Multianalyte immunoassay based on insulating-controllable PoPD film at arrayed electrodes integrated on a silicon chip

A novel, simple and label-free multianalyte immunoassay system is presented here by integrating arrayed electrodes on a silicon chip via MEMS. The chip is consisted of six Au disk electrodes, an Au counter electrode and an Ag/AgCl reference electrode. Semi-insulating poly(o-phenylenediamine) (PoPD) was utilized to co-polymerize and immobilize antibodies at the arrayed Au electrodes, and wider linear detection range was obtained than those prepared with completely insulating PoPD. Electrochemical cyclic voltammogram (CV), AC impedance spectroscopy, AFM and fluorescence microscopy were employed to characterize the system. The arrayed electrodes offered exact control of deposition position via electrochemical operation, allowing selectively immobilization of different antibodies at desired positions on a single chip. Specific recognition of antibody (Ab) to corresponding antigen (An) was quantitatively monitored by cyclic voltammograms in the presence of electrochemical redox probe, ferrocene methanol. The proposed immunoassay chips showed sensitive response to three liver fibrosis markers, hyaluronic acid (HA), collagen type IV (IV-C) and lamin (LN) at ng/mL level simultaneously and specifically in a tiny amount of volume, usually 50 μL. The results obtained via chips were well consistent with those obtained by commercial radio immunoassays (RIA).     

Microbead-based electrochemical immunoassay with interdigitated array electrodes

The objective of this study was to develop a sensitive and miniaturized immunoassay by coupling a microbead-based immunoassay with an interdigitated array (IDA) electrode. An IDA electrode amplifies the signal by recycling an electrochemically redox-reversible molecule. The microfabricated platinum electrodes had 25 pairs of electrodes with 1.6-microm gaps and 2.4-microm widths. An enzyme-labeled sandwich immunoassay on paramagnetic microbeads with mouse IgG as the analyte and beta-galactosidase as the enzyme label was used as the model system. beta-Galactosidase converted p-aminophenyl beta-D-galactopyranoside to p-aminophenol (PAP). This enzyme reaction was measured continuously by positioning the microbeads near the electrode surface with a magnet. Electrochemical recycling occurred with PAP oxidation to p-quinone imine (PQI) at +290 mV followed by PQI reduction to PAP at -300 mV vs Ag/AgCl. Dual-electrode detection amplified the signal fourfold compared to single-electrode detection, and the recycling efficiency reached 87%. A calibration curve of PAP concentration vs anodic current was linear between 10(-4) and 10(-6)M. A signal from 1000 beads in a 20-microL drop was detectable and the immunoassay was complete within 10 min with a detection limit of 3.5x10(-15)mol mouse IgG.     

编码悬浮微块片上多组分并行免疫检测的方法

采用热压印光刻技术制备了一种多金属构成、带数字标识图形的悬浮微块,其中的镍层与金层可分别实现微块的磁控靶向与生物探针的联接。借助微块表面的数字微通孔标识符号,实现了微块的生物探针编码;用异硫氰酸荧光素荧光标记编码的悬浮微块,通过悬浮微块的多组分并行免疫荧光检测,实现了微块的生物探针解码及生物分子的定量检测。这种编码的地址数取决于微块表面的微通孔数,理论上可以成千上万。因此,表面经过生物探针修饰的悬浮微块是建立生物分子编码库的理想途径,可作为基于高通量悬浮阵列技术的免疫分析平台。     

Fabrication and characterization of a multiwell array SERS chip with biological applications

Uniform, large surface area substrates for surface-enhanced Raman spectroscopy (SERS) are fabricated by oblique angle deposition. The SERS-active substrates are patterned by a polymer-molding technique to provide a uniform array for high throughput biosensing and multiplexing. Using a conventional SERS-active molecule, 1,2-di(4-pyridyl)ethylene (BPE) ≥98%, we show that this device provides a uniform Raman signal enhancement from well to well with a detection limit of at least 10⁻⁸ M of the BPE solution or 10⁻¹⁸ mol of BPE. The SERS intensity is also demonstrated to vary logarithmically with the log of BPE concentration and the apparent sensitivity of the patterned substrate is compared to previous reports from our group on non-patterned substrates. Avian influenza is analyzed to demonstrate the utility of SERS multiwell patterned substrates for biosensing. The spectra acquired from patterned substrates show better reproducibility and less variation compared to the unpatterned substrates according to multivariate analysis. Our results highlight potential advantages of the patterned substrate.     

Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.     

