Highly Fluorescent Magnetic Nanobeads with a Remarkable Stokes Shift as Labels for Enhanced Detection in Immunoassays

Fluorescence immunoassays are popular for achieving high sensitivity, but they display limitations in biological samples due to strong absorption of light, background fluorescence from matrix components, or light scattering by the biomacromolecules. A powerful strategy to overcome these problems is introduced here by using fluorescent magnetic nanobeads doped with two boron‐dipyrromethane dyes displaying intense emission in the visible and near‐infrared regions, respectively. Careful matching of the emission and absorption features of the dopants leads to a virtual Stokes shift larger than 150 nm achieved by an intraparticle Förster resonance energy transfer (FRET) process between the donor and the acceptor dyes. Additionally, the magnetic properties of the fluorescent beads allow preconcentration of the sample. To illustrate the usefulness of this approach to increase the sensitivity of fluorescence immunoassays, the novel nanoparticles are employed as labels for quantification of the widely used Tacrolimus (FK506) immunosuppressive drug. The FRET‐based competitive inhibition immunoassay yields a limit of detection (LOD) of 0.08 ng mL^?1, with a dynamic range (DR) of 0.15–2.0 ng mL^?1, compared to a LOD of 2.7 ng mL^?1 and a DR between 4.1 and 130 ng mL^?1 for the immunoassay carried out with direct excitation of the acceptor dye.     

Immobilization method for the preparation of biosensors based on pH shift-induced deposition of biomolecule-containing polymer films.

Miniaturization of amperometric biosensors is crucially dependent on the availability of methods for the nonmanual immobilization of biological recognition elements on the transducer surface. From an aqueous polymer suspension, the precipitation of a polymer film with entrapped biological recognition elements is initiated by electrochemically induced oxidation of H20 at the electrode surface. Using the locally generated H+ gradient, acidic side chains of the polymer are titrated, leading to a change in the polymer solubility and hence to the controlled deposition of a polymer film. To investigate the properties and limitations of this immobilization technology, the specific features of a glucose biosensor based on polymer-entrapped glucose oxidase and amperometric detection of enzymatically generated H202 were investigated. Besides the reproducibility of the immobilization procedure, the sensitivity (14.59 mA cm(-2) M(-1) at pH 7), long-term stability (up to 5000 measurements in a sequential-injection analyzer), dependence on enzyme concentration, polymer thickness, and possibilities to fabricate multilayer sensor architectures were exploited. In addition, the miniaturization potential of this nonmanual immobilization technology was evaluated by investigating the modification of microband electrode arrays and cross talk between the neighboring microsensors.     

Extreme ultra-violet resists development concepts and performances

Recently published experimental results indicate that current extreme ultra-violet lithography (EUVL) patterning process seems to be very hard to meet the device manufacturing specification goals, such as resolution, line-width roughness, and sensitivity (RLS) simultaneously. To overcome trade-off limitations between RLS performances of resist, we have approached the problem in several ways. Regarding materials, to make a uniform resist film we applied living radical polymerization and purification to obtain evenly interacting polymer chains. To obtain perfectly miscible resist components, such as polymer, photo acid generator (PAG) and quencher, we have optimized their structures to have similar polarity range. Acid diffusivity factors are also controlled by the resist components properties, including polymer T(g) and photo-acid polarity. In EUVL process, we applied surfactant rinse process to reduce line-width roughness and pattern collapse. In this paper, we discuss the performance of our EUV according to our material development concepts, that is, resist film homogeneity and acid diffusion control in order to meet the device manufacturing specification goals, such as resolution, line-width roughness (LWR), and sensitivity.     

Nanocomposite hydrogels

Hydrogels, which consist of three-dimensional polymer networks and large amounts of water, have long been believed to be interesting but mechanically fragile materials limited to specific uses. Recently, important breakthroughs have been made as a result of the creation of nanocomposite hydrogels (NC gels), and most of the traditional limitations of hydrogels have been overcome. NC gels are prepared by in situ free-radical polymerization at high yield under mild conditions (near ambient temperature, without stirring), and various shapes and surface forms are readily obtained. Because of their unique organic (polymer)/inorganic (clay) network structure, high toughness and excellent optical properties and stimulus-sensitivity are simultaneously realized in NC gels. Furthermore, NC gels exhibit a number of interesting new characteristics. In this paper, the fundamental and recent developments related to NC gels are reviewed.     

