Regulation of anti-LDL immobilization on self-assembled protein G layer using CHAPS and its application to immunosensor

In antibody-based immunoassay, it is important to fabricate a structured base plate with high activity for antibody immobilization. To prevent the reduction of anti-LDL binding capacity due to protein G aggregation, detergent was introduced to control the size of protein G aggregate. The self-assembled monolayer of 11-mercaptoundecanoic acid (11-(MUA)) and hexanethiol mixture was fabricated to form the stable protein G layer. 3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) was used for the regulation of protein G aggregate immobilized on the 11-(MUA) surface. The effect of various CHAPS concentrations on the amount of anti-low density lipoprotein (LDL) immobilized on protein G layer was investigated. Twelve millimolars of CHAPS were determined as the optimal concentration to maximize the binding capacity of anti-LDL due to the control of protein G density on the surface. The anti-LDL layer on self-assembled protein G using CHAPS was applied to surface plasmon resonance immunosensor for detection of LDL and its detection limit was 100 pM.     

Ellipsometric and X-ray specular reflection studies on naturally grown overlayers on aluminium thin films

The results of detailed investigations on the natural surface layer formed at room temperature on aluminium films exposed to air are presented. Aluminium films of high perfection, deposited onto very smooth glass substrates, have been studied over a 2 year period using ellipsometry. Soft X-ray specular reflection analysis revealed a composite surface layer structure composed by a thin (d₂ = 0.8 nm) very compact alumina layer in contact with the aluminium substrate and by a thick (d₃ = 3 nm) hydrated oxide layer. A new computer procedure was applied for this composite layer system, which evaluated 72 ellipsometric experimental data and achieved a best fit of the measured and calculated Ψ and Δ values. The resultant optical constants of the aluminium substrate were n = 1.09, 0.95, 0.535 and 0.370 for λ = 579 nm, 546 nm, 436 nm and 365 nm respectively, whereas k = 6.72, 6.40, 4.96 and 4.23 respectively for the same mercury lines. Among widely scattered data from the literature, these are in good agreement with results of Hass on the assumption of a similar surface layer structure, using n₂ = 1.77 (alumina) and n₃ = 1.58 (hydrated oxide).

Our optical constants for aluminium were applied for evaluating ellipsometric experimental data obtained during the 2 year period. A slight systematic change in the d₂ and d₃ values of the samples was found, owing to hydration.     

Quantitative dosing of surfaces with fluorescent molecules: characterization of fractional monolayer coverages by counting single molecules.

Quantitative deposition of dye molecules onto a substrate has been achieved at very low surface concentrations, in the range of 5 x 10(-8) - 1 x 10(-6) monolayer, using the technique of controlled substrate withdrawal from solution. These small surface populations were determined with high (>96%) efficiency by single-molecule counting using an epi-illumination, fluorescence microscope with charge-coupled device detector. The fluorescence imaging resolution (3sigma) is 0.78 microm; over a uniform excitation area of 67 x 67 microm2, a large number (>7,500) of spatially resolved channels are available for counting individual molecules. At low coverages, the number density of fluorescence spots on the surface agrees with the expected surface concentration of molecules, based on the concentration of dye in solution and the solution film thickness predicted from theory. When the surface density of molecules is high enough that fluorescence spot overlap is likely to occur within the optical resolution of the instrument, the observed fewer number of spots can be corrected for overlap through a site occupation model based on Poisson statistics.     

Influence of incoherent superposition of light on ellipsometric coefficients

Reflections from the back surface of a transparent substrate influence the evaluation of optical constants of thin films from ellipsometric measurements. If the thickness of the substrate is large compared with the coherence length of the light, the relative phase between the p and s mode, which commonly is measured by ellipsometry, cannot be defined properly. We show how the reflections from the back surface of the substrate are taken into account in ellipsometric measurements by calculating the intensities of reflections for arbitrary angles of polarization. Applications of the new method, such as transmittance ellipsometry, ellipsometry at the back surface of the substrate, and the determination of the optical constants at the substrate-layer interface, are compared with measurements.     