Luminescence catalyst immunoassay of β2-microglobulin with haemin as a chemically amplifiable label

Luminescence catalyst immunoassay, a nonradioactive and nonenzymatic immunoassay, has been applied to the determination of β₂-microglobulin (β₂-MG). The method is characterized by high sensitivity and simple handling, since the analytical method employs a catalytically amplifiable label and solid-phase sandwich technique, respectively. In the first stage, an antibody-immobilized plate is reacted with the analyte (β₂-MG). In the second stage, the bound β₂-MG undergoes successive binding with haemin-labelled antibody. In the last stage, the plate is placed in the luminol-H₂O₂ system to generate luminescence. A calibration curve with a β₂-MG concentration at the midpoint of 100 ng ml^?1 was obtained. The 10 and 90% response levels in the curve are 10 and 1000 ng ml^?1.     

An enhanced chemiluminescent enzyme immunoassay for serum carcinoembryonic antigen based on a modification of a commercial kit

A conventional colorimetric peroxidase end-point (ortho-phenylenediamine substrate), used in an enzyme immunoassay for carcinoembryonic antigen, employing plastic beads as solid support, has been replaced by a much faster (30 seconds versus 30 minutes) enhanced chemiluminescent assay for the peroxidase label. Para-iodophenol was used to enhance the light emission from the peroxidase catalysed chemiluminescent reaction between luminol and hydrogen peroxide. Values for precision and carcinoembryonic antigen concentration obtained with the chemiluminescent and colorimetric versions of the immunoassay on 62 serum specimens were in good agreement.     

Extended standard curve stability on the CCD magic lite immunoassay system using a two-point adjustment

Ciba Corning Diagnostics has developed a two-point adjustment algorithm for use with the Magic Lite System which allows for extended stability of a full 7–10 point calibration curve over the life of a kit. This adjustment is accomplished by using a master calibration curve established during manufacturing along with two calibrators for each assay. Most conventional non-automated immunoassays contain anywhere from 6 to 10 calibrators which are included with each run of an assay. Eliminating the need to run full standard curves and using the two-point adjustment algorithm results in significant savings in both labour and reagent usage.     

Immobilization of single-stranded DNA by self-assembled polymer on gold substrate for a DNA chip

We developed a self-assembling polymer based on polyallylamine (PAH) for use in DNA chips. Thioctic acid (TA) was covalently attached to PAH in sidechains to immobilize the polymer on a gold surface by self-assembly. N-hydroxysuccinimide-ester terminated probe single-stranded (ss) DNA is easily covalently immobilized onto a TA-PAH-coated gold surface. Finally, the surface was covered with polyacrylic acid, which formed ion complexes with the TA-PAH, to reduce the cationic charge. This ssDNA on a polymer-coated surface recognized a fully matched DNA sequence and restrained nonspecific adsorption of target DNA. The selectivity and efficiency of hybridization was affected by adjusting the ionic strength of sodium chloride.     

Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA–bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I₅₀ and I₁₀ values of purified mAbs were 0.63 and 0.13 μg/ml, respectively, with a dynamic range between 0.19 and 2.04 μg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 μg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.     

Miniaturized amperometric flow immunoassay system using a glass fiber membrane modified with anion

This paper describes a miniaturized amperometric flow immunoassay system using a glass fiber membrane modified with anion. The glass fiber membrane was functionally modified with gamma-glycidoxypropyltrimethoxysilane and sodium thiosulfate and was used for separation of protein. Anti-human chorionic gonadotrophin (HCG) immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc), namely, Fc-conjugated IgG (Fc-IgG), was used as a novel analytical reagent. HCG and Fc-IgG complexes were separated from free Fc-IgG based on differences in isoelectric point (pI) using the glass fiber membrane modified with a thiosulfonyl acid functional group. The assay yields a linear relationship between current and HCG concentration in the range of 0-2000 mIU/mL. This simple technique enables the assay of HCG within 2 min. The modified glass fiber membrane was regenerated by occasional elution with malonate buffer (pH 6.0) containing 0.5 M NaCl, to remove free Fc-IgG. Free Fc-IgG recovered in this manner could be reused up to eight times without significant decreases in sensitivity. This miniaturized amperometric flow immunoassay requires only minute quantities of serum and generates highly reproducible results.     

Detection of tetanus-induced effects in linearly lined-up micropatterned neuronal networks: application of a multi-electrode array chip combined with agarose microstructures

K-7174, a GATA-specific inhibitor, is a putative anti-inflammatory agent that attenuates effects of inflammatory cytokines in certain cell types. However, molecular mechanisms involved have not been elucidated. We found that, in glomerular podocytes, induction of monocyte chemoattractant protein 1 (MCP-1) and inducible nitric oxide synthase (iNOS) by TNF-alpha was abrogated by K-7174. It was correlated with unexpected induction of unfolded protein response (UPR) evidenced by: (1) induction of endogenous indicators 78 kDa glucose-regulated protein and CCAAT/enhancer-binding protein-homologous protein, and (2) suppression of an exogenous indicator, endoplasmic reticulum stress-repressive alkaline phosphatase. In podocytes, induction of UPR by either tunicamycin, thapsigargin, A23187 or AB5 subtilase cytotoxin completely reproduced the suppressive effect of K-7174. Furthermore, K-7174-elicited UPR abrogated induction of MCP-1 and iNOS not only by TNF-alpha but also by medium conditioned by activated macrophages. These results suggested a novel, UPR-dependent mechanism underlying the anti-inflammatory potential of K-7174.     