Supercritical angle fluorescence immunoassay platform

An inexpensive and easy-to-use immunoassay platform for the sensitive detection of analytes is presented. It comprises single-use polymer test tubes and a compact fluorescence reader. The optics for the capture of supercritical angle fluorescence (SAF) has been built into the tubes allowing for the extremely sensitive readout of solid phase immunoassays in real time and without washing steps. One-step sandwich immunoassays with interleukin 2 (IL-2) were carried out with capture antibodies immobilized in the tubes. At a turn around time of 12 min, the limit of detection for IL-2 was 0.27 pM (4.5 pg/mL) and the linear range covered 3 orders of magnitude. The developed technology is also adaptable to well plates and has great potential of replacing the work-intensive and time-consuming enzyme-linked immunosorbant assay (ELISA).     

Interfacial Assembly of Signal Amplified Multienzymes and Biorecognized Antibody into Proteinosome for an Ultrasensitive Immunoassay

Enzyme as signal tag has been widely employed in colorimetric immunoassays for decades. Nevertheless, it remains a great challenge to substantially improve the detection sensitivity of enzyme‐based immunoassays, which inhibits further critical applications. To circumvent this confinement, a multifunctional self‐assembled proteinosome based on the integration of signal amplification elements (enzyme) and biorecognition unit (antibody) is proposed for fabricating an immunoassay strategy with significantly enhanced sensitivity. Owing to the self‐assembly technique, this proteinosome not only efficiently loads abundant enzymes to possess high catalytic activity, but also enhances enzymatic stability and maintains recognition ability of antibody. Using imidacloprid as a model target, the proteinosome‐based immunoassay reaches a limit of detection down to the picogram mL^?1 level, which is 150‐fold lower than that of conventional enzyme‐linked immunosorbent assay. This method provides a versatile approach for constructing spherical proteinosome as a recognizer and amplifier for profiling a broad range of target antigen.     

Disposable noncompetitive immunosensor for free and total prostate-specific antigen based on capacitance measurement

This work reports on the successful integration of a one-step lateral flow immunoassay format and impedance detection of the specific affinity event using an electrochemical transducer coated with a pH-sensitive polymer layer. This approach was particularly applied to the development of a rapid single-use immunosensor for the sensitive detection of free and total prostate-specific antigen (f-PSA, t-PSA) tumor marker. Strips of nitrocellulose membrane were coated with appropriate antibodies to f-PSA and t-PSA and used as solid supports for the performance of noncompetitive immunoassays where PSA was allowed to react with both immobilized anti-PSA antibody and anti-PSA urease enzyme conjugate for less than 1 min. An additional piece within the device consisting of a storage blister filled with a urea solution allowed the rapid washing of unbound species from the membrane strips and simultaneous urea hydrolysis catalyzed by the bound urease conjugate in an automatic fashion. The hydrolysis of urea increased the pH of the reaction media, which in turn induced a breakdown of the polymer layer on the transducer and a consequent measurable change in capacitance of the system. This was easily recorded at a given frequency over a 30-min period. Overall, we describe a one-step immunosensor prototype that exhibits enough sensitivity to detect both forms of PSA at concentration levels down to 3 ng/mL. With the possibility of being portable and considering its ease of use, robustness, and simplicity, this device has great potential as a tool for the screening and early detection of prostate cancer.     

Microwave-accelerated metal-enhanced fluorescence: platform technology for ultrafast and ultrabright assays

We describe an exciting assay platform technology that promises to fundamentally address two underlying physical constraints of modern assays and immunoassays, namely, assay sensitivity and rapidity. By combining the use of metal-enhanced fluorescence with low-power microwave heating, we can indeed significantly increase the sensitivity of surface assays as well as >95 % kinetically complete the assay within a few seconds. Subsequently, this new technology promises to fundamentally change the way we currently employ immunoassays in clinical medicine. This new model platform system can be potentially applied to many other important assays, such as to the clinical assessment of myoglobin, where both assay speed and sensitivity is paramount for the assessment and treatment of acute myocardial infarction. To demonstrate the utility of microwave-accelerated metal-enhanced fluorescence (MAMEF), we show that a simple protein-based assay system can be optically amplified approximately 10-fold by using silver nanostructures, while being kinetically complete in less than 20 s. This new platform approach is subsequently over 10-fold more sensitive and approximately 90 times faster than a control assay that operates both at room temperature and without the use of metal-enhanced fluorescence. Finally, we show that low-power heating by microwaves in our model system does not denature proteins, as evidenced by no protein structural changes, probed by fluorescence resonance energy transfer.     

