Point-of-Need bioanalytics based on planar optical interferometry.

This review brings about a comprehensive presentation of the research on interferometric transducers, which have emerged as extremely promising candidates for viable, truly-marketable solutions for PoN applications due to the attested performance that has reached down to 10(-8) in term of effective refractive index changes. The review explores the operation of the various interferometric architectures along with their design, fabrication, and analytical performance aspects. The issues of biosensor functionalization and immobilization of receptors are also addressed. As a conclusion, the comparison among them is attempted in order to delve into and acknowledge their current limitations, and define the future trends.

Plasma nanotextured polymeric lab-on-a-chip for highly efficient bacteria capture and lysis.

We describe the design, fabrication, and successful demonstration of a sample preparation module comprising bacteria cell capture and thermal lysis on-chip with potential applications in food sample pathogen analysis. Plasma nanotexturing of the polymeric substrate allows increase of the surface area of the chip and the antibody binding capacity. Three different anti-Salmonella antibodies were directly and covalently linked to plasma treated chips without any additional linker chemistry or other treatment. Then, the Ab-modified chips were tested for their capacity to bind bacteria in the concentration range of 10(2)-10(8) cells per mL; the module exhibited 100% efficiency in Salmonella enterica serovar Typhimurium bacteria capture for cell suspensions below 10(5) cells per mL (10(4) cells injected with a 100 µL sample volume) and efficiency higher than 50% for 10(7) cells per mL. Moreover, thermal lysis achieved on-chip from as low as 10 captured cells was demonstrated and shown to compare well with off-chip lysis. Excellent selectivity (over 1 : 300) was obtained in a sample containing, in addition to S. Typhimurium and E. coli bacteria.

Direct Covalent Biomolecule Immobilization on Plasma-Nanotextured Chemically Stable Substrates.

A new method for direct covalent immobilization of protein molecules (including antibodies) on organic polymers with plasma-induced random micronanoscale topography and stable-in-time chemical functionality is presented. This is achieved using a short (1-5 min) plasma etching and simultaneous micronanotexturing process, followed by a fast thermal annealing step, which induces accelerated hydrophobic recovery while preserving important chemical functionality created by the plasma. Surface-bound biomolecules resist harsh washing with sodium dodecyl sulfate and other detergents even at elevated temperatures, losing less than 40% of the biomolecules bound even at the harshest washing conditions. X-ray photoelectron spectroscopy, secondary-ion mass spectrometry, and electron paramagnetic resonance are used to unveil the chemical modification of the plasma-treated and stabilized surfaces. The nanotextured and chemically stabilized surfaces are used as substrates for the development of immunochemical assays for the sensitive detection of C-reactive protein and salmonella lipopolysaccharides through immobilization of the respective analyte-specific antibodies onto them. Such substrates are stable for a period of 1 year with ambient storage.

Improved DNA microarray detection sensitivity through immobilization of preformed in solution streptavidin/biotinylated oligonucleotide conjugates.

A novel immobilization approach involving binding of preformed streptavidin/biotinylated oligonucleotide conjugates onto surfaces coated with biotinylated bovine serum albumin is presented. Microarrays prepared according to the proposed method were compared, in terms of detection sensitivity and specificity, with other immobilization schemes employing coupling of biotinylated oligonucleotides onto directly adsorbed surface streptavidin, or sequential coupling of streptavidin and biotinylated oligonucleotides onto a layer of adsorbed biotinylated bovine serum albumin. A comparison was performed employing biotinylated oligonucleotides corresponding to wild- and mutant-type sequences of seven single point mutations of the BRCA1 gene. With respect to the other immobilization protocols, the proposed oligonucleotide immobilization approach offered the highest hybridization signals (at least 5 times higher) and permitted more elaborative washings, thus providing considerably higher discrimination between complimentary and non-complementary DNA sequences for all mutations tested. In addition, the hybridization kinetics were significantly enhanced compared to two other immobilization protocols, permitting PCR sample analysis in less than 40 min. Thus, the proposed oligonucleotide immobilization approach offered improved detection sensitivity and discrimination ability along with considerably reduced analysis time, and it is expected to find wide application in DNA mutation detection.

Monolithically integrated broad-band Mach-Zehnder interferometers for highly sensitive label-free detection of biomolecules through dual polarization optics.

