One- and two-photon induced fluorescence of Pacific Blue-labeled human serum albumin deposited on different core size silver colloids

We studied one- and two-photon induced fluorescence of Pacific Blue (PB)-labeled human serum albumin (HSA) in the presence of different size silver colloids. The PB fluorescence emission intensity was observed with small (30-40 nm) and large (about 120 nm) colloids and compared with PB emission in absence of colloids. For the system with a small core size colloids we did not detect any fluorescence enhancement with one-photon excitation and the enhancement observed with two-photon excitation was about 2.5-fold. In contrast, for large silver colloids we observed about a 2-fold increase in PB fluorescence brightness for one-photon excitation, and the enhancement with two-photon excitation excided 13-folds. Much stronger increases in brightness observed with two-photon excitation, compared to one-photon excitation, indicate a dominant role of enhanced local field in fluorescence enhancement on silver colloids in solutions.     

Fluorescence Enhancement Caused by Plasmonics Coupling Between Silver Nano-Cubes and Silver Film

In this article, we experimentally investigated the plasmonics interaction in the system composed of Ag nano-cubes on Ag film with controlled distance. The distance is controlled by Rhodamine B (RhB)-doped polymethylmethacrylate (PMMA) film as the spacer, whose fluorescence intensity was then enhanced by the plasmonics interaction. Experimental results show that the fluorescence enhancement is sensitive to the thickness of the spacer. The largest enhancement factor obtained is 521 with the RhB-doped PMMA film of 10 nm thickness. For comparison, we also presented the fluorescence enhancement caused by only the localized surface plasmons from Ag nano-cubes on glass substrate coated with RhB-doped PMMA film, which gives out lower enhancement factors at the same thick spacer. Our experimental results are consistent with previous theoretical investigation and shows promising applications in fluorescence based bio-sensing or bio-imaging.     

Fluorescence enhancement of fluorophores tethered to different sized silver colloids deposited on glass substrate

We studied fluorescence enhancements of fluorescein tethered to silver colloids of different size. Thiolated 23-mer oligonucleotide (ss DNA-SH) was bound selectively to silver colloids deposited on 3-aminopropyltriethoxysilane (APS)-treated quartz slides. Fluorescein-labeled complementary oligonucleotide (ss Fl-DNA) was added in an amount significantly lower than the amount of unlabeled DNA tethered to the colloids. The hybridization kinetics, observed as an increase in fluorescence emission, on small (30-40 nm) and large (> 120 nm) colloids were similar. However, the final fluorescence intensity of the sample with large colloids was about 50% higher than that observed for the sample with small colloids. The reference sample without ss DNA-SH was used to estimate the fluorescence enhancements of fluorescein tethered to the small colloids (E = 2.7) and to the large colloids (E = 4.1) due to its steady fluorescence signal. The proposed method, based on controlled hybridization with minimal amount of fluorophore labeled ss DNA, can be used to reliably estimate the fluorescence enhancements on any silver nanostructures.     

Optimisation of a silicon/silicon dioxide substrate for a fluorescence DNA microarray

This paper presents a comprehensive theory and experimental characterisation of the modulation of the fluorescence intensity by the construction of optical interferences on oxidised silicon substrates used for DNA microarrays. The model predicts a 90-fold variation of the fluorescence signal depending on the oxide thickness. For a Cy3 dye, the signal is maximal for a 90 nm oxide thickness corresponding to a 7.5-fold enhancement with respect to a standard glass substrate. For experimental validation of the model, we have prepared Si/SiO2 substrates with different parallel steps of decreasing oxide thicknesses on the same sample using a buffered oxide etch (BOE) etching process after thermal oxidation. The SiO2 surface has been functionalized by a silane monolayer before in situ synthesis of L185 oligonucleotide probes. After hybridisation with complementary targets, the variations of the fluorescence intensity versus oxide thickness are in very good accordance with the theoretical model. The experimental comparison against a glass substrate shows a 10-fold enhancement of the detection sensitivity. Our results demonstrate that a Si/SiO2 substrate is an attractive alternative to standard glass slides for the realisation of fluorescence DNA microarrays whenever detection sensitivity is an important issue.     