Rapid determination of hazardous compounds in food based on a competitive fluorescence microsphere immunoassay

Development of a microsphere-based competitive fluorescence immunoassay for the determination of hazardous low-molecular-weight compounds in food is described. In this method, antigens are covalently bound to carboxy-modified microspheres to compete monoclonal antibody with low-molecular-weight compounds in food samples; mouse IgG/fluorescein isothiocyanate conjugate is used as the fluorescent molecular probe. Thus, the hazardous low-molecular-weight compounds are quantified using a multiparameter flow cytometer. This method has been evaluated using clenbuterol as a model compound. It has a sensitivity of 0.01 ng/mL with dynamic range of 0.01-100 ng/mL, and the concentration of clenbuterol providing 50% inhibition (IC₅₀) is 1.1 ng/mL. The main advantages of this method are its high efficiency, biocompatibility, and selectivity, as well as ultralow trace sample consumption and low cost.     

Application of an automated immunomagnetic separation-enzyme immunoassay for the detection of Salmonella spp during an outbreak associated with a retail premises

AIMS: The application of an automated immunomagnetic separation-enzyme immunoassay (AIMS-EIA) during the investigation of a suspected outbreak of Salmonella food poisoning at a retail premises. METHODS AND RESULTS: Six food samples and 24 environmental swabs were taken from the retail premises and six food handlers' submitted faecal samples during the investigation of the outbreak. Isolation and identification of Salmonella from these samples was performed according to established standard operating procedures and by AIMS-EIA. Twelve of the 18 (67%) Salmonella culture positive samples were AIMS-EIA positive on testing pre-enrichment samples after 24 h, whilst 17 (94%) samples were AIMS-EIA positive following selective enrichment for a further 48 h. One food handler was found to be positive for Salmonella by both culture and AIMS-EIA. All Salmonella isolates were confirmed as Salmonella Enteritidis phagetype 21c. CONCLUSIONS: The AIMS-EIA protocol compliments the conventional culture approach to produce more timely results for the management of the risk to public health without significantly increasing the workload of the laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: The food production premise investigated in this study was heavily contaminated with Salmonella Enteritidis. Application of the AIMS-EIA was significant in the effective intervention of control measures for the protection of public health.     

Direct protein detection with a nano-interdigitated array gate MOSFET

A new protein sensor is demonstrated by replacing the gate of a metal oxide semiconductor field effect transistor (MOSFET) with a nano-interdigitated array (nIDA). The sensor is able to detect the binding reaction of a typical antibody Ixodes ricinus immunosuppressor (anti-Iris) protein at a concentration lower than 1 ng/ml. The sensor exhibits a high selectivity and reproducible specific detection. We provide a simple model that describes the behavior of the sensor and explains the origin of its high sensitivity. The simulated and experimental results indicate that the drain current of nIDA-gate MOSFET sensor is significantly increased with the successive binding of the thiol layer, Iris and anti-Iris protein layers. It is found that the sensor detection limit can be improved by well optimizing the geometrical parameters of nIDA-gate MOSFET. This nanobiosensor, with real-time and label-free capabilities, can easily be used for the detection of other proteins, DNA, virus and cancer markers. Moreover, an on-chip associated electronics nearby the sensor can be integrated since its fabrication is compatible with complementary metal oxide semiconductor (CMOS) technology.     

Enhanced fluorescence resonance energy transfer immunoassay with improved sensitivity based on the Fab'-based immunoconjugates

Fluorescence resonance energy transfer (FRET) is a powerful technique to monitor protein-protein interaction. Recently, we developed homogeneous and noncompetitive immunoassay based on the enhanced FRET by leucine zipper interaction. Here we improved the assay by establishing a general method for preparation of the Fab'-based immunoconjugate. Anti-human serum albumin Fab' numbers 11 and 13 were chemically conjugated with recombinant proteins consisting of thioredoxin, flexible linker, and green fluorescent protein color variant tethered with a leucine zipper motif. Compared with single chain antibody variable region-based fusion proteins prepared by the gene fusion method in our previous study, the resultant Fab'-based immunoconjugates accomplished an assay with nearly 10 times greater sensitivity. Furthermore, the conjugation method enabled us to apply the assay generally to measurement of another high-molecular weight antigen for which antibodies prepared for sandwich immunoassay are commercially available. Because of the facility and generality of the preparation method for the immunoconjugate, the assay is expected to be applied to many antigens that require rapid diagnosis and moderate measurement range.     

