Sampling-resolution strategy for one-way multiplexed immunoassay with sequential chemiluminescent detection

A sampling-resolution strategy was designed by using a multichannel flow-injection technique for rapid one-way multiplexed immunoassay. The multichannel sampling combined an incubation process in batch with a simple magnetic collection. After incubation for 6 min, free enzyme conjugates could be separated from the formed enzyme-labeled sandwich immunocomplexes with a magnet for simultaneously stopping the immunoreaction. With the help of two valves, the chemiluminescence (CL) substrate was then sequentially mixed with the immunocomplexes in different channels for sequentially triggering the CL reaction in a time interval of 15 s. After triggering for 5 min, the mixtures were sequentially injected into a one-way detection channel in the same interval to form the analyte zones separated with HCl solution and washing buffer for avoiding cross talk. With the use of alpha-fetoprotein, carcinoma antigen 125, carcinoma antigen 199, and carcinoembryonic antigen as proof-of-principle analytes, the sequential CL detection could be completed within 1 min with the linear calibration ranges of 1.0-80 microg/L, 1.0-60 kU/L, 1.0-120 kU/L, and 1.0-100 microg/L, respectively. This system showed acceptable detection and fabrication reproducibility, and the assay results were in acceptable agreement with those from single-analyte tests of clinical sera, showing a promise of automated clinical application.     

Static two-dimensional aperture coding for multimodal, multiplex spectroscopy

We propose a new class of aperture-coded spectrometer that is optimized for the spectral characterization of diffuse sources. The instrument achieves high throughput and high spectral resolution by replacing the slit of conventional dispersive spectrometers with a more complicated spatial filter. We develop a general mathematical framework for deriving the required aperture codes and discuss several appealing code families. Experimental results validate the performance of the instrument.     

Encoded silica colloidal crystal beads as supports for potential multiplex immunoassay

We developed a new kind of suspension array for multiplexed immunoassays using silica colloidal crystal beads (SCCBs) as coding carriers. The monodisperse and size-controlled SCCBs were fabricated by a microfluidic device. Calcination was employed to improve the mechanical stability and lower the fluorescent background of the SCCBs. Immobilization of protein molecules on the surface of the SCCBs through chemical bonds was studied, and the modification condition was optimized to increase the detection sensitivity. Results indicated that the SCCBs as supports were more sensitive (0.92 ng/mL IgG) than the glass beads (27 ng/mL IgG) and the planar carriers (140 ng/mL IgG). A multiplex immunoassay showed the flexibility and feasibility of SCCBs array in clinical applications.     

Microfabricated renewable beads-trapping/releasing flow cell for rapid antigen-antibody reaction in chemiluminescent immunoassay

A renewable flow cell integrating a microstructured pillar-array filter and a pneumatic microvalve was microfabricated to trap and release beads. A bead-based immunoassay using this device was also developed. This microfabricated device consists of a microfluidic channel connecting to a beads chamber in which the pillar-array filter is built. Underneath the filter, there is a pneumatic microvalve built across the chamber. Such a device can trap and release beads in the chamber by "closing" or "opening" the microvalve. On the basis of the pneumatic microvalve, the device can trap beads in the chamber before performing an assay and release the used beads after the assay. Therefore, this microfabricated device is suitable for "renewable surface analysis". A model analyte, 3,5,6-trichloropyridinol (TCP), was chosen to demonstrate the analytical performance of the device. The entire fluidic assay process, including beads trapping, immuno binding, beads washing, beads releasing, and chemiluminesence signal collection, could be completed in 10 min. The immunoassay of TCP using this microfabricated device showed a linear range of 0.20-70 ng/mL with a limit of detection of 0.080 ng/mL. The device was successfully used to detect TCP spiked in human plasma at the concentration range of 1.0-50 ng/mL, with an analytical recovery of 81-110%. The results demonstrated that this device can provide a rapid, sensitive, reusable, low-cost, and automatic tool for detecting various biomarkers in biological fluids.     

High-performance immunoaffinity chromatography and chemiluminescent detection in the automation of a parathyroid hormone sandwich immunoassay

An automated sandwich immunoassay was developed based on high-performance immunoaffinity chromatography and chemiluminescent detection, using the determination of parathyroid hormone (PTH) in plasma as a model system. In this method, injections of plasma and acridinium ester-labeled anti-(1-34 PTH) antibodies were made onto a column containing immobilized anti-(44-68 PTH) antibodies. Upon elution, PTH and its associated labeled antibody were combined with an alkaline peroxide postcolumn reagent, and the resulting light production was measured. Factors considered in optimizing this system included the column's dissociation properties, the rate of light production in the postcolumn reactor, and the use of sequential vs simultaneous injection of sample and labeled antibody. The final system developed required 6 min per plasma injection, following a 1-h incubation of sample with labeled antibody. The response was linear over 2-3 orders of magnitude and the lower limit of detection for a 66-microL plasma sample was only 16 amol, or 2.4 x 10(-13) M. Overall, this method had a precision and response similar to those of manual PTH methods but required 24-fold less time to perform. By using different immobilized and labeled antibodies, this method could easily be adapted for use with other analytes.     

