was previously demonstrated to favor electrical coupling between azurin (AZ) and the metal electrode, is also found to facilitate AZ interaction with the redox partner, if compared to the case of AZ directly adsorbed on bare gold. Our findings confirm the key role of a well-designed immobilization strategy, capable of optimizing both biorecognition capabilities and electrical coupling with the conductive substrate at the single-molecule level, as a starting point for advanced applications of redox proteins for ultrasensitive biosensing.

Azurin↗

Hydrodynamic dimensions, electrophoretic mobility, and stability of hydrophilic quantum dots.

Luminescent semiconductor quantum dots (QDs) have great potential for use in biological assays and imaging. These nanocrystals are capped with surface ligands (bifunctional molecules, amphiphilic polymers, phospholipids, etc.) that render them hydrophilic and provide them with functional properties. These coatings alters their hydrodynamic radii and surface charge, which can drastically affect properties such as diffusion within the cell cytoplasm. Heavy atom techniques such as transmission electron microscopy and X-ray scattering probe the inorganic core and do not take into account the ligand coating. Herein we use dynamic light scattering to characterize the hydrodynamic radius (R(H)) of CdSe-ZnS QDs capped with various hydrophilic surface coatings (including dihydrolipoic acid and amphiphilic polymers) and self-assembled QD-protein bioconjugates. Experiments were complemented with measurements of the geometric size and zeta potential using agarose gel electrophoresis and laser Doppler velocimetry. We find that the effects of surface ligands on the hydrodynamic radius and on the nanoparticle mobility are complex and strongly depend on a combination of the inorganic core size and nature and lateral extension of the hydrophilic surface coating. These properties are critical for the design of QD-based biosensing assays as well as QD bioconjugate diffusion in live cells.

Biosensing Techniques↗

Electrochemically tunable surface-plasmon-enhanced diffraction gratings and their (bio-)sensing applications.

Electrochemistry was combined with surface-plasmon-enhanced diffraction (ESPD) to investigate a redox-switchable polymer grating and its (bio-)sensing applications. Patterned arrays of polyaniline (PANI)/ poly(styrenesulfonate) (PSS) were fabricated by the combination of electropolymerization and micromolding in capillaries (MIMIC) and were used as an optical grating for surface-plasmon-enhanced diffraction experiments. The diffraction efficiency (DE) could be tuned by changes in the applied potential, and by changes in the pH of the surrounding solution (dielectric medium). The response of the DE to the pH depends strongly on the redox state of the PANI/PSS grating. If the polymer grating is mainly in its reduced state, the DE shows a linear dependence on the pH. The DE of the PANI/PSS grating can also be modulated by an electrocatalytic event: by keeping PANI/PSS in its oxidized form, the addition of beta-nicotinamide adenine dinucleotide (NADH) increases the DE with the increase of NADH concentration, which points to the possibility of the use of ESPD technologies for biosensing.

Aniline Compounds↗

Cast thin film biosensor design based on a Nafion backbone, a multiwalled carbon nanotube conduit, and a glucose oxidase function.

Novel electroanalytical sensing nanobiocomposite materials are reported. These materials are prepared by mixing multiwalled carbon nanotubes (MWNTs), a Nafion cation exchanger, and glucose oxidase (GOD) in appropriate amounts. The MWNTs are cylindrical with a diameter in the range 40-60 nm and with a length of up to several micrometers, and they provide electrical conductivity. Nafion acts as a polymer backbone to give stable and homogeneous cast thin films. Both MWNTs and Nafion provide negative functionalities to bind to positively charged redox enzymes such as glucose oxidase. The resulting biosensing composite material is inexpensive, reliable, and easy to use. The homogeneity of the MWNT-Nafion-GOD nanobiocomposite films was characterized by atomic force microscopy (AFM). Amperometric transducers fabricated with these materials were characterized electrochemically using cyclic voltammetry and amperometry in the presence of hydrogen peroxide and in the presence of glucose. Their linear response to hydrogen peroxide was demonstrated. The glucose biosensor sensitivity was strongly influenced by the glucose oxidase concentration within the nanobiocomposite film. The optimized glucose biosensor (2.5 mg/mL GOD) displayed a sensitivity of 330 nA/mM, a linear range of up to 2 mM, a detection limit of 4 microM, and a response time of <3 s.

