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The potential of fluorescence resonance energy transfer (FRET) in a photonic crystal (PC) nanostructured array to enhance the speed and sensitivity of a protein-based immunoassay was tested. Forty-nanometer carboxylated particles conjugated with donor-labeled capture antibodies were trapped by electrophoresis and used as a FRET energy donor. The PC array was able to enhance fluorescent excitation and emission by phase matching. To provide a proof of concept for this FRET-based homogeneous assay on a PC chip, an immunoassay was tested with a simple immunoglobulin G (IgG)-based reaction. A standard curve was generated by testing two different antibody reaction times: 20 min. and 1 min. The results were compared directly to those obtained from a FRET assay that used a modern, high-sensitivity plate reader with a 96-well plate and a reaction time of 1 h. The rabbit-IgG detection limits of the FRET-based homogeneous assay on the PC were 0.001 and 0.1 μg/mL for incubation times of 20 and 1 min, respectively; the sensitivities were 10(3) and 10 times better than the 96-well plate reader, respectively. Thus, FRET on a PC immunoplatform was shown to be a facile, effective, rapid, and sensitive detection technology.

One of the challenges in shrinking immunoassays to smaller sizes is to immobilize the biological molecules to nanometer-scaled spots. To overcome this complication, we have employed a particle-based immunoassay to create a nanostructured platform with a regular array of sensing elements. The technique makes use of an electrophoretic particle entrapment system (EPES) to immobilize nanoparticles that are coated with biological reagents into wells using a very small trapping potential. To provide useful information for controlling the trapping force and optimal design of the nanoarray, electrophoretic trapping of a nanoparticle was modeled numerically. The trapping efficiency, defined as the fraction of wells occupied by a single particle, was 91%. The performance of the array was demonstrated with a competitive immunoassay for a small molecule analyte, 3-phenoxybenzoic acid (214.2 g mole(-1)). The limit of detection determined with a basic fluorescence microscope was 0.006 μg l(-1) (30 pM); this represented a sixteen-fold improvement in sensitivity compared to a standard 96-well plate-based ELISA; the improvement was attributed to the small size of the sample volume and the presence of light diffraction among factors unique to this structure. The EPES/nanoarray system promises to offer a new standard in applications that require portable, point-of-care and real-time monitoring with high sensitivity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Electrophoretic particle entrapment system (EPES) is employed to generate 2D array of nanoparticles coated with biological molecules (i.e., antibodies). Phase matching of the excitation and the emission in the 2D arrays with particles produces a highly enhanced fluorescence signal that was shown to improve the limit of detection in immunoassays. The phase matching is achieved when the particle are in the sub-100 nm range. A comparison between different size particles shows that the sensitivity of an immunoassay is extended to a range that is difficult to achieve with standard technology (e.g., enzyme-linked immunosorbent assay-ELISA). The effectiveness of this novel configuration of particle-in-a-well was demonstrated with an assay for human epidermal growth factor receptor 2 (HER2; breast cancer biomarker), with a detection limit as low as 10 attomolar (aM) in less than 10 μL of serum-based sample. The limit of detection of HER2 indicated far superior assay performance compared to the corresponding standard 96-well plate-based ELISA. The particle-based photonic platform reduces the reagent volume and the time for performing an assay in comparison to competing methods. The simplicity of operation and the level of sensitivity demonstrated here can be used for rapid and early stage detection of biomarkers.

A rapid and simple magnetic particle-based immunoassay has been demonstrated in a capillary mixing system. Antibody-coated micrometer size superparamagnetic polystyrene (SPP) particles were used in an assay for rabbit IgG in a sandwich (noncompetitive) format. The kinetics of the assay was compared between a plate-based system and a single capillary tube. The interaction between the antigen (R-IgG) and the antibody (anti-R-IgG) that was carried by the SPP particles in a rotating capillary was tested under a stationary magnetic field. Competing magnetic and viscous drag forces helped to enhance the interaction between the analyte and the capture antibodies on the particles. The dimensionless Mason number (Mn) was employed to characterize the magnetic particle dynamics; a previously determined critical Mason number (Mn(c)) was employed as a guide to the appropriate experimental conditions of magnetic field strength and rotational speed of the capillary. The advantage of the rotating capillary system included a short assay time and a reduced reactive volume (20 μL). The results show that the immunoassay kinetics were improved by the formation of chains of the SPP particles for the conditions that corresponded to the critical Mason number.