In-depth resolution for a strip source in the Fresnel zone

The problem of determining the achievable resolution limits in the reconstruction of a current distribution is considered. The analysis refers to the one-dimensional, scalar case of a rectilinear, bounded electric current distribution when data are collected by measurement of the radiated field over a finite rectilinear observation domain located in the Fresnel zone, orthogonal and centered with respect to the source. The investigation is carried out by means of analytical singular-value decomposition of the radiation operator connecting data and unknown, which is made possible by the introduction of suitable scalar products in both the unknown and data spaces. This strategy permits the use of the results concerning prolate spheroidal wave functions described by B. R. Frieden [Progress in Optics Vol. IX, E. Wolf, ed. (North-Holland, Amsterdam 1971), p. 311.] For values of the space-bandwidth product much larger than 1, the steplike behavior of the singular values reveals that the inverse problem is severely ill posed. This, in turn, makes it mandatory to use regularization to obtain a stable solution and suggests a regularization scheme based on a truncated singular-value decomposition. The task of determining the depth-resolving power is accomplished with resort to Rayleigh's criterion, and the effect of the geometrical parameters of the measurement configuration is also discussed.     

Multistatic-multiview resolution from Born fields for strips in Fresnel zone

We address the inverse scattering problem of estimating the resolution limits achievable in the reconstruction of a dielectric strip object within a two-dimensional and scalar geometry. The scattered field is observed over a bounded rectilinear domain located in the Fresnel zone, and a single-frequency multistatic-multiview configuration is considered. The analysis is performed by casting the problem as the inversion of the linearized scattering operator arising from the Born approximation and by means of its singular-value decomposition. Finally, the role of the geometrical parameters of the measurement configuration is highlighted.     

Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers

A novel flow-through immunosensing system for performing a multianalyte chemiluminescent determination in a single run was designed. A new analytical strategy of substrate zone-resolved technique was proposed. Using carcinoma antigen 125 (CA 125) and carcinoembryonic antigen (CEA) as model analytes, the capture antibodies for CA 125 and CEA were immobilized on an UltraBind aldehyde-activated membrane to act as an immunoreactor, to which the mixture of CA 125, CEA, and their corresponding tracers, horseradish peroxidase (HRP)-labeled anti-CA 125 and alkaline phosphatase (ALP)-labeled anti-CEA, was introduced for on-line incubation. The substrates for HRP and ALP were then delivered into the detection cell sequentially to perform substrate zone-resolved immunoassay by a sandwich format. Under optimal conditions, CA 125 and CEA could be assayed in the ranges of 5.0-100 units/mL and 1.0-120 ng/mL, respectively. The whole assay process including incubation, wash, detection, and regeneration could be completed in 35 min. The serum samples from the clinic were assayed with the proposed method, and the results were in acceptable agreement with the reference values. This method and the strategy of substrate zone-resolved technique could be further developed for high-throughput multianalyte immunoassay.     

A numerical simulation for two-dimensional moving boundary problems with a mushy zone

For materials such as alloy, organic phase-change materials and many others, the change of phases may take place over a temperature range. This leads to phase-change problems with the mushy zone in which the solid and liquid phases coexist. The present study introduces a numerical method combining the Laplace transform technique and the control volume method to solve two-dimensional phase-change problems with the mushy zone. The hybrid numerical method involves the control volume formulation for the space domain and the Laplace transform technique for the time domain. The Taylor's series approximation is applied to linearize nonlinear terms in the governing equation. The transfinite mapping method is used to generate control-volume meshes in each region. The growth of the mushy zone is unknown a priori and is predicted by using the least-square iteration scheme. It will be found that the present hybrid numerical method can be efficiently applied to solve two-dimensional phase-change problems with a mushy zone.     

Advanced multiple-angle incidence resolution spectrometry for thin-layer analysis on a low-refractive-index substrate

A long-time issue that thin films supported on a substrate with low refractive index cannot be subjected to multiple-angle incidence resolution spectrometry (MAIRS) has been resolved, and an advanced MAIRS technique has been developed. Infrared multiple-angle incidence resolution spectroscopy (IR MAIRS) is being recognized as a powerful spectroscopic tool for revealing molecular orientation in thin films or molecular adsorbates. MAIRS has been, however, employed with only IR spectroscopy thus far, since MAIRS requires high refractive index substrates such as germanium and silicon, and shorter wavelength regions have no appropriate substrates. Although one of the most commonly useful substrates for IR spectroscopy is CaF2, unfortunately it cannot be employed for IR MAIRS because of the low refractive index. To overcome this experimental limitation, the principle of MAIRS has been studied theoretically, and it has been found that polarization effects on transmission intensity is the main cause that disturbs a proper MAIRS analysis. As a result, now it is possible to employ IR MAIRS for analysis of thin films on CaF2 by removing the s-polarization and by use of the advanced MAIRS algorithm.     