Internal reflection ellipsometry for real-time monitoring of polyelectrolyte multilayer growth onto tantalum pentoxide

Internal reflection ellipsometry was used for detection of the consecutive coating of two polyelectrolytes, poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA), onto a tantalum pentoxide (Ta₂O₅) substrate until the 10th bilayer. The UV patterned PAH-PAA-multilayer was characterized in air via ellipsometry and atomic force microscopy. Suited optical models enabled the determination of the layer thicknesses in wet and dry states. Linear multilayer formation could be proved by Attenuated Total Reflection — Fourier Transformed Infrared Spectroscopy measurements following the increase of the ν(C=O) band depending on the adsorption of the PAA. Streaming potential measurements after each layer deposition step indicated a change in surface charge after each layer deposition due to the consecutive coating of PAH and PAA. In this article the internal reflection ellipsometry is shown to be a convenient possibility to analyze the modification of a thin transparent Ta₂O₅ substrate.     

Macro-/nanoporous silicon as a support for high-performance protein microarrays

The present work demonstrates the possibilities of using macroporous silicon as a substrate for highly sensitive protein chip applications. The formation of 3D porous silicon structures was performed by electrochemical dissolution of monocrystalline silicon. The fabricated macroporous silicon network has a rigid spongelike structure showing high uniformity and mechanical stability. The microfluidic properties of the substrates were found to be essential for a good bioassay performance. Small spot area, good spot reproducibility, and homogeneous spot profiles were demonstrated on the substrates for immobilized aRIgG. Water contact angles were measured on the porous surface and compared to that of planar silicon, silanized glass, and ordinary microscope glass slides. The effect of the porous surface on the performance of a model IgG-binding immunoassay is presented. aRIgG was microdispensed onto the chip surface forming a microarray of spots with high affinity for the target analyte. The dispensing was performed using an in-house-developed piezoelectric flow-through dispenser. Each spot was formed by a single droplet (100 pL) at each position. The macroporous silicon allowed a high-density microarraying with spot densities up to 4400 spots/cm2 in human plasma samples without cross-talk and consumption of only 0.6 pmol of antibodies/1-cm2 array. Antigen levels down to 70 pM were detected.     

A localized surface plasmon resonance based immunosensor for the detection of casein in milk

In this research, a localized surface plasmon resonance (LSPR) immunosensor based on gold-capped nanoparticle substrate for detecting casein, one of the most potent allergens in milk, was developed. The fabrication of the gold-capped nanoparticle substrate involved a surface-modified silica nanoparticle layer (core) on the slide glass substrate between bottom and top gold layers (shell). The absorbance peak of the gold-capped nanoparticle substrate was observed at ～520 nm. In addition, the atomic force microscopy (AFM) images demonstrated that the nanoparticles formed a monolayer on the slide glass. After immobilizing anti-casein antibody on the surface, our device, casein immunosensor, could be applied easily for the detection of casein in the raw milk sample without a difficult pretreatment. Under the optimum conditions, the detection limit of the casein immunosensor was determined as 10 ng/mL. Our device brings several advantages to the existing LSPR-based biosensors with its easy fabrication, simple handling, low-cost, and high sensitivity.     

A label-free multisensing immunosensor based on imaging ellipsometry

An immunosensor based on imaging ellipsometry and its potential applications was demonstrated in this paper. It has been proven a fast, reliable, and convenient method to quantify the thickness distribution of protein layers or detect protein concentration in solution. Combined with a protein chip, the immunosensor was able to detect multiple analytes simultaneously without any labeling. Preliminary results demonstrated how this immunosensor could be used to monitor several independent biospecific binding processes in real-time and in situ conditions.     

A localized surface plasmon resonance based immunosensor for the detection of casein in milk

In this research, a localized surface plasmon resonance (LSPR) immunosensor based on gold-capped nanoparticle substrate for detecting casein, one of the most potent allergens in milk, was developed. The fabrication of the gold-capped nanoparticle substrate involved a surface-modified silica nanoparticle layer (core) on the slide glass substrate between bottom and top gold layers (shell). The absorbance peak of the gold-capped nanoparticle substrate was observed at ∼520 nm. In addition, the atomic force microscopy (AFM) images demonstrated that the nanoparticles formed a monolayer on the slide glass. After immobilizing anti-casein antibody on the surface, our device, casein immunosensor, could be applied easily for the detection of casein in the raw milk sample without a difficult pretreatment. Under the optimum conditions, the detection limit of the casein immunosensor was determined as 10 ng/mL. Our device brings several advantages to the existing LSPR-based biosensors with its easy fabrication, simple handling, low-cost, and high sensitivity.     