Lanthanide oxide nanoparticles are promising luminescent probes in bioanalysis, because of their unique spectral properties, photostability, and low-cost synthesis. We report for the first time the application of europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles to the optical imaging of antibody micropatterns. The nanoparticles were synthesized by spray pyrolysis and coated with antibody (IgG) molecules by physical adsorption. Our experiments showed that the Eu:Gd2O3 is a good biocompatible solid support for antibody immobilization. The antibodies (anti-rabbit IgG) immobilized on the nanoparticles had excellent biological activity in the specific recognition reaction with rabbit IgG patterned in line strips (10 μm×10 μm) on a glass substrate by use of a micro-contact printing technique. The specific immunoreaction was confirmed by two independent microscopic techniques—fluorescence and scanning electron microscopy (SEM). Both microscopic images revealed that the nanoparticles were organized into designated structures as defined by the microcontact printing process with negligible non-specific binding. The nanoparticles can be used as fluorescent markers in a variety of immunosensing applications in a microscale format. [ABSTRACT FROM AUTHOR]

A rapid and simple magnetic particle-based immunoassay has been demonstrated in a capillary mixing system. Antibody-coated micrometer size superparamagnetic polystyrene (SPP) particles were used in an assay for rabbit IgG in a sandwich (noncompetitive) format. The kinetics of the assay was compared between a plate-based system and a single capillary tube. The interaction between the antigen (R-IgG) and the antibody (anti-R-IgG) that was carried by the SPP particles in a rotating capillary was tested under a stationary magnetic field. Competing magnetic and viscous drag forces helped to enhance the interaction between the analyte and the capture antibodies on the particles. The dimensionless Mason number (Mn) was employed to characterize the magnetic particle dynamics; a previously determined critical Mason number (Mnc) was employed as a guide to the appropriate experimental conditions of magnetic field strength and rotational speed of the capillary. The advantage of the rotating capillary system included a short assay time and a reduced reactive volume (20 μL). The results show that the immunoassay kinetics were improved by the formation of chains of the SPP particles for the conditions that corresponded to the critical Mason number. [ABSTRACT FROM AUTHOR]

Nanoscale wells have been fabricated in a chip to construct a photonic crystal that is used for enhanced immunoassays of a common food-borne toxin, Staphylococcal enterotoxin B (SEB). The nanostructure of the photonic crystal (PC) in the array enhanced the fluorescent signal due to a guided mode resonance. Nanoparticles were used as the solid substrate for attachment of capture antibodies; the particles were then isolated in individual wells of the chip by using an electrophoretic particle entrapment system (EPES). The standard curve generated from the chip consisted of two log-linear regions: the first region with a greater sensitivity, limited by the Kd of the antibody, resembling the 96-well plate ELISA and the other that shows greater than six orders of linearity extending to attomolar concentrations, which is unique to the device we have developed. SEB dissolved in phosphate buffered saline was resolved to levels as low as 35 aM with 106-fold better limit of detection than a conventional 96-well-ELISA. Different concentrations of SEB spiked into milk were tested to assess the reliability of the device and the efficacy of the extended log-linear regime in a "real" food matrix. The presence of the milk did not significantly alter the limit of detection. With very low amounts of sample (less than 10 μL) and fast read-out time, the PC-based system shows great promise for the detection of a wide range of target molecules with close to a single molecule level of sensitivity. [ABSTRACT FROM AUTHOR]

