Automated microarray system for the simultaneous detection of antibiotics in milk

A parallel affinity sensor array (PASA) for the rapid automated analysis of 10 antibiotics in milk is presented, using multianalyte immunoassays with an indirect competitive ELISA format. Microscope glass slides modified with (3-glycidyloxypropyl)trimethoxysilane were used for the preparation of hapten microarrays. Protein conjugates of the haptens were immobilized as spots on disposable chips, which were processed in a flow cell. Monoclonal antibodies against penicillin G, cloxacillin, cephapirin, sulfadiazine, sulfamethazine, streptomycin, gentamicin, neomycin, erythromycin, and tylosin allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with horseradish peroxidase generating enhanced chemiluminescence, which was recorded with a sensitive CCD camera. All liquid handling and sample processing was fully automated, and one analysis was carried out in milk within less than 5 min. The detection limits ranged from 0.12 (cephapirin) to 32 microg/L (neomycin). Penicillin G could be detected at the maximum residue limit (MRL); the detection limits for all other analytes were far below the respective MRLs. The PASA system proved to be the first immunochemical biosensor platform having the potential to test for numerous antibiotics in parallel, such being of considerable interest for the control of milk in the dairy industry.     

Competitive immunoassays for simultaneous detection of metabolites and proteins using micromosaic patterning

New high-throughput immunoassay methods for rapid point-of-care diagnostic applications represent an unmet need and current focus of numerous innovative methods. We report a new micromosaic competitive immunoassay developed for the analysis of the thyroid hormone thyroxine (T4), inflammation biomarker C-reactive protein (CRP), and the oxidative damage marker 3-nitrotyrosine (BSA-3NT) on a silicon nitride substrate. To demonstrate the versatility of the method, both direct and indirect format competitive immunoassays were developed and could be applied simultaneously for single samples. Signals from standard solutions were fit to a logistic equation, allowing simultaneous detection of T4 (7.7-257.2 nM), CRP (0.3-4.2 microg/mL), and BSA-3NT (0.03-22.3 microg/mL). Total assay time including sample introduction, washing, and fluorescence measurement was less than 45 min. Dissociation constants for affinity pairs in the system have been estimated using regression. This proof-of-concept experiment shows that both small and macromolecular biomarkers can be quantified from a single sample using the method and suggests that groups of clinically related analytes may be analyzed by competitive micromosaic immunoassay techniques.     

Multiple and simultaneous detection of specific bacteria in enriched bacterial communities using a DNA microarray chip with randomly generated genomic DNA probes

A DNA microarray chip for detecting the presence of specific bacterial strains was developed using random genomic probes derived from genomic DNA, i.e., without any sequence information. Thirteen bacteria from different genuses were selected as targets. For the fabrication of the random genomic probes, genomic DNA from pure cultures of each bacterium was fractionated using several pairs of restriction endonucleases. After size fractionation of the genomic DNA fragments, random genomic libraries for each bacterium were constructed. From the library, specific probes were amplified by PCR and the probes were affixed to a slide glass to fabricate the DNA microarray chip. The results from tests with pure and mixed cultures of the bacteria used in the fabrication of the chips showed specific responses and only a small portion of cross-hybridization. This DNA microarray chip was also tested to detect the presence of specific bacteria in mixed populations. In these tests, it was demonstrated that this system provided a fast and specific response to the presence of bacterial species in mixed samples, even in activated sludge samples. This indicates that any DNA microarray chip for the detection of specific bacteria can be fabricated using the same protocols as presented in this study without requiring any genus level sequence information from pure isolates.     