Electroactive oligoaniline-containing self-assembled monolayers for tissue engineering applications

A novel electroactive silsesquioxane precursor, N-(4-aminophenyl)-N'-(4'-(3-triethoxysilyl-propyl-ureido) phenyl-1,4-quinonenediimine) (ATQD), was successfully synthesized from the emeraldine form of amino-capped aniline trimers via a one-step coupling reaction and subsequent purification by column chromatography. The physicochemical properties of ATQD were characterized using mass spectrometry as well as by nuclear magnetic resonance and UV-vis spectroscopy. Analysis by cyclic voltammetry confirmed that the intrinsic electroactivity of ATQD was maintained upon protonic acid doping, exhibiting two distinct reversible oxidative states, similar to polyaniline. The aromatic amine terminals of self-assembled monolayers (SAMs) of ATQD on glass substrates were covalently modified with an adhesive oligopeptide, cyclic Arg-Gly-Asp (RGD) (ATQD-RGD). The mean height of the monolayer coating on the surfaces was approximately 3 nm, as measured by atomic force microscopy. The biocompatibility of the novel electroactive substrates was evaluated using PC12 pheochromocytoma cells, an established cell line of neural origin. The bioactive, derivatized electroactive scaffold material, ATQD-RGD, supported PC12 cell adhesion and proliferation, similar to control tissue-culture-treated polystyrene surfaces. Importantly, electroactive surfaces stimulated spontaneous neuritogenesis in PC12 cells, in the absence of neurotrophic growth factors, such as nerve growth factor (NGF). As expected, NGF significantly enhanced neurite extension on both control and electroactive surfaces. Taken together, our results suggest that the newly electroactive SAMs grafted with bioactive peptides, such as RGD, could be promising biomaterials for tissue engineering.     

Strong Fluorescence Enhancement with Silica-Coated Au Nanoshell Dimers

Fluorescence intensity is vital for fluorescence sensing and imaging because it determines the sensing sensitivity and imaging brightness. This study reports plasmon-enhanced fluorescence by engineering plasmonic nanostructures, that are SiO₂-coated Au nanoshell dimers with a high yield exceeding 60 %. With this elaborately designed nanostructure, we show that the thin SiO₂ shell can conveniently distance the fluorophore from the underneath metal, thereby effectively avoiding fluorescence quenching. Meanwhile, the inner Au nanoshell dimers create abundant hot spots at particle-particle junctions and enable near-infrared fluorescence enhancement. The largest fluorescence enhancement achieved is 69 times for the design with a 9 nm external SiO₂ shell, as is also confirmed by three-dimensional finite-difference time-domain simulations. This dramatically increased fluorescence has great significance in fluorescence-based sensing and imaging.     

Monolayers of silver nanoparticles decrease photobleaching: application to muscle myofibrils

Studying single molecules in a cell has the essential advantage that kinetic information is not averaged out. However, since fluorescence is faint, such studies require that the sample be illuminated with the intense light beam. This causes photodamage of labeled proteins and rapid photobleaching of the fluorophores. Here, we show that a substantial reduction of these types of photodamage can be achieved by imaging samples on coverslips coated with monolayers of silver nanoparticles. The mechanism responsible for this effect is the interaction of localized surface plasmon polaritons excited in the metallic nanoparticles with the transition dipoles of fluorophores of a sample. This leads to a significant enhancement of fluorescence and a decrease of fluorescence lifetime of a fluorophore. Enhancement of fluorescence leads to the reduction of photodamage, because the sample can be illuminated with a dim light, and decrease of fluorescence lifetime leads to reduction of photobleaching because the fluorophore spends less time in the excited state, where it is susceptible to oxygen attack. Fluorescence enhancement and reduction of photobleaching on rough metallic surfaces are usually accompanied by a loss of optical resolution due to refraction of light by particles. In the case of monolayers of silver nanoparticles, however, the surface is smooth and glossy. The fluorescence enhancement and the reduction of photobleaching are achieved without sacrificing the optical resolution of a microscope. Skeletal muscle myofibrils were used as an example, because they contain submicron structures conveniently used to define optical resolution. Small nanoparticles (diameter ∼60 nm) did not cause loss of optical resolution, and they enhanced fluorescence ∼500-fold and caused the appearance of a major picosecond component of lifetime decay. As a result, the sample photobleached ∼20-fold more slowly than the sample on glass coverslips.     