Identification of proteins bound to a thioaptamer probe on a proteomics array

A rapid method to screen and identify unknown bound proteins to specific nucleic acid probes anchored on ProteinChip array surfaces from crude biological samples has been developed in this paper. It was demonstrated with screening specific binding proteins from LPS-stimulated mouse 70Z/3 pre-B cell nuclear extracts by direct coupling of thioaptamer XBY-S2 to the pre-activated ProteinChip array surfaces. With pre-fractionation of crude nuclear extracts by ion exchange method, specific "on-chip" captured proteins have been obtained that were pure enough to do "on-chip" digestion and the subsequent identification of the "on-chip" bound proteins by microsequencing of the trypsin digested peptide fragments through tandem MS. Five mouse heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2/B1, A3, A/B, and D0 were identified. To verify those bound hnRNPs, a novel thioaptamer/antibody sandwich assay provides highly sensitive and selective identification of proteins on ProteinChip arrays.     

A novel automated assay with dual-color hybridization for single-nucleotide polymorphisms genotyping on gold magnetic nanoparticle array

A high-throughput and cost-effective single-nucleotide polymorphism (SNP) genotyping method based on a gold magnetic nanoparticle (GMNP) array with dual-color hybridization has been designed. Biotinylated single-strand polymerase chain reaction (PCR) products containing the SNP locus were captured by the GMNPs that were coated with streptavidin. The GMNP array was fabricated by immobilizing single-stranded DNA (ssDNA)-GMNP complexes onto a glass slide using a magnetic field, and SNPs were identified with dual-color fluorescence hybridization. Three different SNP loci from 24 samples were genotyped successfully using this platform. This procedure allows the user to directly analyze the bead fluorescence to determine the SNP genotype, and it eliminates the need for background subtraction for signal determination. This method also bypasses tedious PCR purification and concentration procedures, and it facilitates large-scale SNP studies by using a method that is highly sensitive, simple, labor-saving, and potentially automatable.     

A multipixel diffuse correlation spectroscopy system based on a single photon avalanche diode array

The autocorrelation of laser speckles from coherent near infrared light is used for noninvasive estimates of relative changes in blood perfusion in techniques such as laser Doppler flowmetry (LDF) and diffuse correlation spectroscopy (DCS). In this study, a 2D array of single photon avalanche diodes (SPADs) was used to combine the strengths of multiple detectors in LDF with high light sensitivity in DCS. The system was tested on milk phantoms with varying detector fiber diameter (200 and 600 μm), source‐detector fiber separation (4.6‐10.2 mm), fiber‐SPAD distance (2.5‐36.5 mm), contiguous measurement time per repetition for the autocorrelation (1‐33 ms) and temperature (15.6‐46.7°C). An in vivo blood occlusion test was also performed. The multipixel approach improved signal‐to‐noise ratio (SNR) and, in our setup, the use of a multimode detector fiber was beneficial for SNR. In conclusion, the multipixel system works, but improvements and further studies regarding, for example, the data acquisition and optimal settings are still needed.      

Construction of Agropyron Gaertn. genetic linkage maps using a wheat 660K SNP array reveals a homoeologous relationship with the wheat genome

Agropyron Gaertn. (P genome) is a wild relative of wheat that harbours many genetic variations that could be used to increase the genetic diversity of wheat. To agronomically transfer important genes from the P genome to a wheat chromosome by induced homoeologous pairing and recombination, it is necessary to determine the chromosomal relationships between Agropyron and wheat. Here, we report using the wheat 660K single nucleotide polymorphism (SNP) array to genotype a segregating Agropyron F₁ population derived from an interspecific cross between two cross‐pollinated diploid collections ‘Z1842’ [A. cristatum (L.) Beauv.] (male parent) and ‘Z2098’ [A. mongolicum Keng] (female parent) and 35 wheat–A. cristatum addition/substitution lines. Genetic linkage maps were constructed using 913 SNP markers distributed among seven linkage groups spanning 839.7 cM. The average distance between adjacent markers was 1.8 cM. The maps identified the homoeologous relationship between the P genome and wheat and revealed that the P and wheat genomes are collinear and relatively conserved. In addition, obvious rearrangements and introgression spread were observed throughout the P genome compared with the wheat genome. Combined with genotyping data, the complete set of wheat–A. cristatum addition/substitution lines was characterized according to their homoeologous relationships. In this study, the homoeologous relationship between the P genome and wheat was identified using genetic linkage maps, and the detection mean for wheat–A. cristatum introgressions might significantly accelerate the introgression of genetic variation from Agropyron into wheat for exploitation in wheat improvement programmes.