Protein detection and characterization based on Broad-band Mach-Zehnder Interferometry is analytically outlined and demonstrated through a monolithic silicon microphotonic transducer. Arrays of silicon light emitting diodes and monomodal silicon nitride waveguides forming Mach-Zehnder interferometers were integrated on a silicon chip. Broad-band light enters the interferometers and exits sinusoidally modulated with two distinct spectral frequencies characteristic of the two polarizations. Deconvolution in the Fourier transform domain makes possible the separation of the two polarizations and the simultaneous monitoring of the TE and the TM signals. The dual polarization analysis over a broad spectral band makes possible the refractive index calculation of the binding adlayers as well as the distinction of effective medium changes into cover medium or adlayer ones. At the same time, multi-analyte detection at concentrations in the pM range is demonstrated.

All-silicon monolithic Mach-Zehnder interferometer as a refractive index and bio-chemical sensor.

A complete Mach-Zehnder interferometer monolithically integrated on silicon is presented and employed as a refractive index and bio-chemical sensor. The device consists of broad-band light sources optically coupled to photodetectors through monomodal waveguides forming arrays of Mach-Zehnder interferometers, all components being monolithically integrated on silicon through mainstream silicon technology. The interferometer is photonically engineered in a way that the phase difference of light travelling through the sensing and reference arms is approximately wavelength independent. Consequently, upon effective medium changes, it becomes feasible even with a broad-band source to induce sinusoidal-type of detector photocurrents similar to the classical monochromatic counterparts. The device is completed with its fluidic and interconnect components so that on chip interferometric measurements can be performed. Examples of refractive index and protein sensing are presented to establish the potential of the proposed device for real-time in situ monitoring applications. This is the only silicon device that has achieved complete on-chip interferometry.

Plasma-assisted nanoscale protein patterning on Si substrates via colloidal lithography.

Selective immobilization of proteins in well-defined patterns on substrates has recently attracted considerable attention as an enabling technology for applications ranging from biosensors and BioMEMS to tissue engineering. In this work, a method is reported for low-cost, large scale and high throughput, selective immobilization of proteins on nanopatterned Si, based on colloidal lithography and plasma processing to define the areas (<300 nm) where proteins are selectively immobilized. A close-packed monolayer of PS microparticles is deposited on oxidized Si and, either after microparticle size reduction or alternatively after metal deposition through the PS close-packed monolayer, is used as etching mask to define SiO2 nanoislands (on Si). C4F8 plasma was used to selectively etch and modify the SiO2 nanoislands while depositing a fluorocarbon layer on the Si surface. The plasma-treated surfaces were chemically characterized in terms of functional group identification through XPS analysis and reaction with specific molecules. Highly selective protein immobilization mainly through physical adsorption on SiO2 nanoislands and not on surrounding Si was observed after C4F8 plasma-induced chemical modification of the substrate. The thickness of the immobilized protein monolayer was estimated by means of AFM image analysis. The method reported herein constitutes a cost-efficient route toward rapid, large surface, and high-density patterning of biomolecules on solid supports that can be easily applied in BioMEMS or microanalytical systems.

Protein adsorption and covalent bonding to silicon nitride surfaces modified with organo-silanes: comparison using AFM, angle-resolved XPS and multivariate ToF-SIMS analysis.

Organo-silanes provide a suitable interface between the silicon-based transducers of various biosensing devices and the sensing proteins, immobilized through physical adsorption, as for (3-aminopropyl)triethoxysilane (APTES), or covalent binding, e.g. via protein amine groups to (3-glycidoxypropyl)trimethoxysilane (GOPS) modified surface. Immobilization of rabbit gamma globulins (RgG) to silicon nitride surfaces, modified either with APTES or GOPS, was examined as a function of incubation time using atomic force microscopy (AFM), angle-resolved X-ray photoelectron spectroscopy (ARXPS) and time of flight secondary ion mass spectrometry (ToF-SIMS). Multivariate technique of principal component analysis was applied to ToF-SIMS spectra in order to enhance sensitivity of immobilized RgG detection. Principal component regression shows a linear relationship with surface density determined rigorously from ARXPS following an organic bilayer approach, allowing for protein coverage quantification by ToF-SIMS. Taking it overall the surface immobilized amount of RgG is higher and develops faster on the surfaces silanized with APTES rather than with GOPS. Similar, although less distinct, difference is observed between the two surface types concerning the temporal evolution of average AFM height. The average height of protein overlayer correlates well with ARXPS and ToF-SIMS data expressed in terms of protein surface density. However, determined linear regression coefficients are distinctively higher for the surfaces modified with epoxy- rather than amino-silane, suggesting different surface density and conformation of the proteins immobilized through to covalent binding and physical adsorption.