A homogeneous noncompetitive immunoassay for the detection of small haptens

We describe a noncompetitive homogeneous immunoassay for small haptens based on the antigen-dependent reassociation of antibody variable domains and beta-galactosidase (beta-gal) complementation (open sandwich enzymatic complementation immunoassay). As a model system, the reassociation of two fusion proteins, an anti 4-hydroxy-3-nitrophenylacetyl (NP) antibody heavy-chain variable-region fragment fused to an N-terminal deletion mutant of beta-gal (V(H)delta alpha) and the light-chain variable-region fragment fused to a C-terminal deletion mutant of beta-gal (V(L)delta omega), was monitored by the enzymatic complementation between the two. Upon simple mixing of the reagents with the sample, an antigen (NP)-dependent increase in enzymatic activity was observed. When 5-iodo-NP was measured, a 10 times higher sensitivity was observed, probably due to its higher affinity. Compared with our corresponding heterogeneous open sandwich enzyme-linked immunosorbent assay, approximately 1000-fold improvement in the sensitivity was attained, probably due to lower background V(H)-V(L) association. In addition, the assay required less time, handling, sample volume, and assay reagents.     

A sensitive and quantitative element-tagged immunoassay with ICPMS detection

We report a set of novel immunoassays in which proteins of interest can be detected using specific element-tagged antibodies. These immunoassays are directly coupled with an inductively coupled plasma mass spectrometer (ICPMS) to quantify the elemental (in this work, metal) component of the reacted tagged antibodies. It is demonstrated that these methods can detect levels of target proteins as low as 0.1-0.5 ng/mL and yield a linear response to protein concentration over 3 orders of magnitude.     

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.     

A free-labeled method for DNA-binding protein detection using a double-stranded DNA microarray

We have developed a new type of double-stranded DNA microarray to perform detection of sequence-specific DNA-binding proteins. The DNA-binding site of a DNA-binding protein is divided into two fragments. One fragment was immobilized on an aldehyde-coated glass microscope slide surface via chemical bonds. The other fragment was labeled with a fluorescent molecule. When using this kind of double-stranded DNA microarray, the labeled DNA fragment was pre-incubated with detection sample for 5 to 10 minutes and then hybridized with the microarray. In our experiment, six different concentrations of Nuclear Factor kappa-B P50 homodimer in detection samples were tested. The microarray fluorescence intensity was obtained and the relationship between the intensity and the protein concentration was calculated. The detection results suggested that this free-labeled detection system could have the ability to be used in research and medical diagnosis and for high-throughput screening of drugs targeted to DNA-binding proteins.     

Hybrid surface platform for the simultaneous detection of proteins and DNAs using a surface plasmon resonance imaging sensor

In this work, we present a novel surface and assay for the simultaneous detection of DNA and protein analytes on a surface plasmon resonance (SPR) imaging sensor. A mixed DNA/oligo (ethylene glycol) (OEG) self-assembled monolayer (SAM) is created using a microarrayer. Thiol-modified single-stranded DNA sequences are spotted onto a gold-coated glass substrate. Backfilling with an OEG-modified alkanethiol creates a protein-resistant surface background. Antibodies conjugated to complementary single-stranded DNA sequences are immobilized on the surface through DNA hybridization. By converting only part of the DNA array into a protein array, simultaneous detections of DNA and protein analytes are possible. A model system of two cDNA sequences and two human pregnancy hormones are used to demonstrate the assay. No cross-reactivity was observed between DNA or protein analytes and nontargeted immobilized cDNA sequence or antibodies. A response from a detection of a single analyte in a mixture of protein and DNA analytes corresponds well with that of a single-analyte solution.     

A porous silicon optical microcavity for sensitive bacteria detection

A porous silicon microcavity (PSM) is highly sensitive to subtle interface changes due to its high surface area, capillary condensation ability and a narrow resonance peak (～10 nm). Based on the well-defined optical properties of a PSM, we successfully fabricated a bacteria detection chip for molecular or subcellular analysis by surface modification using undecylenic acid (UA), and the specific recognition binding of vancomycin to the D-alanyl-D-alanine of bacteria. The red shift of the PSM resonance peak showed a good linear relationship with bacteria concentration ranging from 100 to 1000 bacteria ml( - 1) at the level of relative standard deviation of 0.994 and detection limit of 20 bacteria ml( - 1). The resulting PSM sensors demonstrated high sensitivity, good reproducibility, fast response and low cost for biosensing.     