141 DNA origami as a platform for assembly of nanophotonic elements

Achieving DNA-functionalized semiconductor quantum dots (QDs) that are robust enough to be compatible with the DNA nanotechnology that withstand precipitation at high temperature and ionic strength is a challenge. Here we report a method that facilitates the synthesis of stable core/shell (1–20 monolayers) QD-DNA conjugates in which the end part (5–10 nucleotides) of the phosphorothiolated oligonucleotides is embedded within the shell of the QD. These reliable QD-DNA conjugates exhibit excellent chemical, colloidal and photonic stability over a wide pH range (4–12) and at high salt concentrations (>100?mM Na⁺ or Mg²⁺), bright fluorescence emission with quantum yields of upto 70%, and broad spectral tunability with emission ranging from UV to NIR (360–800?nm). The assembly of these different QDs into DNA origami in a well-controlled pattern was demonstrated (Deng, Samanta, Nangreave, Yan, & Liu, 2012). We also used DNA origami as a platform to co-assemble a gold nanoparticle with 20?nm diameter (AuNP) and an organic fluorophore (TAMRA) and studied the distance dependent plasmonic interactions between the particle and the dye using steady state fluorescence and lifetime measurements. Greater fluorescence quenching was found at smaller inter-particle distances, which was accompanied by an enhancement of the decay rate. We further fabricated 20?nm and 30?nm AuNP homodimers with different inter-particle distances using DNA origami scaffolds and positioned a Cy3 fluorophore between the AuNPs in both the assemblies. Up to 50% enhancement of the Cy3 fluorescence quantum efficiency was observed for the dye between the 30?nm AuNPs. These results are in good agreement with the theoretical simulations (Pal et al., 2013).     

Determination of the Optical Thickness of sub 10-nm Thin Metal Films by SPR Experiments

We discuss the experimental data of surface plasmon resonance (SPR) occurring at the interface between air and single and bimetallic thin layers of Au and Ag prepared on glass substrates. The bilayer configuration allowed for the measurements of the optical constants of metallic films that are ultra thin; e.g., below 10 nm of thickness since SPR modes on such thin films in a single-layer configuration are shallow. We also discuss the effect of film thickness on SPR coupling. Thickness and refractive index of the films were determined by matching experimental SPR curves to the theoretical ones. Thickness and roughness of the films were also measured by atomic force microscopy. The results obtained by experimental measurements are in good agreement with AFM analysis.     

Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission

Metallic particles and surfaces display diverse and complex optical properties. Examples include the intense colors of noble metal colloids, surface plasmon resonance absorption by thin metal films, and quenching of excited fluorophores near the metal surfaces. Recently, the interactions of fluorophores with metallic particles and surfaces (metals) have been used to obtain increased fluorescence intensities, to develop assays based on fluorescence quenching by gold colloids, and to obtain directional radiation from fluorophores near thin metal films. For metal-enhanced fluorescence it is difficult to predict whether a particular metal structure, such as a colloid, fractal, or continuous surface, will quench or enhance fluorescence. In the present report we suggest how the effects of metals on fluorescence can be explained using a simple concept, based on radiating plasmons (RPs). The underlying physics may be complex but the concept is simple to understand. According to the RP model, the emission or quenching of a fluorophore near the metal can be predicted from the optical properties of the metal structures as calculated from electrodynamics, Mie theory, and/or Maxwell's equations. For example, according to Mie theory and the size and shape of the particle, the extinction of metal colloids can be due to either absorption or scattering. Incident energy is dissipated by absorption. Far-field radiation is created by scattering. Based on our model small colloids are expected to quench fluorescence because absorption is dominant over scattering. Larger colloids are expected to enhance fluorescence because the scattering component is dominant over absorption. The ability of a metal's surface to absorb or reflect light is due to wavenumber matching requirements at the metal-sample interface. Wavenumber matching considerations can also be used to predict whether fluorophores at a given distance from a continuous planar surface will be emitted or quenched. These considerations suggest that the so called "lossy surface waves" which quench fluorescence are due to induced electron oscillations which cannot radiate to the far-field because wavevector matching is not possible. We suggest that the energy from the fluorophores thought to be lost by lossy surface waves can be recovered as emission by adjustment of the sample to allow wavevector matching. The RP model provides a rational approach for designing fluorophore-metal configurations with the desired emissive properties and a basis for nanophotonic fluorophore technology.     