Multivariate curve resolution applied to the analysis and resolution of two-dimensional [1H,15N] NMR reaction spectra

Multivariate curve resolution is proposed for the study of complex chemical reactions monitored by two-dimensional (2D) NMR spectroscopy. In particular, in this work, multivariate curve resolution is applied to the study of the reaction between (15)N-labeled cisplatin and the amino acid-nucleotide hybrid (Phac-Met-linker-p(5')dG). At several stages of the reaction, 2D [(1)H,(15)N] HSQC NMR spectra were acquired and stored in data matrices. In a first step, multivariate curve resolution was applied to analyze individually each one of these 2D spectra, allowing the resolution of the corresponding (1)H and (15)N one-dimensional correlation spectra. In a second step, the whole set of 2D spectra recorded along the reaction were simultaneously analyzed by multivariate curve resolution, allowing the resolution of the kinetic concentration profiles and of the pure 2D NMR spectra of each of the species detected along the reaction. Results finally obtained confirmed previously postulated reaction mechanisms involving the existence of two monofunctional adducts and of two bifunctional adducts, with the structure of one of them not completely resolved.     

Lab-on-valve system integrating a chemiluminescent entity and in situ generation of nascent bromine as oxidant for chemiluminescent determination of tetracycline

A novel configuration of a lab-on-valve (LOV) system was fabricated and applied for chemiluminescence (CL) detection by integrating a demountable Z-type flow cell onto the LOV unit. A bismuthate immobilized microcolumn was incorporated in one port of the LOV for in situ oxidation of KBr and generation of bromine as oxidant for the bromine-hydrogen peroxide-tetracycline (TC) chemiluminescent reaction. The nascent bromine reacts with hydrogen peroxide and produces a weak CL signal, the intensity of which was significantly enhanced in the presence of TC following an energy-transfer mechanism. A novel procedure for tetracycline quantification was therefore developed based on the present system. When compared with the reported flow injection-CL methods for TC, this procedure not only provided an improved detection limit of 2.0 microg L(-)(1) but also minimized sample and reagent consumption. A linear range of 6.0-10 000 microg L(-)(1) was derived along with RSD values of 5.9 (at the concentration level of quantification limit) and 2.2% (at 50 microg L(-)(1)), and a sampling frequency of 120 h(-)(1) was achieved. The system was validated with a National Standard Procedure (GB/T 18932.4-2002, HPLC with UV detection) by measuring TC contents in commercial milk samples.     

A two-dimensional in situ fatigue cohesive zone model for crack propagation in composites under cyclic loading

In this paper a two-dimensional fatigue cohesive zone model (CZM) for crack propagation in composites under cyclic loading has been formulated and validated through successful predictions of fatigue crack growth under pure and mixed mode conditions for several different composites. The proposed fatigue CZM assumes simple power-law functions for fatigue damage accumulation of which the damage parameters can be calibrated from simple fatigue tests under pure mode I and mode II conditions. The model relies solely on the in situ cohesive responses for fatigue damage rate calculation, enabling the differentiation of the local elemental load history from the global load history. An effective cycle jump strategy for high-cycle fatigue has also been proposed. It has been demonstrated that once calibrated, the fatigue CZM can predict the Paris laws for the pure modes. Furthermore, it can predict the Paris laws of any mixed-mode conditions without the need of additional empirical parameters. This is of significant practical importance because it leads to greatly reduced experimental needs for mixed mode crack propagation widely observed in composites under cyclic loads.     

High resolution, two-dimensional image mapping of ZnO nanowires by confocal microphotoluminescence and microraman spectroscopy

High-quality ZnO nanowires were synthesized using both Au catalysts and ZnO seeds by chemical vapor depositionon basal plane sapphire substrates. The nanowires were hexagonal and aligned with their c-axis closely perpendicular to the sapphire substrate surface. The structural characteristics of the nanowiresgrown using the different catalysts/seeds were compared using scanning electron microscopyand X-ray diffraction. Their optical properties were assessed using microphoto-luminescence and confocal microRaman spectroscopy and compared. The nanowires exhibited a strong near band-edge related UV luminescence emission along with a defect related visible emission. The dependence of the luminescence as a function of incident excitation power and depth along the axis of the nanowires was studied. The wurtzite structure of the ZnO was confirmed from the Raman measurements. Two-dimensional mappings of the microphotoluminescence emission at different wavelengths and microRaman scattering from the nanowire samples were carried out using a confocal laser scanning microscope. This enabled the ability to correlate the near band-edge UV and visible emissions over the mapped area.     