Chemiluminescence imaging ELISA using an imprinted polymer as the recognition element instead of an antibody

An imaging assay analogous to competitive enzyme immunoassays has been developed using a molecularly imprinted polymer instead of an antibody. The antigen 2,4-dichlorophenoxyacetic acid (2,4-D) was labeled with tobacco peroxidase, and the chemiluminescence reaction of luminol was used for detection. Microtiter plates (96 or 384 wells) were coated with polymer microspheres imprinted with 2,4-D, which were fixed in place by using poly(vinyl alcohol) as glue. In a competitive mode, the analyte-peroxidase conjugate was incubated with the free analyte in the microtiter plate, after which the bound fraction of the conjugate was quantified. After addition of the chemiluminescent substrates, light emission was measured in a high-throughput imaging format with a CCD camera. Calibration curves corresponding to analyte concentrations ranging from 0.01 to 100 microg/mL were obtained.     

Acoustic emission for monitoring the mechanical behaviour of natural fibre composites: A literature review

In recent years, natural fibres are increasingly used as reinforcements for the production of low-cost and lightweight polymer composites: other advantages include non-abrasive nature, high specific properties, and biodegradability. However, their limitations, including moisture absorption, poor wettability and large scattering in mechanical properties, and the not sufficient understanding of mechanisms controlling their mechanical behaviour and failure modes, still confine the use of natural fibre reinforced composites in non-structural applications. Acoustic emission (AE) proved useful for its capability of real-time monitoring over the whole material volume and high sensitivity to any process generating stress waves.This paper presents a literature review of AE applications in studies on natural fibre composites. The following fields of application are covered: (1) interface studies in single fibre composite (SFC) tests, (2) damage evolution and failure mechanisms detection and (3) crack propagation, including also current limitations of existing literature and future work.     

Plasmonic Au islands on polymer nanopillars

The refractive index sensitivity of localized surface plasmon resonance sensors can be improved by placing the plasmonic metal particles on pillars instead of on a planar substrate. In this paper, a simple and versatile colloidal lithography method for the fabrication of plasmonic Au islands on top of polymer nanopillars is described. The pillar height is controlled by varying the thickness of the initial polymer film. An increased pillar height results in a blue shift of the absorption spectrum of the Au islands. This is explained by a decreased effective refractive index around the islands. For pillars higher than approximately 40 nm no further blue shift is observed, in agreement with the decay length of the electromagnetic field around the islands. Pillar-supported Au islands were also fabricated on a flexible foil, demonstrating the potential of the method described here for the fabrication of flexible plasmonic substrates. Benefits and limitations of the method and of using polymers as the pillar material are discussed.     

Performance evaluation of the phosphorescent porphyrin label: solid-phase immunoassay of alpha-fetoprotein

Phosphorescent conjugates of antibodies, neutravidin, and biotin (pentylamine derivative) were synthesized using previously described monofunctional labeling reagent of platinum(II) coproporphyrin-I with isothiocyanate reactive group (PtCP-NCS). These conjugates, which can be considered as standard reagents for a range of bioanalytical applications, were evaluated in solid-phase immunoassay schemes with the clinical analyte a-fetoprotein (AFP). A custom-designed time-resolved phosphorescence plate reader based on a compact and low-cost 532-nm laser and optimized for measurement of porphyrin labels was used. Using optimized tracers, instrumentation and assay protocols, subpicomolar detection limits were obtained both for PtCP label in solution and for AFP in solid-phase immunoassay. This sensitivity is comparable with standard time-resolved fluorescence immunoassays with lanthanide labels. The performance of metalloporphyrin labels, instrumentation, and solid-phase immunoassays as an alternative to the established detection platforms is discussed.     