Microfabricated linear hydrogel microarray for single-nucleotide polymorphism detection

A platform is developed for rapid, multiplexed detection of single-nucleotide polymorphisms using gels copolymerized with oligonucleotide capture probes in a linear microchannel array. DNA samples are analyzed by electrophoresis through the linear array of gels, each containing 20-40 μM of a unique oligonucleotide capture probe. Electrophoresis of target DNA through the capture sites and the high concentration of capture probes within the gels enables significantly shorter incubation times than standard surface DNA microarrays. These factors also result in a significant concentration of target within the gels, enabling precise analysis of as little as 0.6 femtomoles of DNA target. Differential melting of perfectly matched and mismatched targets from capture probes as a function of electric field and temperature enables rapid, unambiguous identification of single-nucleotide polymorphisms.     

Magnetic bead based immunoassay for autonomous detection of toxins

We are developing an automated system for the simultaneous, rapid detection of a group of select agents and toxins in the environment. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads and a photoactive porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactive group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags. Released eTags are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated in a flow-through format with higher LODs of 32 ng/mL (or 640 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.     

Ganglioside-liposome immunoassay for the ultrasensitive detection of cholera toxin

An extremely sensitive bioassay has been developed for cholera toxin (CT) detection, using ganglioside-incorporated liposomes. Cholera is a diarrheal disease, often associated with water or seafood contamination. Ganglioside GM1 was used to prepare the liposomes by spontaneous insertion into the phospholipid bilayer. CT recognition and signal generation is based on the strong and specific interaction between GM1 and CT. In a sandwich immunoassay, CT was detected as a colored band on the nitrocellulose membrane strip, where CT bound to GM1-liposomes can be captured by immobilized antibodies. The intensity of the band could be visually estimated or measured by densitometry, using computer software. The limit of detection (LOD) of CT in the assay system was found to be 10 fg/mL which is equivalent to 8 zmol in the 70-microL sample. The assay was also tested with water samples spiked with CT, providing a LOD of 0.1-30 pg/mL, which is much better than previously reported limits of detection from other assays. The assay could be completed within 20 min. These results demonstrate that the bioassay developed for CT is rapid and ultrasensitive, suggesting the possibility for detecting CT, simply and reliably, in field screening.     

Nanoparticle probes and mid-infrared chemical imaging for DNA microarray detection

To date most mid-infrared spectroscopic studies have been limited, due to lack of sensitivity, to the structural characterization of a single oligonucleotide probe immobilized over the entire surface of a gold-coated slide or other infrared substrate. By contrast, widely used and commercially available glass slides and a microarray spotter that prints approximately 120-μm-diameter DNA spots were employed in the present work. To our knowledge, mid-infrared chemical imaging (IRCI) in the external reflection mode has been applied in the present study for the first time to the detection of nanostructure-based DNA microarrays spotted on glass slides. Alkyl amine-modified oligonucleotide probes were immobilized on glass slides that had been prefunctionalized with succinimidyl ester groups. This molecular fluorophore-free method entailed the binding of gold-nanoparticle-streptavidin conjugates to biotinylated DNA targets. Hybridization was visualized by the silver enhancement of gold nanoparticles. The adlayer of silver, selectively bound only to hybridized spots in a microarray, formed the external reflective infrared substrate that was necessary for the detection of DNA hybridization by IRCI in the present proof-of-concept study. IRCI made it possible to discriminate between diffuse and specular external reflection modes. The promising qualitative results are presented herein, and the implications for quantitative determination of DNA microarrays are discussed.     

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.     