Abstract: Suspension arrays present a promising tool for multiplexed assays in large-scale screening applications. A simple and robust platform for quantitative multiprotein immunoanalysis has been developed with the use of magnetic Co:Nd:Fe2O3/luminescent Eu:Gd2O3 core/shell nanoparticles (MLNPs) as a carrier. The magnetic properties of the MLNPs allow their manipulation by an external magnetic field in the separation and washing steps in the immunoassay. Their optical properties enable the internal calibration of the detection system. The multiplexed sandwich immunoassay involves dual binding events on the surface of the MLNPs functionalized with the capture antibodies. Secondary antibodies labeled with conventional organic dyes (Alexa Fluor) are used as reporters. The amount of the bound secondary antibody is directly proportional to the concentration of the analyte in the sample. In our approach, the fluorescence intensity of the reporter dye is related to the luminescence signal of the MLNPs. In this way, the intrinsic luminescence of the MLNPs serves as an internal standard in the quantitative immunoassay. The concept is demonstrated for a simultaneous immunoassay for three model proteins (human, rabbit, and mouse IgGs). The method uses a standard bench plate reader. It can be applied to disease diagnostics and to the detection of biological threats. [Copyright &y& Elsevier]

Currently, detection in microarray bioanalysis is based mainly on the use of organic dyes. To overcome photo-bleaching and spectral overlaps we applied a new type of fluorophore, crystalline europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles, as labels in immunoassay microarrays. The Eu:Gd2O3 nanoparticles synthesized by spray pyrolysis offer narrow red emission, large Stokes shift, photostable laser-induced fluorescence with a long lifetime (1 ms). The amino functionalization of the particles was achieved by poly(L-lysine) (PL) encapsulation. The formation of a stable PL shell was confirmed by TEM analysis, colloidal stability studies, and quantification of the surface reactive amino groups. The PL-encapsulatecl particles were covalently conjugated to antibodies and successfully applied as reporters in a competitive fluorescence microimmunoassay for phenoxybenzoic acid (PBA), a generic biomarker of human exposure to pyrethroid insecticides. Microarrays were fabricated by microcontact printing of BSA-PBA in line patterns (10 × 10 μm). Confocal fluorescence microscopy combined with internal standard (fluorescein) calibration was used for quantitative measurements. The microarray immunoassay demonstrated a limit of detection of 1.4 μg L-1 PBA. This work suggests the potential application of lanthanide oxide nanoparticles as fluorescent probes in microarray and biosensor technology, immunodiagnostics, and high-throughput screening. [ABSTRACT FROM AUTHOR]

A method for simply and cheaply preparing inorganic phosphor nanoparticles of Eu203 as labels in biology has been demonstrated with a simple microwave-assisted surface chemistry. The capping process adds a silane layer to the surface of the particles and provides amine groups that can be used for biological conjugation. The surface layer also protects the particles during conjugation chemistry. The particles retain their desirable optical properties that are typical of europium, that is, a spectrally narrow, red emission and a long fluorescence lifetime. The application of the nanoparticle labels in an immunoassay yields very good sensitivity in an immunoassay for atrazine (sub-parts-per-billion detection limit) without optimization of the detection system. The microwave functionalization technique will permit a broad range of inorganic nanophase phosphors to be used in high-throughput assays for environmental monitoring. [ABSTRACT FROM AUTHOR]

A fluorescence quenching competitive immunoassay in micro droplets was applied to the sensitive detection of the pyrethroid insecticide, esfenvalerate. Laser induced fluorescence from rhodamine dye was used as a marker. The competitive immunoreaction was performed in micro droplets generated by a vibrating orifice aerosol generator system with a 10-μm diameter orifice. Fluorescence that was emitted from the droplets was detected by a 1/8 m imaging spectrograph with a 512×512 thermoelectrically cooled, charged-coupled device camera. The conjugate of esfenvalerate with rhodamine exhibited similar fluorescence to that of pure rhodamine 6G. When anti-esfenvalerate antibodies were added to the droplets, the fluorescence decreased. The reduction in emission was due to a strong quenching effect that arises from the interaction between the protein and rhodamine molecules following the antigen–antibody reaction. When a sample of esfenvalerate was added to the droplets, the release of the conjugated rhodamine from the antigen–antibody complex allowed the fluorescence signal to recover. An assay in a picoliter droplet sample was shown to enable detection down to approximately 0.1 nM. A very small mass of analyte could be detected with this method. A sample of river water was used to gauge the impact of matrix effects and was shown to give rise to negligible interference with the immunoassay. [Copyright &y& Elsevier]