Photochemical patterning of biological molecules inside a glass capillary

A simple way for photochemical patterning of biological molecules onto the inner wall of fused-silica capillary is described. The method is based on a modification of the inner capillary surface with photoactive benzophenone (BP) derivative. The UV irradiation at 365 nm of the capillary filled with a sample solution results in cross-linking of the solutes to the BP moiety via a stable covalent bond. As a proof of concept, oligonucleotides and proteins were arrayed inside the capillary using an inverted microscope as an irradiation device. We demonstrated that the capillary arrays produced in this way are functional and could be used in different bioassays including DNA hybridization, protein interaction studies, and immunoassays. Having a sensitivity comparable to the fluorophore-based assays in a planar format, the capillary array possesses several advantages including submicroliter sample volume and a short assay time. The capillary format should therefore be considered as a possible alternative to a planar format in a number of low-density array applications such as mutation detection and diagnostic immunoassays.     

Carbon nanotubes with enhanced chemiluminescence immunoassay for CCD-based detection of Staphylococcal enterotoxin B in food

Enhanced chemiluminescence (ECL) detection can significantly enhance the sensitivity of immunoassays but often requires expensive and complex detectors. The need for these detectors limits broader use of ECL in immunoassay applications. To make ECL more practical for immunoassays, we utilize a simple cooled charge-coupled device (CCD) detector combined with carbon nanotubes (CNTs) for primary antibody immobilization to develop a simple and portable point-of-care immunosensor. This combination of ECL, CNT, and CCD detector technologies is used to improve the detection of Staphylococcal enterotoxin B (SEB) in food. Anti-SEB primary antibodies were immobilized onto the CNT surface, and the antibody-nanotube mixture was immobilized onto a polycarbonate surface. SEB was then detected by an ELISA assay on the CNT-polycarbonate surface with an ECL assay. SEB in buffer, soy milk, apple juice, and meat baby food was assayed with a LOD of 0.01 ng/mL using our CCD detector, a level similar to the detection limit obtained with a fluorometric detector when using the CNTs. This level is far more sensitive than the conventional ELISA, which has a LOD of approximately 1 ng/mL. Our simple, versatile, and inexpensive point-of-care immunosensor combined with the CNT-ECL immunoassay method described in this work can also be used to simplify and increase sensitivity for many other types of diagnostics and detection assays.     

Theory of equidistant three-dimensional radiance measurements with optical microprobes

Fiber-optic radiance microprobes, increasingly applied for measurements of internal light fields in living tissues, provide three-dimensional radiance distribution solids and radiant energy fluence rates at different depths of turbid samples. These data are, however, distorted because of an inherent feature of optical fibers: nonuniform angular sensitivity. Because of this property a radiance microprobe during a single measurement partly underestimates light from the envisaged direction and partly senses light from other directions. A theory of three-dimensional equidistant radiance measurements has been developed that provides correction for this instrumental error using the independently obtained function of the angular sensitivity of the microprobe. For the first time, as far as we know, the measurements performed with different radiance microprobes are comparable. An example of application is presented. The limitations of this theory and the prospects for this approach are discussed.     

Detection of cardiac biomarkers using micellar electrokinetic chromatography and a cleavable tag immunoassay

Biomarkers provide clinicians with an important tool for disease assessment. Many different biomarkers have been discovered, but few of them suffice as stand-alone indicators for disease presence or prognosis. Because no single biomarker can be relied upon for accurate disease detection there has been a substantial push for new multianalyte screening methods. Furthermore, there is a need to push assays toward a point-of-care technology to reduce the time between clinical analysis and medical intervention and minimize artifacts created during sample storage. There currently are, however, few inexpensive multianalyte methods for disease detection that can function in a point-of-care setting. A new approach which bridges the gap between traditional immunoassays and high-density microarrays by utilizing microfluidics, immunoassays, and micellar electrokinetic chromatography (MEKC) is discussed here. This chemistry, the cleavable tag immunoassay (CTI), is a low- to medium-density heterogeneous immunoassay designed to detect 1-20 analytes simultaneously. Although similar to traditional sandwich immunoassays, this approach is unique because the signal is not imaged on the surface; instead, a fluorescent tag is chemically cleaved from the antibody and analyzed by microchip MEKC. In this report, the CTI chemistry is used for the detection of four cardiac biomarkers elevated in acute myocardial infarction. Limit of detection (LOD) and dynamic range are reported for all biomarkers with LODs on the order of low nanograms per milliliter to low picograms per milliliter. Most importantly, the dynamic range for each of the biomarkers spans the boundary between normal and elevated levels. Finally, elevated marker levels were measured in spiked human serum samples.     