Model immunoassay on silicon surfaces: vertical and lateral nanostructure vs. protein coverage.

To provide complete characterization of immunoassay on silicon biosensor surfaces, atomic force microscopy, (angle-resolved) X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry were applied to examine Si(3)N(4) surfaces modified with (3-aminopropyl)triethoxysilane, coated with gamma globulins (IgG), blocked with bovine serum albumin and then reacted with anti-IgG antibody for two complementary pairs (rabbit and mouse IgG) at various concentrations (from 0.3 nM to 330 nM). Protein coverage, as reflected in (amine to total N1s) XPS signal ratio and determined from ARXPS, decreases slightly due to blocking and then increases monotonically for anti-IgG antibody concentrations higher than 1 nM. AFM images reveal hardly any change of lateral nanostructure due to blocking but response to antibody solutions, based on both the mean size (from autocorrelation) and dominant spacing (from Fourier analysis) of surface features, similar to that given by ARXPS. AFM height histograms provided information about the vertical nanostructure and the parameters of height distribution (average height, spread - roughness and skewness) were distinctly influenced by coating, blocking and immunoreaction. Average protein layer thickness values determined based on protein structure (molecular weight, dimensions) and surface coverage provided from ARXPS were in accord with average height of protein layer determined from AFM. TOF-SIMS analysis indicated that BSA blocks free surface sites and in addition replaces some already adsorbed IgGs.

High-capacity and high-intensity DNA microarray spots using oxygen-plasma nanotextured polystyrene slides.

Commercially available polystyrene (PS) slides were plasma nanotextured (nano-roughened) through treatment in oxygen plasma discharges to create substrates with increased surface area for microarray applications. Conditions of plasma treatment were determined for maximum and uniform oligonucleotide immobilization on these nanotextured PS slides. Oligonucleotides were immobilized onto the surface in the form of biotinylated oligonucleotide/streptavidin conjugates to take advantage of increased protein binding capacity of the substrate. It was found that the amount of oligonucleotides that could be immobilized was increased up to ten times on plasma treated as compared with untreated slides. The sensitivity of detection of labelled hybridized probes was improved by a factor of 20. Optimized nanotextured PS slides were subsequently used to develop a microarray for the detection of three deleterious BRCA1 gene mutations by immobilizing oligonucleotides corresponding to wild and mutant-type sequences. The microarray developed on the nanotextured PS slides provided higher specific hybridization signal and discrimination ratios as compared with flat untreated PS slides.

Creating highly dense and uniform protein and DNA microarrays through photolithography and plasma modification of glass substrates.

We demonstrate a method to create high density protein microarrays with excellent spot uniformity using photolithography and plasma processing on low cost commercially available microscope glass slides. Protein deposition and fluorescence signal evaluation on these substrates are performed by standard arrayers and scanners. To this end, spots of commercial photoresists (AZ5214, SU8 and Ormocomp(®)) were defined through lithography on glass substrates followed by short SF(6) plasma treatment and selective protein adsorption on these spots with respect to glass (spot to background fluorescence signal ratios 30:1 to 40:1) was demonstrated using model protein binding assays. Among the photoresists tested, Ormocomp was selected since it provided the highest protein binding capacity. No ageing of Ormocomp/glass substrates in terms of protein binding capacity was observed for at least two months. Besides to protein microarrays, DNA microarrays were also developed by spotting streptavidin-biotinylated oligonucleotide conjugates corresponding to wild- and mutant-type sequences of four deleterious BRCA1 gene mutations. For all of the examined mutations, higher specific hybridization signals (1.5-4 times) and improved discrimination ratios between wild- and mutant-type sequences as well as higher spot uniformity and repeatability were demonstrated on Ormocomp/glass substrates with intra- and inter-spot CVs of 8.0% and 4.5%, respectively, compared to commercial polystyrene (intra- and inter-spot CVs 36% and 18%) and epoxy-coated glass (intra- and inter-spot CVs 26% and 20%) slides. Thus, the proposed substrates can be readily applied to protein and DNA microarrays fabrication and, moreover, the described method for selective protein adsorption can be advantageously implemented in various analytical microdevices for multi-analyte detection.

Spectroscopic and microscopic characterization of biosensor surfaces with protein/amino-organosilane/silicon structure.