Kinetic and equilibrium binding analysis of protein-ligand interactions at poly(amidoamine) dendrimer monolayers

The interaction of streptavidin (SA) with a biotinylated surface has been of great interest in the development of an interfacial layer for protein immobilization based on self-assembled monolayers (SAMs) and polymeric layers. Here, we demonstrate the unique characteristics of protein-ligand interactions on dendrimer monolayers based on kinetic and equilibrium binding analyses. With amine-ended poly(amidoamine) dendrimers from the first (G1) to fourth (G4) generation, the formation of even, compact dendrimer monolayers on gold was confirmed using FT-IR spectroscopy and ellipsometry. For the SA-biotin interaction, quantitative analysis of bound SA using surface plasmon resonance showed that the saturation binding level of SA was fairly higher in all dendrimer layers when compared to other tested systems of 11-mercaptoundecylamine SAMs and a poly(L-lysine) layer. Kinetic studies revealed that the initial binding rate of SA up to the saturation level was 2-fold higher in all dendrimer layers than in the SAMs regardless of the surface density of functionalized biotin. Concurrently, the dendrimer layers led to much higher values of sticking probability, which is defined as the probability that the SA molecule adsorbs upon collision with a biotinylated surface, at a fixed SA coverage, and prolonged the significant levels around the maximum probability with increasing SA coverage. Plots of the saturation coverage of SA versus the SA concentration in solution showed that SA binding onto the biotinylated G1 and G3 layers fit to a Langmuir isotherm model. Taken together, faster binding of SA and highly ordered packing of the molecules seems to be achieved through typical properties of the dendrimer monolayers such as surface distribution of functionalized biotin, surface corrugation, and flexibility of highly branched larger dendrimers, which provides a guideline for the construction and analysis of an interfacial layer in biosensing applications.     

Influence of nanosecond laser irradiation on optical properties of surface layers of CdTe crystals

The effect of surface laser processing on the optical properties and structure of the surface region of (111) oriented CdTe single crystals have been studied by multiple-angle-of-incidence single-wavelength ellipsometry. The measurements were performed both on Cd and Te surfaces of CdTe(111) crystals and morphology was monitored by AFM. CdTe crystals were subjected to various treatments including chemical and laser etching. The ellipsometric parameters Δ and Ψ were obtained at several light incidence angles and data were interpreted based on the model of an absorbing surface layer located on an absorbing substrate. Surface roughness was taken into account and the surface layer was considered as an equivalent film with flat boundaries and effective optical parameters. The refraction n and absorption k indexes, and thickness d of the modified surface layer were calculated and features of nanosecond laser irradiation on CdTe(111) polar surfaces were discussed.     

Optical properties of soda-lime float glass from spectroscopic ellipsometry

Optical properties of soda-lime glass manufactured by the float process were investigated using spectroscopic ellipsometry and intensity transmission measurements. Thickness and optical properties of surface layers on the air and tin sides were determined with ellipsometry. The tin side surface layer shows a graded refractive index with a non-linear profile. Intensity transmission data were used to quantify absorption in the bulk glass. Transmission-mode generalized ellipsometry characterized residual birefringence in the bulk glass. Birefringence effects on ellipsometric delta data were corrected using a simple empirical offset with 1/wavelength dependence. A general optical model for soda-lime glass is presented which can be used for subsequent analysis of coated soda-lime glass and other transparent substrates.     

Optical analysis of absorbing double layers by combined reflection and transmission ellipsometry

A method known as combined ellipsometry, which utilizes the simultaneous interpretation of the ellipsometric parameters determined for light reflected and transmitted by the air side and by the substrate side of thin films, was used to obtain an optical analysis of absorbing double layers deposited onto a non-absorbing substrate. It is shown that the use of this method enables the evaluation of all the optical parameters characterizing absorbing double layers consisting of a non- absorbing and an absorbing thin film. Combined ellipsometry can be used for analysing the double layers formed by two highly absorbing thin films in a reasonable way if this method is complemented by a precise auxiliary method, e.g. the Fizeau method. These conclusions are demonstrated by experimental results obtained for samples of the following double layers: MgF₂/Al, Al/MgF₂, TiO/Au and Au/Ni. The ellipsometric parameters were measured at a wavelength λ of 632.8 nm.     