Characterization of oligodeoxyribonucleotide synthesis on glass plates

Achieving high fidelity chemical synthesis on glass plates has become increasingly important, since glass plates are substrates widely used for miniaturized chemical and biochemical reactions and analyses. DNA chips can be directly prepared by synthesizing oligonucleotides on glass plates, but the characterization of these micro-syntheses has been limited by the sub-picomolar amount of material available. Most DNA chip syntheses have been assayed using in situ coupling of fluorescent molecules to the 5′-OH of the synthesized oligonucleotides. We herein report a systematic investigation of oligonucleotide synthesis on glass plates with the reactions carried out in an automated DNA synthesizer using standard phosphoramidite chemistry. The analyses were performed using ³²P gel electrophoresis of the oligonucleotides cleaved from glass plates to provide product distribution profiles according to chain length of oligonucleotides. 5′-Methoxythymidine was used as the chain terminator, which permits assay of coupling reaction yields as a function of chain length growth. The results of this work reveal that a major cause of lower fidelity synthesis on glass plates is particularly inefficient reactions of the various reagents with functional groups close to glass plate surfaces. These problems cannot be detected by previous in situ fluorescence assays. The identification of this origin of low fidelity synthesis on glass plates should help to achieve improved synthesis for high quality oligonucleotide microarrays.     

Reduced steric hindrance and optimized spatial arrangement of carbohydrate ligands in imprinted monolayers for enhanced protein binding

Imprinted monolayers provide several advantages over bulk imprinting methods. This is especially important for large templates such as proteins. Concanavalin A (Con A)-imprinted binary monolayers consisting of glycolipids with oligo(ethylene glycol) (OEG) spacers and zwitterionic phospholipids (DPPC) were constructed and investigated. The shorter phosphorylcholine (PC) headgroups with an almost flat-on orientation in the binary monolayers gave rise to reduced steric hindrance favorable to the accommodation of Con A with greater ease and facilitated the access of the OEG-linked mannose moieties for enhanced protein binding. Further enhanced binding resulted from optimized spatial rearrangement of the glycolipids at the air–water interface directed by Con A in the subphase to create bivalent binding sites and to minimize steric crowding of neighboring mannose ligands. The combination of the exposed carbohydrate ligands from biologically inert surfaces and the optimized ligand arrangement is the most reasonable solution to enhancement of protein affinity. The bivalent carbohydrate binding sites protruding from the imprinted monolayers were created to be complementary to the Con A binding pockets. This strategy generates tailor-made surfaces with enhanced protein binding and opens the possibility of controlled assembly of intellectual biomaterials and preparation of biosensors.     

Gold Nanowire and Nanorod Plasmonic Mechanisms for Increasing Ultra-Thin Organic Photovoltaic Active Layer Absorption

We theoretically investigate the effect of incorporating gold cylindrical- and ellipsoidal-shaped nanowires and gold nanorods situated centrally within the active layer of organic bulk-heterojunction photovoltaic devices, on the optical absorption performance using finite element electromagnetic simulations. Gold cylindrical nanowire-embedded devices show increased active layer absorption enhancement with increasing radius; however, this effect decreases with the introduction of a polystyrene dielectric capping layer around the nanowires. Active layer absorption, with respect to changes in the orientation, aspect ratio, periodicity, and spacing between ellipsoidal nanowires were optimized. A maximum absorption enhancement weighted by AM 1.5 solar spectrum of 17 % is predicted for gold ellipsoidal nanowires of aspect ratio of 1.167 with in-plane horizontal orientation and arranged with periodicity of 35 nm within a 30-nm thin active layer. We attribute this enhancement primarily to interparticle electromagnetic coupling between adjacent nanowires, where, a maximum spatial and spectral overlap of the electromagnetic field with the absorption band of the active layer material is achieved. This effect increases with decreasing aspect ratio as well as decreasing periodicity with a trade-off observed between nanowire packing density and the active layer absorption enhancement. For gold nanorod-embedded organic photovoltaic devices, the inter-particle electromagnetic coupling effects are weaker and longitudinal surface–plasmon resonances supported by the nanorods are more pronounced. However, since the longitudinal surface–plasmon resonances occur at wavelengths greater than the absorption edge of the photovoltaic active layer, a mere 3.4 % increase in absorption enhancement is achieved for the photovoltaic device incorporating gold nanorods with aspect ratio of 1.167 and periodicity of 35 nm.     