Acetal-functionalized polymer particles useful for immunoassays. III: Preparation of latex-protein complexes and their applications

Monodisperse polymer colloids with dimethyl and diethyl acetal functionalities synthesized by a two-step emulsion polymerization process were chosen as the polymeric support to carry out covalent coupling with the antibody IgG anti C-reactive protein, and to test the utility of the latex-protein complexes formed in immunoassays with the specific CRP antigen. More than the 80% of the initially linked protein was covalently coupled in all of the latexes. The agglutination reaction was followed by turbidimetry. With the aim of analyzing the effect of some of the variables of the immunological reaction, the reaction time, the particle concentration and the coverage degree of protein in the complexes were varied.     

Covalent micropatterning of poly(dimethylsiloxane) by photografting through a mask

A new photografting method to micropattern a covalent surface modification on poly(dimethylsiloxane) (PDMS) provides advantages in simplicity and efficiency. To accomplish the entire process on the benchtop, the PDMS was initially treated with benzophenone dissolved in a water/acetone mixture. This process permitted limited diffusion of the photoinitiator into the PDMS surface. Polymerization of acrylic acid was initiated by exposure of the benzophenone-implanted PDMS to UV radiation through a photomask with a thin aqueous layer of acrylic acid sandwiched between the PDMS and photomask. This procedure resulted in patterned poly(acrylic acid) (PAA) on the PDMS surface. In the modified regions, PAA and PDMS formed an interpenetrating polymer network extending 50 microm into the PDMS with an X-Y spatial resolution of 5 microm. The carboxyl groups of the PAA graft could be derivatized to covalently bond other molecules to the patterned PAA. Two bioanalytical applications of this micropatterned surface were demonstrated: (1) a guide for cell attachment and growth and (2) a substrate for immunoassays. 3T3 cells were shown to selectively localize to modified surface regions where they could be cultured for up to 7 days. Additionally, the micropatterned surface was used to immobilize either protein A or antibody for heterogeneous immunoassays.     

Improving the sensitivity of immunoassays by tuning gold nanoparticles to the tipping point

Conventional lateral flow immunoassays are based on labeled antibodies. In this paper we describe an alternative design based on gold nanoparticles labeled with haptens. The haptens are conjugated to gold nanoparticles by a method that allows the number per particle to be tuned to the point of maximum sensitivity. This leads to improvements compared with conventional lateral flow devices without relinquishing any of their advantages. In parallel assays for the environmental pollutant 2,4-dinitrophenol the alternative devices were 50% more sensitive.     

Analysis of temperature/pressure sensitivity of the resonant wavelength of long-period channel waveguide gratings

A theoretical study on the sensitivity of the resonant wavelength of long-period waveguide gratings (LPWGs) to temperature and pressure is reported. Starting with the phase-matching condition of the LPWG, general expressions for the temperature and pressure sensitivities are derived. The temperature sensitivity considers the thermo-optic and thermal expansion effects, and the pressure sensitivity takes into account the elasto-optic and elastic deformation effects of the materials involved, as well as the modal dispersion effect. Focusing on the extensively studied glass and polymer waveguides, the contributions of these effects to the temperature or pressure sensitivity were roughly evaluated and illustrated in the form of histograms in order to show the roles of these effects straightforwardly. The results show that a LPWG based on a polymer waveguide is preferred to that based on a glass waveguide for obtaining high temperature or pressure sensitivity. The temperature sensitivity is dominated by the modal dispersion effect and the difference between the thermo-optic coefficients of the waveguide and the cover layer materials, while the thermal expansion effects make only a minor contribution to the sensitivity for the cases of both glass and polymer waveguides. The pressure sensitivity is dominated by the modal dispersion effect and the difference between the elasto-optic coefficients of the channel waveguide and the cover layer materials. In particular, in the case of the polymer LPWG the elastic deformation effects of the waveguide and grating materials make a moderate contribution to the pressure sensitivity and cannot be ignored. The minor contributions from the thermal expansion effects or the elastic effects may play a role in designing a temperature- or a pressure-insensitive LPWG device. Finally, the possibility that the waveguide loss affects the LPWG temperature/pressure sensitivity is discussed.