Biosensor based on imaging ellipsometry for serotype-specific detection of Riemerella anatipestifer

In this study, a practicable method for the detection of Riemerella anatipestifer (R. anatipestifer) using biosensor based on imaging ellipsometry (BIE) is described. The method is performed by immobilizing anti-R. anatipestifer egg yolk immunoglubilin (IgY) onto modified chemistry surface to form sensing layer. Antigen captured by sensing layer can then be quantitatively measured through imaging ellipsometry in grayscale format. The results demonstrate that it can detect R. anatipestifer as low as 5.2 × 10³ CFU/mL. Furthermore, the proposed method can realize specific discrimination for multiple serotypes of 1, 2, 4, and 14 of R. anatipestifer. It has the advantages of rapid, simple, high sensitivity and low cost.     

Electrochemical impedance immunosensor for the detection of cardiac biomarker Myogobin (Mb) in aqueous solution

A label-free, electrochemical impedance immunosensor based on surface modified thin flat gold wire electrode is reported for the quantitative detection of cardiac biomarker Myoglobin in aqueous solution. The protein antibody, ab-Mb, was covalently immobilized through a self assembled monolayer of 11-mercaptoundecanoic acid (MUA) and 3-mercapto propionic acid (MPA) via carbodiimide coupling reaction using N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC) and N-Hydroxy Succinamide (NHS). The immunosensor (ab-Mb/MUA-MPA/Au) was characterized by electrochemical techniques. The electrochemical performance of the immunosensor was studied by electrochemical impedance spectroscopy. The immunosensor showed an increased electrontransfer resistance on coupling with biomarker protein antigen, ag-Mb, in the presence of a redox probe [Fe (CN)₆]^3−/4−. The modified Au electrode immunosensor exhibits an electrochemical impedance response to antigen, ag-Mb concentrations in a linear range from 10 ng to 650 ng mL⁻¹ with a lowest detection limit of 5.2 ng mL⁻¹.     

Miniaturized electrospraying as a technique for the production of microarrays of reproducible micrometer-sized protein spots

Electrospraying in a stable cone-jet mode at <400 microm above a substrate is shown to be a powerful technique to produce arrays of identical micrometer-sized spots consisting of biologically active substances. Aqueous solutions with a surface tension of 0.04 N m(-1) and conductivities ranging from 0.04 to 2.2 S m(-1) were sprayed at ultralow flow rates ranging from 100 to 300 pL s(-1). The charged jet that emanates from the cone tip breaks up into a spray of charged droplets that are deposited in the form of a uniform spot of 130-350 microm in diameter by spraying during 0.5-3 s at 220-400 microm above a substrate, respectively. After a spot was deposited, spraying was stopped instantaneously by increasing the distance between the capillary tip and the substrate by an additional 100 microm using a computer-controlled x-y-z table. This was immediately followed by a rapid shift of the substrate 400 microm sideways and 100 microm upward, thus causing spraying to resume instantaneously because of the increased electric field strength, which resulted in the deposition of the next spot. It is shown here that spraying of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6P-DH), and pyruvate kinase (PK) on a liquid layer resulted in the complete preservation of their activities despite the high solution conductivity of 3.3 S m(-1) and high currents ranging from 300 to 500 nA. LDH and PK activities were fully preserved after spraying onto dry aluminum by adding 0.05 M buffer and 0.5 and 1 wt % of trehalose, respectively, to the spray solutions. Electrospraying allows for accurate dispensing of liquid volumes as small as 50 pL. Enzymatic activities of LDH and PK are fully preserved after spraying.     