Composition and structure of biorecognition protein layers created on silicon substrates modified with amino-organosilanes determine the sensitivity and specificity of silicon based biosensing devices. In the present work, diverse spectroscopic and microscopic methods were applied to characterize model biosensor surfaces, formed on Si(3)N(4) or SiO(2) by modification with (3-aminopropyl)triethoxysilane, coating with rabbit gamma-globulins (IgGs) through physical adsorption, blocking with bovine serum albumin (BSA) and specific binding of an anti-rabbit IgG antibody. In addition, silanized substrates with directly adsorbed BSA or anti-rabbit IgG antibody were examined as reference surfaces. The protein/amino-organosilane/silicon structure of all surfaces was confirmed by X-ray photoelectron spectroscopy. Homogeneity of protein coverage was verified with near-field scanning optical microscope, working in reflection and fluorescence mode. Surface coverage with proteins was determined with angle-resolved XPS using a previously established bilayer approach. Inner structure of protein layers was examined with atomic force microscopy. Vertical arrangement of carbon functional groups was revealed by high resolution ARXPS. Combined spectroscopic and microscopic data reveal the complex character of interactions with the immobilized IgG molecules during blocking with BSA and immunoreaction with anti-IgG antibody. Within experimental error, neither surface coverage nor lateral structural scales of protein layer (provided by Fourier and auto-correlation analysis of topographic and phase images) increase during blocking procedure. On the other hand, coverage and all structural measures rise considerably after immunoreaction. In addition, it was found that polar functional groups orient towards substrate for all protein layers, independently of coverage, prior to and after both blocking and specific binding.

Plasma nanotextured PMMA surfaces for protein arrays: increased protein binding and enhanced detection sensitivity.

Poly(methyl methacrylate) (PMMA) substrates were nanotextured through treatment in oxygen plasma to create substrates with increased surface area for protein microarray applications. Conditions of plasma treatment were found for maximum uniform protein adsorption on these nanotextured PMMA surfaces. Similar results were obtained using both a high-density plasma (HDP) and a low-density reactive ion etcher (RIE), suggesting independence from the plasma reactor type. The protein binding was evaluated by studying the adsorption of two model proteins, namely, biotinylated bovine serum albumin (b-BSA) and rabbit gamma-globulins (RgG). The immobilization of these proteins onto the surfaces was quantitatively determined through reaction with fluorescently labeled binding molecules. It was found that the adsorption of both proteins was increased up to 6-fold with plasma treatment compared to untreated surfaces and up to 4-fold compared to epoxy-coated glass slides. The sensitivity of detection was improved by 2 orders of magnitude. Moreover, highly homogeneous protein spots were created on optimized plasma-nanotextured surfaces through deposition with an automated microarray spotter, revealing the potential of plasma-nanotextured surfaces as protein microarray substrates.

Multi-analyte capillary immunosensor for the determination of hormones in human serum samples.

In this work we present the development of a multi-analyte immunosensor for the determination of follitropin, human chorionic gonadotropin and prolactin in human serum. The immunosensor is based on plastic capillaries. According to the methodology, discrete areas of the internal capillary surface are coated with different antibodies, which are highly specific for each one of the analytes to be determined. The sample that will be analyzed along with a mixture of analyte-specific biotinylated antibodies is introduced into the capillary. The coated and the detection antibodies react with different epitopes of the analytes in the sample to form a 'sandwich'. The detection is based on reaction of the immobilized biotinylated antibody with streptavidin labeled with R-phycoerythrin. The fluorescent areas formed were quantified by scanning the capillary with a light beam of appropriate wavelength. A light sensor placed at the end of the capillary detects the emitted photons, that are trapped and waveguided into the capillary walls. The multi-analyte immunosensor assays were characterized by high specificity and short analysis time. In addition, the results obtained by the multi-analyte optical capillary immunosensor were comparable to those obtained by immunofluorimetric assays performed in microtitration wells. Potential applications of the proposed immunosensor include determination of several analyte panels in a broad spectrum of disciplines such as endocrinology, hematology, and oncology.

Electron beam patterning of biomolecules.

