Analysis of free hormone fractions by an ultrafast immunoextraction/displacement immunoassay: studies using free thyroxine as a model system

A system was developed for measuring the noncomplexed or free fraction of a hormone in serum based on the combined use of ultrafast immunoextraction with a chromatographic displacement immunoassay. This approach was tested using L-thyroxine as a model analyte. Items considered in the development of this technique included the choice of immunoassay format and the selection of conditions for removal of thyroxine's free fraction from samples without significant interference from its protein-bound fraction. The final method had an effective extraction time of 90 ms and allowed the amount of free thyroxine to be determined within 30 s after sample injection. The limit of detection was 6 pM (S/N = 3) for a 100-microL sample, and the linear response extended up to at least 100 pM. This technique gave good correlation versus reference methods when used for the determination of free thyroxine in serum samples. Advantages of this method included its speed and its ability to analyze a sample with no pretreatment other than standard filtration. The same approach could be adapted for other hormones or drugs by using appropriate antibodies and labeled analogues for such agents.     

Analysis of free drug fractions using near-infrared fluorescent labels and an ultrafast immunoextraction/displacement assay

A chromatographic method was developed for measuring free drug fractions based on the use of an ultrafast immunoextraction/displacement assay (UFIDA) with near-infrared (NIR) fluorescent labels. This approach was evaluated by using it to determine the free fraction of phenytoin in serum or samples containing the binding protein human serum albumin (HSA). Items considered in the design of this method included the dissociation rate of HSA-bound phenytoin, the rate of capture of free phenytoin by immunoextraction microcolumns, the behavior of NIR fluorescent labels in a displacement format, and the overall response and stability of the resulting assay. In the final UFIDA method, the free fraction of phenytoin was extracted in approximately 100 ms by a microcolumn containing a small layer of anti-phenytoin antibodies. This gave a displacement peak for a NIR-fluorescent-labeled analogue of phenytoin that appeared within 2-3 min of sample injection, creating a signal proportional to the amount of free phenytoin in the sample. The UFIDA method provided results within 1-5% of those determined by ultrafiltration for reference samples. The lower limit of detection was 570 pM, and the linear range extended up to 10 microM. This approach is not limited to phenytoin but can be adapted for other analytes through the use of appropriate antibodies and labeled analogues.     

Analysis of free drug fractions by ultrafast immunoaffinity chromatography

A chromatographic method was developed for measuring the nonbound (or free) fraction of drugs by using millisecond-scale extractions on small immunoaffinity columns. The design of this system was developed by considering the dissociation rates of (R)- and (S)-warfarin from the binding protein human serum albumin (HSA) and by performing computer simulations of the immunoaffinity extraction of these drugs. The final system was tested by using it to measure the free fractions of (R)- or (S)-warfarin in samples with known concentrations of these agents and HSA. The free warfarin fraction was extracted in 180 ms by a 2.1-mm-i.d. sandwich microcolumn that contained a 1.1-mm layer of an anti-warfarin antibody support. The nonretained peaks from this immunoaffinity column were passed through a series internal surface reversed-phase columns and a fluorescence detector for the analysis of any protein-bound warfarin that remained in the sample. The experimental results were found to have good agreement with those predicted from the known equilibrium constants for the binding of (R)- and (S)-warfarin with HSA. This approach can be modified for other analytes by changing the types of antibodies that are used in the immunoaffinity column and by using an appropriate detector for the nonretained drug fraction.     

Development of a sensitive microarray immunoassay for the quantitative analysis of neuropeptide y

A direct competitive immunoassay in an antibody microarray format was developed for the sensitive detection of neuropeptide Y (NPY) and employed in the analysis of NPY in human sweat samples. This is the first demonstration that antibody microarray, as a powerful multiplex analysis tool, can be used for the sensitive determination of NPY and potentially other neuropeptides. 400 pg/mL of dibiotinylated NPY and 0.1 mg/mL spotting capture antibody were found to offer the best performance, yielding a sensitivity of 50 pg/mL and a linear dynamic range of 0.1-100 ng/mL for NPY. Evaluation of matrix effects by using artificial sweat revealed that dialysis is necessary for analyzing NPY in human sweat samples with microarray immunoassay. In a preliminary application, 50-210 pg/mL of NPY was detected in sweat samples collected with Macroduct collectors. This study indicates that antibody microarrays can be used for NPY analysis and that human sweat could be a valuable sample source for biomarker and proteomics studies, especially when noninvasive human sample collection is preferable.     