Biosensing Techniques↗

Characterization of aldehyde-PEG tethered surfaces: influence of PEG chain length on the specific biorecognition.

A functionalized poly(ethylene glycol) (PEG) layer possessing a reactive aldehyde group at the free end of the tethered PEG chain was constructed by simple coating on the substrate, using the acetal-PEG/poly(DL-lactide) block copolymer, followed by the hydrolysis of the acetal end group by an acid treatment. The reactivity of the aldehyde group at the distal end of the PEG tethered chain was evaluated via a reductive amination using 4-amino-2,2,6,6-tetramethylpiperidinyloxy as the model compound. Further conjugation of the aldehyde group with sugar moieties has demonstrated an increased recognition ability with lectins with an increasing PEG chain length, which was attributable to the mobility of the chain end. These results provide a novel idea for highly sensitive biorecognition, suggesting a method to create highly selective biosensing surfaces that are able to prevent the undesired nonspecific adsorption of biocomponents.

Aldehydes↗

Amplified electrocatalysis at DNA-modified nanowires.

Arrayed gold nanowires are a novel and useful platform for electrochemical DNA detection. Pilot studies testing the use of these templated structures with an electrocatalytic reporter system revealed that very low detection thresholds for target DNA sequences can be obtained. One factor contributing to the heightened sensitivity is the high signal-to-noise ratio achieved with the large electrocatalytic signals observed at DNA-modified nanowires. Here, we explain the improved sensitivity with evidence illustrating that electrocatalysis at DNA-modified nanostructures generates amplified signals that are significantly larger than those observed at bulk gold surfaces. The results presented strongly suggest that the three-dimensional architectures of the nanowires facilitate the electrocatalytic reaction because of enhanced diffusion occurring around these structures. Effects unique to the nanoscale are shown to underlie the utility of nanowires for DNA biosensing.

Biosensing Techniques↗

A biosensor that uses ion-channel switches.

Biosensors are molecular sensors that combine a biological recognition mechanism with a physical transduction technique. They provide a new class of inexpensive, portable instrument that permit sophisticated analytical measurements to be undertaken rapidly at decentralized locations. However, the adoption of biosensors for practical applications other than the measurement of blood glucose is currently limited by the expense, insensitivity and inflexibility of the available transduction methods. Here we describe the development of a biosensing technique in which the conductance of a population of molecular ion channels is switched by the recognition event. The approach mimics biological sensory functions and can be used with most types of receptor, including antibodies and nucleotides. The technique is very flexible and even in its simplest form it is sensitive to picomolar concentrations of proteins. The sensor is essentially an impedance element whose dimensions can readily be reduced to become an integral component of a microelectronic circuit. It may be used in a wide range of applications and in complex media, including blood. These uses might include cell typing, the detection of large proteins, viruses, antibodies, DNA, electrolytes, drugs, pesticides and other low-molecular-weight compounds.

Biosensing Techniques↗

Computational design of receptor and sensor proteins with novel functions.

The formation of complexes between proteins and ligands is fundamental to biological processes at the molecular level. Manipulation of molecular recognition between ligands and proteins is therefore important for basic biological studies and has many biotechnological applications, including the construction of enzymes, biosensors, genetic circuits, signal transduction pathways and chiral separations. The systematic manipulation of binding sites remains a major challenge. Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, l-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or l-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally. The biological and biosensing activities of the designed receptors illustrate potential applications of computational design.

Amino Acid Sequence↗

Protein-modified nanocrystalline diamond thin films for biosensor applications.