Self-assembled layer of thiolated protein G as an immunosensor scaffold

In this study, our goal was to produce a self-assembled layer on a gold electrode that would enable the capture of antibodies orientated for maximum binding to their specific antigen in an immunosensor. To achieve this, the amine groups from lysine residues in protein G were initially converted to thiol groups with 2-iminothiolane. The high affinity of thiols for a gold surface facilitates the direct formation of a self-assembled protein G layer. Following this, the coated gold electrode was exposed to a solution of capture antibody (mAb1) so that these antibodies could attach to the protein G layer through their nonantigenic regions, leaving antigen binding sites available with minimal steric hindrance for binding of target analyte. A comparative study between this method and the more conventional strategy of covalently attaching a layer of nonthiolated protein G on an alkanethiol self-assembled monolayer-coated gold electrode has been performed. Based on a reduced preparation time, and an enhanced capacity for immobilized capture antibody to bind its target analyte due to a more favorable orientation, the layer of thiolated protein G was found to be a more suitable backbone for an electrochemical immunosensor.     

High-resolution mapping of redox-immunomarked proteins using electrochemical-atomic force microscopy in molecule touching mode

We explore the possibility of using molecule touching atomic force electrochemical microcopy (Mt/AFM-SECM) for high-resolution mapping of proteins on conducting surfaces. The proposed imaging strategy relies on making surface-immobilized proteins electrochemically "visible" via redox-immunomarking by specific antibodies conjugated to poly(ethylene glycol) (PEG) chains terminated by redox ferrocene (Fc) heads. The flexibility and length of the PEG chains are such that, upon approaching a combined AFM-SECM microelectrode tip toward the surface, the Fc moieties can efficiently shuttle electrons from the surface to the tip. The so-generated SECM positive feedback tip current allows the specific localized detection of the sought protein molecules on the surface. This new electrochemical imaging scheme is validated experimentally on the basis of a model system consisting of mouse IgGs adsorbed onto electrode surfaces and recognized by Fc-PEG-labeled antimouse antibodies. In order to estimate the resolution of Mt/AFM-SECM for protein imaging, regular arrays of submicrometer-sized spots of mouse IgGs are fabricated onto gold electrode surfaces using particle lithography. The Fc-PEG-immunomarked mouse IgG spots are imaged by Mt/AFM-SECM operated in tapping mode. Both an electrochemical image, reflecting the surface distribution of the redox-labeled IgGs, and a topography image are then simultaneously and independently acquired, with a demonstrated resolution in the ~100 nm range. The strength of Mt/AFM-SECM imaging is to combine the nanometric resolution of AFM with the selectivity of the electrochemical detection, potentially allowing individual target proteins to be identified amidst similarly sized "nano objects" present on a conducting surface.     

Optical and electronic characterization of transition layer in thin film Au-GaAs Schottky barrier

The possibility of determination of optical parameters and thickness of interfacial oxide (IO) layer by multiple-angle-of-incidence (MAI) ellipsometry has been studied for Au-IO-GaAs structures with different oxide thickness. The films parameters have been calculated by the original program of inverse ellipsometric problem solution. Parameters of oxide layer have been determined both for free GaAs surface and for that covered by Au film. Gold film was prepared by vacuum evaporation on the heated n-n+GaAs substrate. Its parameters have been determined by MAI ellipsometry and transparency measurements of Au-quartz satellite system. The investigation showed the change of optical parameters of GaAs oxide layer after the Au evaporation. In the assumption of invariable thickness of IO layer its optical parameters are : n = 1.0±0.1, k = 0.4 ÷ 0.6. The mechanism of current transport and electrical parameters of Au-IO-GaAs structures have been determined from I-V and C-V characteristics of Schottky diodes. The results of the determination of transition layer thickness to its dielectric constant ratio (d\_i)) from electrical characteristics of barrier also indicate its difference from initial parameters of oxide layer probably due to interdiffusion of Au, Ga, and As.     

Detection of organophosphorus compounds by covalently immobilized organophosphorus hydrolase

As a consequence of organophosphorus (OP) toxins posing a threat to human life globally, organophosphorus hydrolase (OPH) has become the enzyme of choice to detoxify such compounds. Organophosphorus hydrolase was covalently immobilized onto a quartz substrate for utilization in paraoxon detection. The substrate was cleaned and modified prior to chemical attachment. Each modification step was monitored by imaging ellipsometry as the thickness increased with each modification step. The chemically attached OPH was labeled with a fluorescent dye (7-isothiocyanato-4-methylcoumarin) for the detection of paraoxon in aqueous solution, ranging from 10(-9) to 10(-5) M. UV-visible spectra were also acquired for the determination of the hydrolysis product of para-oxon, namely p-nitrophenol.