Development of a multichannel microfluidic analysis system employing affinity capillary electrophoresis for immunoassay

A six-channel microfluidic immunoassay device with a scanned fluorescence detection system is described. Six independent mixing, reaction, and separation manifolds are integrated within one microfluidic wafer, along with two optical alignment channels. The manifolds are operated simultaneously and data are acquired using a singlepoint fluorescence detector with a galvano-scanner to step between separation channels. A detection limit of 30 pM was obtained for fluorescein with the scanning detector, using a 7.1-Hz sampling rate for each of the reaction manifolds and alignment channels (57-Hz overall sampling rate). Simultaneous direct immunoassays for ovalbumin and for anti-estradiol were performed within the microfluidic device. Mixing, reaction, and separation could be performed within 60 s in all cases and within 30 s under optimized conditions. Simultaneous calibration and analysis could be performed with calibrant in several manifolds and sample in the other manifolds, allowing a complete immunoassay to be run within 30 s. Careful chip conditioning with methanol, water, and 0.1 M NaOH resulted in peak height RSD values of 3-8% (N = 5 or 6), allowing for cross-channel calibration. The limit of detection (LOD) for an anti-estradial assay obtained in any single channel was 4.3 nM. The LOD for the cross-channel calibration was 6.4 nM. Factors influencing chip and detection system design and performance are discussed in detail.     

Development and optimization of a novel drug free nanolipid vesicular system for treatment of osteoarthritis

Objective: The goal of this study is to improve the transdermal delivery of phosphatidylcholine (PC) via constructing a novel nanolipid vesicular system (NLVS) with high level of permeability through the stratum corneum (SC).

Significance: In our study, a novel drug free NLVS was developed. The system depends on PC boundary cartilage lubrication to relieve osteoarthritic pain without developing gastrointestinal problems associated with anti-inflammatory drug.

Materials and methods: A full two-level (2³) factorial design is applied to optimize the quality of the prepared NLVS. The selected independent variables are the concentration of PC, the concentration of edge activator (EA), and EA type. The developed NLVS was evaluated for in-vitro, ex-vivo as well as in-vivo efficacy in rat animal model.

Results: Based on the factorial design, the selected formulation variables significantly affect the tested responses. The prepared NLV formulations have a particle size (PS)in the range of 10.34 to 496.3?nm, polydispersity index (PdI) values less than one, and negative zeta potential (ZP) range of ?1.42 to ?32.01?mV. In-vitro and ex-vivo study results reveal that the designed NLVS is effective in sustaining PC release and enhancing its transdermal permeation over 24?h. The optimal permeation flux through ex-vivo study is 0.415?mg/cm²/h following zero-order kinetics. Moreover, in-vivo study of the optimized formulations demonstrated remarkable reduction in inflammatory mediators associated with osteoarthritis (OA).

Conclusion: The results indicate that the optimized drug free NLVS significantly augment transdermal delivery of PC and have a potential role in treatment of OA without the risk of systemic side effects.     

Buckling failure mode analysis of buried X80 steel gas pipeline under reverse fault displacement

High strength steel pipeline is widely used in long distance transportation of natural gas. These pipelines are vulnerable under active faults in strong seismic areas. The buckling failure modes of high strength X80 gas pipeline crossing reverse fault were analyzed systematically in this paper. Based on the nonlinear finite element method, a pipe-elbow hybrid model was developed for buckling failure analysis of X80 steel pipeline under reverse fault displacement. The pipe soil interaction relationship was simulated by a series of elastic-plastic soil springs. The nonlinearity of pipe material and large deformation were also considered. The non-linear stabilization algorithm was selected due to the convergence of the numerical model. Engineering parameters used in the Second West to East Gas Pipeline in China were selected in this study. Typical features for beam buckling and local buckling failure in the proposed numerical model were derived. Based on a series of parametric studies, the influences of the fault displacement, fault dip angle, pipe wall thickness, buried depth of pipe and soil conditions on the buckling failure modes were discussed in detail. The proposed methodology can be referenced for failure analysis and strength evaluation of pipelines subjected to reverse fault displacement.     

Development of a differential optical-fiber displacement sensor

A novel optical-fiber displacement sensor is proposed and demonstrated. It consists of a laser diode light source, an optical-fiber probe, and two photodetectors. The bundling of the probe is sectioned into three parts: a centrally positioned fiber in the bundle for illumination, the first-neighbor fibers for receiving (part I), and the remaining fibers for receiving (part II). The ratio of the difference to the sum of the output signals from the part I and the part II receiving fibers can eliminate the variation in the sensitivity of the sensor to reflectivity of the target. The performance of the sensor is geometrically analyzed. The working distance is determined by the distance from the centered illuminating fiber to the boundary between the part I and the part II receiving fibers. The experimental measurements made with three different reflectivity targets confirm that the sensor performance is independent of the three reflectivities, as predicted by the analysis.     