Diamond exhibits several special properties, for example good biocompatibility and a large electrochemical potential window, that make it particularly suitable for biofunctionalization and biosensing. Here we show that proteins can be attached covalently to nanocrystalline diamond thin films. Moreover, we show that, although the biomolecules are immobilized at the surface, they are still fully functional and active. Hydrogen-terminated nanocrystalline diamond films were modified by using a photochemical process to generate a surface layer of amino groups, to which proteins were covalently attached. We used green fluorescent protein to reveal the successful coupling directly. After functionalization of nanocrystalline diamond electrodes with the enzyme catalase, a direct electron transfer between the enzyme's redox centre and the diamond electrode was detected. Moreover, the modified electrode was found to be sensitive to hydrogen peroxide. Because of its dual role as a substrate for biofunctionalization and as an electrode, nanocrystalline diamond is a very promising candidate for future biosensor applications.

Biosensing Techniques↗

Design and optimization of a kinase-controlled allosteric switch.

Post-translational control enables rapid and precise regulation of cell behavior. Despite these advantages, general strategies to build phosphorylation-based synthetic circuits are limited. Here we reasoned that engineered allostery, a technique that has been applied to design light- and chemically gated protein switches, could also be used to engineer phosphorylation-controlled protein switches (phospho-switches). Using an allosterically controllable Gal4 transcription factor as a scaffold, we show that a classic kinase F&#xf6;rster resonance energy transfer biosensor architecture can be used as a starting point for phospho-switch design. We optimize all features of the phospho-switch to develop an ERK-controlled transcription factor with a 20-fold phosphorylation-dependent change in transcriptional output. The resulting synthetic ERK-responsive transcription factor responds with comparable sensitivity to the c-fos promoter and reveals spatial ERK signaling patterns in mammalian developmental organoids. We further show that our switch architecture can be generalized to other input kinases and allosterically controlled targets. This work provides a general platform for a new generation of kinase-responsive tools for biosensing and synthetic biology applications.

Allosteric Regulation↗

Biochemically functionalized silica nanoparticles.

In this report, we demonstrate the biochemical modification of silica based nanoparticles. Both pure and dye-doped silica nanoparticles were prepared, and their surfaces were modified with enzymes and biocompatible chemical reagents that allow them to function as biosensors and biomarkers. The nanoparticles produced in this work are uniform in size with a 1.6% relative standard deviation. They have a pure silica surface and can thus be modified easily with many biomolecules for added biochemical functionality. Specifically, we have modified the nanoparticle surfaces with enzyme molecules (glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH)) and a biocompatible reagent for cell membrane staining. Experimental results show that the silica nanoparticles are a good biocompatible solid support for enzyme immobilization. The immobilized enzyme molecules on the nanoparticle surface have shown excellent enzymatic activity in their respective enzymatic reactions. The nanoparticle surface biochemical functionalization demonstrates the feasibility of using nanoparticles for biosensing and biomarking applications.

Biosensing Techniques↗

A polymer-based microfluidic device for immunosensing biochips.

This paper describes the design, fabrication, and test of a PDMS/PMMA-laminated microfluidic device for an immunosensing biochip. A poly(dimethyl siloxane)(PDMS) top substrate molded by polymer casting and a poly(methyl methacrylate)(PMMA) bottom substrate fabricated by hot embossing are bonded with pressure and hermetically sealed. Two inlet ports and an air vent are opened through the PDMS top substrate, while gold electrodes for electrochemical biosensing are patterned onto the PMMA bottom substrate. The analyte sample is loaded from the sample inlet port to the detection chamber by capillary force, without any external intervening forces. For this and to control the time duration of sample fluid in each compartment of the device, including the inlet port, diffusion barrier, reaction chamber, flow-delay neck, and detection chamber, the fluid conduit has been designed with various geometries of channel width, depth, and shape. Especially, the fluid path has been designed so that the sample flow naturally stops after filling the detection chamber to allow sufficient time for biochemical reaction and subsequent washing steps. As model immunosensing tests for the microfluidic device, functionalizations of ferritin and biotin to the sensing surfaces on gold electrodes and their biospecific interactions with antiferritin antiserum and streptavidin have been investigated. An electrochemical detection method for immunosensing by biocatalyzed precipitation has been developed and applied for signal registration. With the biochip, the whole immunosensing processes could be completed within 30 min.

Antibodies↗