Emissive Nanoclusters Based on Subnanometer‐Sized Au38 Cores for Boosting the Performance of Inverted Organic Photovoltaic Cells

In spite of the successful enhancement of the power‐conversion efficiency (PCE) in organic bulk heterojunction (BHJ) solar cells by surface plasmon resonance (SPR), the incorporation of several tens of nanometer‐sized (25–50 nm) metal nanoparticles (NPs) has some limitations to further enhancing the PCE due to concerns related to possibly transferring nonradiative energy and disturbing the interface morphology. Instead of tens of nanometer‐sized metal NPs, here, dodecanethiol stabilized Au nanoclusters (Au:SR, R = the tail of thiolate) with sub‐nm‐sized Au38 cores are incorporated on inverted BHJ solar cells. Although metal NPs less than 5 nm in size do not show any scattering or electric field enhancement of incident light by SPR effects, the incorporation of emissive Au:SR nanoclusters provides effects that are quite similar to those of tens of nanometer‐sized plasmonic metal NPs. Due to effective energy transfer, based on the protoplasmonic fluorescence of Au:SR, the highest performing solar cells fabricated with Au:SR clusters yield a PCE of 9.15%; this value represents an ≈20% increase in the efficiency compared to solar cells without Au:SR nanoclusters.     

Characterization of multilayer-enhanced surface-enhanced raman scattering (SERS) substrates and their potential for SERS nanoimaging

Multilayer gold surface-enhanced Raman scattering (SERS) substrates, which consist of continuous gold films that are separated by self-assembled monolayers (SAMs) and cast over 430-nm diameter silica nanospheres on a glass slide, have been evaluated as a means of further enhancing the SERS signals produced from conventional metal film over nanostructure substrates. Evaluation of the effect of various SAMs, with different terminal functional groups, on the SERS enhancement factor were measured and compared to conventional single-layer gold film over nanostructure substrates, revealing relative enhancements as great as 22.4-fold in the case of 2-mercapto-ethanol spacer layers. In addition to evaluation of the effect of different terminal functionalities, the effect of spacer length was also investigated, revealing that the shorter chain length alcohols provided the greatest signals. Employing the optimal SERS multilayer geometry, SERS nanoimaging probes were fabricated and the SERS enhancement factor and variability in enhancement factor were measured over the SERS active imaging area, providing absolute enhancements similar to previous silver-based SERS nanoimaging probes (i.e., 1.2 × 10⁸). Varying the size of the multilayer gold islands that were deposited on the tip of the SERS active nanoimaging probe, it is possible to tune the optimal SERS excitation wavelength accurately and predictably over the range of approximately 450 to 600 nm, without coating the entire surface of the probe and significantly reducing the transmission and resulting signal-to-noise ratio of the images obtained.     

A Computational Study of the Giant Local Electric Field Enhancement in Al-Au-Ag Trimetallic Three-Layered Nanoshells

The surface plasmon resonance (SPR)-induced local field effect in Al-Au-Ag trimetallic three-layered nanoshells has been studied theoretically. Because of having three kinds of metal, three plasmonic bands have been observed in the absorption spectra and the local electric field factor spectra. The local electric field enhancement and the corresponding resonance wavelength for different plasmon coupling modes and spatial positions of the Al-Au-Ag nanoshells with various geometry dimensions are investigated to find the maximum local electric field enhancement. The calculation results indicate that the giant local electric field enhancement could be stimulated by the plasmon coupling in the middle Au shell or the outer Ag shell and could be optimized by increasing the Ag shell thickness and decreasing the Au shell thickness. What is more, the local electric field enhancement also nonmonotonously depends on the dielectric constant of the environment; the local electric field intensity will be weakened when the surrounding dielectric constant is too small or too large.