Strain imaging using conventional and ultrafast ultrasound imaging: numerical analysis

In elasticity imaging, the ultrasound frames acquired during tissue deformation are analyzed to estimate the internal displacements and strains. If the deformation rate is high, high-frame-rate imaging techniques are required to avoid the severe decorrelation between the neighboring ultrasound images. In these high-frame-rate techniques, however, the broader and less focused ultrasound beam is transmitted and, hence, the image quality is degraded. We quantitatively compared strain images obtained using conventional and ultrafast ultrasound imaging methods. The performance of the elasticity imaging was evaluated using custom-designed, numerical simulations. Our results demonstrate that signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR) and spatial resolutions in displacement and strain images acquired using conventional and ultrafast ultrasound imaging are comparable. This study suggests that the high-frame-rate ultrasound imaging can be reliably used in elasticity imaging if frame rate is critical     

Development of a unified numerical procedure for free vibration analysis of structures

This paper presents the details of a unified numerical algorithm and the associated computer program developed for the efficient determination of natural frequencies and modes of free vibration of structures. Both spinning and nonspinning structures with or without viscous and/or structural damping may be solved by the current procedure; in a addition, the program is capable of solving static problems with multiple load cases as well as the quadratic matric eigenproblem associated with a finite dynamic element structural discretization. A special symmetric matrix decomposition scheme has been adopted for matrix triangularization, which renders the program rather efficient and economical. Also, a novel bisection scheme is described that further accelerates the solution convergence rate, particularly for the case of repeated roots. The associated computer program adopts and out-of-core solution strategy, thereby enabling effective solutions to be achieved for large, complex, practical structures. A complete listing of the program written in FORTRAN V, for the UNIVAC 1100/82 computer, along with the source deck is available for ready use.     

Development of a finite dynamic element for free vibration analysis of two-dimensional structures

The paper develops an efficient free-vibration analysis procedure of two-dimensional structures. This is achieved by employing a discretization technique based on a recently developed concept of finite dynamic elements, involving higher order dynamic correction terms in the associated stiffness and inertia matrices. A plane rectangular dynamic element is developed in detail. Numerical solution results of free-vibration analysis presented herein clearly indicate that these dynamic element combined with a suitable quadratic matrix eigenproblem solution technique effect a most economical and efficient solution for such an analysis when compared with the usual finite element method.     

海水淡化与反渗透技术的发展形势

全世界海水淡化生产能力统计表明，2000年以前蒸馏法(主要是多级闪蒸)占优势，2000年以后则以反渗透法占优势；海水淡化厂已从中东布遍各大洲．当前除中东国家外，都以反渗透法为首选技术．美国已基本上不再发展蒸馏法，在膜法中除海水反渗透外，纳滤、超滤和微滤都已全面进入水脱盐和水处理领域，而且后二者(水处理膜)增长速度远高于脱盐膜技术．我国反渗透淡化技术已经步入成熟发展阶段，有必要在沿海城市发展以海水淡化为基础的补充水源，并开发自有系列技术，积极从事国际市场的开发．     

Development of a "membrane cloaking" method for amperometric enzyme immunoassay and surface plasmon resonance analysis of proteins in serum samples

Detection of trace amounts of target proteins in the presence of high concentrations of matrix proteins (e.g., serum samples) without separation steps is of great significance to biomedical research but remains technically challenging. Here we report a "membrane cloaking" method to overcome nonspecific protein adsorption and fouling problems for label-free surface plasmon resonance detection and heterogeneous immunosensing. A thin, hybrid, self-assembled monolayer on gold was formed with 70 mol % mercaptopropanol and 30 mol % cysteamine/propanedithiol to facilitate membrane fusion and covalent attachment of antibodies. After antibody immobilization, the surface was incubated with lipid vesicles, which fused to form a supported membrane. The analyte spiked in serum was introduced for binding, and the membrane and nonspecifically adsorbed proteins on the membrane were subsequently removed using a nonionic surfactant before the final measurement was carried out. Selection of a suitable surfactant can preserve antibody/antigen binding and selectively remove the membrane, allowing accurate measurement of the captured proteins without interference from nonspecifically adsorbed species. Surface plasmon resonance (SPR) quantification of IgG spiked in undiluted serum ( approximately 75 mg/mL protein) was achieved with the membrane cloaking method, whereas direct measurement without membrane removal resulted in a significantly large error. The cloaking method was also used to develop an enzyme amplified amperometric assay using HRP-conjugated IgG. Detection of concentrations as low as 5 fM proteins was obtained. Finally, a membrane cloaking assay combining SPR and in situ electrochemical measurement was demonstrated on a gold substrate. Similar sensitivity was observed using a continuous flow injection measurement. The method opens new avenues to develop direct assay methods with ultrahigh sensitivity for protein samples using SPR and enzyme-linked amplification mechanisms.     