Dielectric substrate self-bias and plasma confinement in two-dimensional scanning radio frequency plasma-enhanced chemical vapour deposition

A two-dimensional scanning radio frequency (RF) plasma method was developed for large area deposition in order to avoid difficulties encountered in conventional large RF plasma systems, offering precision control of plasma, improved uniformity of thickness and microstructure, and simplicity of system design. Guarded electrode houses were introduced to form localized plasma thus parasitic plasma or parasitic deposition outside the localized plasma region was eliminated. Different electrode configurations were studied and optimized. Self-bias on dielectric substrate was studied for different electrode configurations as a function of RF power, pressure, and gases. Eliminating parasitic plasma by fully shielding the basing electrode resulted in monitoring signal losses of self-bias. Introducing an isolated plasma house for the biasing electrode enabled the recovery of the self-bias on the biasing electrode without parasitic deposition on substrate outside the localized plasma region. Uniformity optimization within the localized plasma region is no longer a concern in this system. Thickness profile control was achieved by scanning the plasma source over the large substrate. TEM analysis showed that homogenous films were deposited in the scanning deposition system.     

Polyamine detection system for high-performance liquid chromatography involving enzymatic and chemiluminescent reactions

We have developed a new specific detection system for polyamines, separated by high-performance liquid chromatography, to replace the usual o-phthalaldehyde (OPA) postlabeling method. This system utilizes a chemiluminescent reaction to determine the amount of hydrogen peroxide generated through the enzymatic oxidation of polyamines. With this method, as little as 5 pmol of putrescine could be accurately determined (S/N = 4), and linearity between the amount of this polyamine and the relative chemiluminescence intensity was observed up to 500 pmol. In the cases of cadaverine, spermidine, and spermine, the linearity continued to 1 nmol. The within-assay coefficients of variation for these four polyamines ranged between 0.97% and 2.03%. As compared with the OPA method, the new method is highly specific for polyamines due to the high specificity of the enzyme used. Another advantage of the new method is the direct determination of polyamines in urine samples that contain various compounds that interfere with the OPA method.     

Influence of silane coupling agent on quality of interfacial transition zone between concrete substrate and repair materials

Silane coupling agent (SCA) was introduced as a modifying material to significantly improve the bond quality of the repaired interfacial transition zone. SCA aqueous solutions with various concentrations were used to coat the surfaces of a granite and of old concrete substrates before applying the repair materials. Both pull-off bond strength test and microstructure observation of the different repair interfacial layers were performed. The test results show that coating a concrete substrate with a SCA aqueous solution with an appropriate concentration can noticeably modify the microstructure of the interfacial transition zone, and therefore, significantly increase the bond strength.     

Micromachining microcarrier-based biomolecular encoding for miniaturized and multiplexed immunoassay

Micromachining techniques, which originated in the microelectronics industry, have been employed to manufacture microparticles bearing an engraved dot-type signature for biomolecular encoding. These metallic microstructures are photolithographically defined and manufactured in a highly reproducible manner. In addition, the code introduced on the particle face is a straightforward visible feature that is easily recognizable with the use of optical microscopy. The number of distinct codes theoretically could be many thousands, depending on the coding element numbers. Such microparticles are, thus, with appropriate surface organic functionalizations, ideal for encoding biomolecular libraries and serving as a platform for developing high-throughput multiplexed bioassay schemes based on suspension array technology. As proof of this statement, we demonstrated that encoded microparticles tagged with antibodies to human immunoglobulin classes are capable, using imaging detection as the interrogating approach, of high sensitivity and high specificity, as well as multiplexed detection of the respective antigens in a microliter-sample volume.     

A microarray immunoassay for simultaneous detection of proteins and bacteria

We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).     

Multiplexed flow cytometric immunoassay for influenza virus detection and differentiation

Microsphere-based immunoassay by flow cytometry has gained popularity lately in protein detection and infectious disease diagnosis due to its capacity for multiplexed analysis and simple assay format. Here, we demonstrated the power of microsphere-based immunoassay for high-sensitivity detection and accurate differentiation of influenza viruses. The effects of sample volume and bead number on the assay sensitivity of viral antigen detection were studied. Compared to enzyme-linked immunosorbent assays, flow-based bead assays provided approximately 10-fold lower detection limit for viral particle detection and performed similarly for recombinant viral hemagglutinin protein detection. A four-plexed assay for influenza virus typing and influenza B virus sublineage characterization was developed to demonstrate the potential for multiplexed viral antigen detection and differentiation.