     

Fluorescent (Au@SiO2)SiC Nanohybrids: Influence of Gold Nanoparticle Diameter and SiC Nanoparticle Surface Density

Gold@silica core–shell nanoparticles were prepared with various gold core diameters (ranging from 20 to 150 nm) and silica thicknesses (ranging from 10 to 30 nm). When the gold diameter is increased, the size dispersion became larger, leading to a broader plasmon band. Then, silicon carbide (SiC) nanoparticles were covalently immobilized onto silica to obtain hybrid (Au@SiO₂) SiC nanoparticles. The absorption properties of these hybrid nanoparticles showed that an excess of SiC nanoparticles in the dispersion can be identified by a strong absorption in the UV region. Compared to SiC reference samples, a blue shift of the fluorescence emission, from 582 to 523 nm, was observed, which was previously attributed to the strong surface modification of SiC when immobilized onto silica. Finally, the influence of several elaboration parameters (gold diameter, silica thickness, SiC concentration) on fluorescence enhancement was investigated. It showed that the highest enhancements were obtained with 10 nm silica thickness, low concentration of SiC nanoparticles, and surprisingly, with a 20-nm gold core diameter. This last result could be attributed to the broad plasmon band of big gold colloids. In this case, SiC emission strongly overlapped gold absorption, leading to possible quenching of SiC fluorescence by energy transfer.     

Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces

We used electron-beam lithography to fabricate chemical nanostructures, i.e. amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of approximately 2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of approximately 1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to approximately 2. The S/B-ratio of the fluorescence signals could be further increased to approximately 4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes.     

Theoretical Considerations on the Responses of Gold-Deposited Surface Plasmon Resonance-Based Glass Rod Sensors Using a Three-Layer Fresnel Equation

The response curves of gold (Au)-deposited surface plasmon resonance-based glass rod sensors were calculated using a three-layer Fresnel equation while considering various parameters for the sensor system calculations. Au films with thicknesses of 30, 45, and 70 nm were deposited on half of the surfaces of the glass rods, which were 2 mm in diameter, with a deposition length of 100 mm. Sensor elements with Au film thicknesses of 45 nm on glass rods with diameters of 1 and 4 mm and with deposition lengths of 10, 20, and 50 mm were also prepared. The sensor system consists of a light-emitting diode (LED) with a wavelength of 654 nm as the light source with a mini-spectrometer as the detector. The LED intensity distribution, the range of the angle of incidence of light into the sensor element, and the thickness distributions of the Au films deposited on the glass rods were considered to be the important parameters for the calculations. The minimum positions of all the theoretical response curves agreed well with those of the experimental response curves within the limits of the experimental and theoretical uncertainties. Most of the overall response characteristics of the theoretical curves agreed well with those of the experimental curves within the limits of both types of uncertainty. It was found that the thickness distribution of the deposited Au film in the cross-sectional direction dominates the sensor response and thus is the most important parameter for calculation of the sensor properties. The agreements between the experimental and theoretical response curves indicate both the potential and the usefulness of the sensor performance estimation process based on the three-layer Fresnel equation.     

Diazo coupling method for covalent attachment of proteins to solid substrates

We describe a process for covalently linking proteins to glass microscope slides and microbeads in a manner that optimizes the reactivity of the immobilized proteins and that is suitable for high-throughput microarray and flow cytometry analysis. The method involves the diazo coupling of proteins onto activated self-assembled monolayers formed from p-aminophenyl trimethoxysilane. Proteins immobilized by this method maintained bioactivity and produced enhanced levels of protein-protein interaction, low background fluorescence, and high selectivity. The binding of immobilized proteins to their specific binding partner was analyzed quantitatively and successfully correlated with solution concentrations. Diazotized surfaces bound more efficiently to proteins containing a hexahistidine tag than those without a his-tag. Moreover, significantly higher reactivity of the immobilized his-tagged proteins was observed on diazotized surfaces than on amine-terminated surfaces. Results suggest that his-tagged proteins are immobilized by reaction of the his-tag with the diazotized surface, thus offering the possibility for preferential orientation of covalently bound proteins.