Latex piezoelectric immunoassay: analysis of C-reactive protein in human serum

We constructed an integrated immunosensor system to combine a quartz crystal microbalance with the agglutination reaction of immunized latex beads. C-reactive protein (CRP) induced an immunoreaction due to the latex bends with anti-CRP antibody. We succeeded in determining the concentration of CRP necessary in human serum for disease diagnosis using a CRP Sensor I.     

Strong displacement discontinuities and Lagrange multipliers in the analysis of finite displacement fracture problems

The finite displacement formulation of a quadrilateral element containing an embedded displacement discontinuity is presented. The formulation is based on the kinematically optimal technique, and some known defects of this technique are addressed. Lagrange multipliers are adopted to ensure the correct crack closure prior to initiation. Six additional degrees of freedom in each element allow the representation of the two states of the crack. A classical enhanced strain technique is employed to improve the bending performance of the element. Numerical examples illustrate both the robustness and the accuracy of the proposed solutions.     

Development and validation of a one-step immunoassay for determination of cadmium in human serum.

A sensitive and simple one-step immunoassay was developed and validated for quantitative determination of Cd(II) in human serum. In this method, a monoclonal antibody that recognizes Cd(II)-EDTA complexes was directly immobilized onto microwell plates. The serum sample containing metallothionein(MT)-bound and non-MT-bound Cd(II) was acidified to displace the Cd(II) from MT. The sample was then treated with metal-free EDTA to convert Cd(II) to Cd(II)-EDTA complexes. A mixture of Cd(II)-EDTA complexes derived from serum samples and Cd(II)-EDTA conjugated with peroxidase enzyme was incubated in the wells to compete for binding sites of the immobilized antibody. After addition of peroxidase substrate, the bound fraction of the enzyme conjugate was measured by a microplate reader, and the signal was inversely proportional to the concentration of the Cd(II) in the sample. The assay limit of detection was 0.24 microg/L, and the effective working range at coefficient of variation of < or = 10% was 0.24-100 microg/L. Analytical recovery of spiked Cd(II), in the concentration range between 0.8 and 50 microg/L, was 97.8 +/- 4.0%. The assay was selective for Cd(II); other metal ions (Mn, Co, Cu, Zn, Mg, Hg, Ca, Ni, Fe, and Pb), tested at concentrations considerably higher than those present in human serum, did not significantly interfere with the assay. The assay results correlated well with those obtained by graphite furnace atomic absorption spectrometry (r = 0.984).     

Development of zeptomole and attomolar detection sensitivity of biotin-peptide using a dot-blot gold nanoparticle immunoassay

An ultrasensitive, simple, and fast immunoassay for biotin-peptide detection using gold nanoparticles conjugated with antibodies has been developed. Biotin was covalently attached to a peptide and the biotin-peptide bound on a nitrocellulose membrane. Antibody-coated gold nanoparticles bound to the biotin-peptide formed red dots. With this method, 100 amol of the biotin-peptide was detected and no immunogold was bound to the membrane in the absence of biotin. The relative intensity of each dot was scored using Quantity One, a quantitative analysis software program. The linear working range of this assay was between 1 pmol and 1 micromol. The assay sensitivity was increased by silver enhancement to 100 zmol, and the linear working range was between 100 zmol and 100 fmol. This assay can be extended to detect target molecules, such as dioxin, digoxin, mercury, and so on, with matched antibodies and has potential broad applications in immunoassay.     

Surfactant free fractions of metallic and semiconducting single-walled carbon nanotubes via optimised gel chromatography

We report the procedure of sorting/purification of carbon nanotubes by electronic type using chromatographic column with sodium dodecylsulfate (SDS) and sodium deoxycholate (DOC) solutions as the eluents. The non-commercial agarose gel in different concentrations has been tested in the process. It was found that in optimal gel concentration the fractionation resulted in ～96.2% yield of semiconducting species. Importantly, to get surfactant-free fractions the post-separation purification procedure has been carried out. The UV–vis–NIR and Raman spectroscopy have been utilised for the samples analysis. High resolution transmission microscopy and thermogravimetric analysis allowed to study the sample morphology and purity, respectively.