The patterning of biomolecules on semiconducting surfaces is of central importance in the fabrication of novel biodevices. In the process of patterning, it is required that the biomolecule preserves its properties and the substrate is not damaged by the chemicals, the temperatures or the patterning beams involved in the procedure. Recently, both DUV and electron beam microlithography have been used in order to deposit protein layers in predefined patterns. Various approaches have been used, some involving photoresists. Contrast between exposed and unexposed regions, resolution of adjacent features and sensitivity to dose variation, are the key issues. The approach followed in this paper consists of a direct patterning of a biotin layer, deposited on an amino-silane primed silicon nitride surface, using an electron beam. After irradiation, the surface is covered by bovine serum albumin (BSA), which acts as a blocking material to protect the exposed areas from streptavidin adsorption. Using 20 keV e-beam energy and doses, in the range 100-1000 microC/cm(2), submicrometer dense lines of 1-microm pitch have been obtained. The results have been tested by fluorescence optical microscopy.

Improved antibody coating protocol using a second antibody antiserum. Application to total thyroxin immunoassay.

A complete antibody coating protocol for the preparation of dry antibody coated tubes is presented. This protocol is based on a recently described antibody immobilization principle. We modify this immobilization principle in order to improve and simplify the coating procedure. In addition, we propose a drying procedure that provides long-term storage stability of the antibody coated tubes. According to the modified protocol, polystyrene plastic tubes are first coated with rabbit gamma-globulins. The tubes are incubated with a sheep anti-rabbitIgG antiserum dilution. After incubation, antigen-specific antibody antiserum raised in rabbits is added directly into the tubes containing the sheep anti-rabbit IgG antiserum solution (difference from the original protocol). Finally, the tubes are washed, blocked, and dried following the drying procedure developed. The suitability of the modified protocol for the development of immunoassays requiring high loading of antibody was exemplified through the development of a RIA for total thyroxin. The estimated assay characteristics (detection limit 4 microg/L, dynamic range up to 210 microg/L, within-run CV 2.7-5.7%, between-run CV 5.1-7.3%, recovery 84.4-112%, cross-reactivity for T3 1.9%) were comparable with those provided by commercially available RIA kits for the determination of thyroxin.

Heterogeneous fluoroimmunoassays using fluorescein as label with measurement of the fluorescence signal directly onto the solid-phase.

A heterogeneous fluoroimmunoassay approach with measurement of the fluorescence signal directly onto the solid support is described. Fluorescein was employed as fluorescent label whereas plastic microtitration wells were used as solid carriers. The influence of the type of the plastic wells on the proposed approach was investigated. Different types of commercially available plastic microtitration wells (white-opaque, black, and transparent) were tested. The solid supports were judged on the grounds of the maximum fluorescence signal, the precision, and the sensitivity they provided in a model non-competitive assay for mouse IgG. Among the solid supports tested, the white-opaque wells provided the most promising results with respect to the parameters in question. This support was also used for the development of a competitive fluoroimmunoassay for the determination of total thyroxin in human serum samples in order to assess the validity of the proposed approach under real immunoassay conditions. The assay developed had appropriate analytical characteristics for the determination of thyroxin and the serum sample values obtained were well correlated with those determined by a commercial solid-phase radioimmunoassay.

Antibody coating approach involving gamma globulins from non-immunized animal and second antibody antiserum.

An alternative protocol for immobilization of antibodies onto plastic solid supports is presented. According to the proposed protocol, tubes are first coated with gamma-globulins from non-immunized animal of the same species as that from which the antigen-specific antibody has been developed. Then, an excess of second antibody is added to the tubes and the anti-species specific antibodies present in the antiserum are immunoadsorbed on the immobilized gamma-globulins. Finally, the antigen specific antibody is immunoadsorbed on the immobilized second antibody. We found that the coating protocol developed allows the use of antigen-specific and second antibody antisera dilutions, thus avoiding the need for affinity purification of antibodies. Additionally, it provides solid-phase second antibody with increased binding capacity compared to the directly adsorbed onto the solid second antibody. The advantages of the proposed coating protocol were demonstrated through the development of a solid-phase radioimmunoassay for the determination of total triiodothyronine in human serum samples.

A multi-band capillary immunosensor.

In the present work we propose a new optical immunosensor based on capillary geometry and capable of multianalyte determinations. The device is made of a polystyrene capillary tube. The inner walls of the capillary are segmented into distinct bands which are coated with appropriate binding molecules. Following excitation, some of the fluorescent photons emitted by the label are trapped and waveguided into the capillary walls provided they are launched towards the walls and within the critical angle. Here, Europium-labeled streptavidin reacted with different amounts of biotinylated bovine serum albumin immobilized onto each one of the bands. Due to the small inner volume of the capillary and the multianalyte feature we expect that the proposed device can be used for fast and inexpensive assays.