Amperometric sensors for simultaneous superoxide and hydrogen peroxide detection

A two-channel sensor capable of almost instantaneous simultaneous detection of superoxide radical and hydrogen peroxide in the concentration range 10(-)(7)-10(-)(4) M is very important for understanding of a number of rapid kinetics processes. A glassy carbon working microelectrode covered by an electrodeposited polypyrrole/horseradish peroxidase (PPy/HRP) membrane was employed as a H(2)O(2) sensor. Another glassy carbon microelectrode covered by a composite membrane of an inside layer of PPy/HRP and an outside layer of superoxide dismutase was employed as a working electrode for superoxide detection. These two working electrodes with Pt counter and tungsten oxide (WO(3)) reference electrodes were contained in one 6 mm diameter Teflon cylinder. Simultaneous measurements were performed at a potential of -60 mV (vs WO(3) reference, pH 5.1). Additional sensor characterization was performed for pH 5.1-9.0. Superoxide sensor behavior as a function of membrane deposition conditions and coating time is reported. Sensors' mutual influence, selectivity, response times, linearity, stability, and sensitivity for hydrogen peroxide and superoxide are presented and discussed. A mathematical model of sensors' responses is proposed, with model calculation corresponding to experiment within 10%.     

An immunoassay for small analytes with theoretical detection limits

A flow-based immunoassay that uses microspheres as the solid phase accomplished the theoretical limit of detectability achievable with the antibody. An equilibrated mixture of anti-estriol monoclonal antibody and estriol was briefly exposed to a bead pack containing immobilized estriol in a flow cell. A small portion of free antibody was separated rapidly from the mixture by binding it to immobilized hormone, but the antibody-hormone complex was kinetically excluded from binding. This rapid separation prevented shift in the equilibrium of the liquid phase binding. Signals were generated by labeling the separated antibodies on the beads with a Cy5-conjugated antispecies secondary antibody. By labeling after the separation step, perturbing the liquid-phase or solid-phase binding was prevented. This assay allowed the reduction of the concentration of primary antibody by continuously accumulating free antibody onto the beads prior to quantification and, thus, offered ideal conditions to achieve theoretical limits of detectability. The optimum achievable dynamic range of this immunoassay was 4-300 pM. Because the proportion of free anti-estriol antibody in the mixture was controlled by the Kd of the antibody-estriol interaction, when the concentration of the antibody was below the Kd, the smallest detectable estriol concentration approached the theoretical limit of detectability achievable with this antibody.     

Magneto-controlled graphene immunosensing platform for simultaneous multiplexed electrochemical immunoassay using distinguishable signal tags

A novel flow-through multiplexed immunoassay protocol for simultaneous electrochemical determination of carcinoembryonic (CEA) and alpha-fetoprotein (AFP) in biological fluids was designed using biofunctionalized magnetic graphene nanosheets (MGO) as immunosensing probes and multifunctional nanogold hollow microspheres (GHS) as distinguishable signal tags. The probes were fabricated by means of co-immobilization of primary anti-CEA (Ab(1)) and anti-AFP (Ab(2)) antibodies on the Fe(3)O(4) nanoparticle-coated graphene nanosheets (MGO-Ab(1,2)). The reverse-micelle method was used for the synthesis of distinguishable signal tags by encapsulation of horseradish peroxide (HRP)-thionine and HRP-ferrocene into nanogold hollow microspheres, respectively, which were utilized as labels of the corresponding GHS-Ab(1) and GHS-Ab(2). A sandwich-type immunoassay format was employed for the online detection of CEA and AFP by coupling a flow-through detection cell with an external magnet. The assay was based on the catalytic reduction of H(2)O(2) at the various peak potentials in the presence of the corresponding mediators. Experimental results revealed that the multiplexed electrochemical immunoassay enabled the simultaneous monitoring of AFP and CEA in a single run with wide working ranges of 0.01-200 ng mL(-1) for AFP and 0.01-80 ng mL(-1) for CEA. The detection limits (LODs) for both analytes at 1.0 pg mL(-1) (at 3s(B)) were very low. No obvious nonspecific adsorption and cross-talk were observed during a series of analyses to detect target analytes. Intraassay and interassay coefficients of variation were <10%. Importantly, the methodology was evaluated for the analysis of clinical serum specimens, receiving a good correlation between the flow-through multiplexed electrochemical immunoassay and an electrochemiluminescence method as a reference.     

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.