Development of an immunobiosensor assay for the beta-adrenergic compound zilpaterol

A surface plasmon resonance biosensor method was developed to measure zilpaterol residues in sheep urine. A CM-5 sensor chip previously reacted with ethylenediamine to produce an aminoethyl group was coupled with 4-carboxybutyl zilpaterol activated using EDC/NHS. Five polyclonal and four monoclonal antibodies were screened for their suitability to detect low levels of zilpaterol using the biosensor technology. Total binding was greater for polyclonal than monoclonal antibodies, but a less diluted antibody solution was required for polyclonal antibodies. A fixed antibody concentration and various concentrations of zilpaterol were injected to obtain a standard curve for each antibody to allow for B-0 and IC50 determination. The stability of the assay was assessed by the consistency of B0 in repeated experiments extending at least six hours. A measure of non-specific binding allowed the assessment of the specificity of the antibody-immobilized ligand interaction. The effect of varying concentrations of urine on B-0 and IC50 was evaluated to assess the degree of

Use of a novel micro-fluidic device to create arrays for multiplex analysis of large and small molecular weight compounds by surface plasmon resonance

There is an increasing demand to develop biosensor monitoring devices capable of biomarker profiling for predicting animal adulteration and detecting multiple chemical contaminants or toxins in food produce. Surface plasmon resonance (SPR) biosensors are label free detection systems that monitor the binding of specific biomolecular recognition elements with binding partners. Essential to this technology are the production of biochips where a selected binding partner, antibody, biomarker protein or low molecular weight contaminant, is immobilised. A micro-fluidic immobilisation device allowing the covalent attachment of up to 16 binding partners in a linear array on a single surface has been developed for compatibility with a prototype multiplex SPR analyser.

The immobilisation unit and multiplex SPR analyser were respectively evaluated in their ability to be fit-for-purpose for binding partner attachment and detection of high and low molecular weight molecules. The multiplexing capability of the dual technology was assessed using phycotoxin concentration analysis as a model system. The parent compounds of four toxin groups were immobilised within a single chip format and calibration curves were achieved. The chip design and SPR technology allowed the compartmentalisation of the binding interactions for each toxin group offering the added benefit of being able to distinguish between toxin families and perform concentration analysis. This model is particularly contemporary with the current drive to replace biological methods for phycotoxin screening.

A Critical Review of Screening Methods for the Detection of Chloramphenicol, Thiamphenicol, and Florfenicol Residues in Foodstuffs

This article gives an extensive overview of the wide range of analytical procedures developed for the detection of amphenicol antibiotic residues (chloramphenicol, thiamphenicol, and florfenicol) in many different types of foodstuffs (milk, meat, eggs, honey, seafood). Screening methods such as microbial inhibition methods, antibody-based immunoassays using conventional and biosensor-based detection systems, and some methods based on alternative recognition systems are described. The relative advantages and disadvantages of these methods are discussed and compared. The current status and future trends and developments in the need for accurate and rapid detection of this group of antimicrobials are also discussed.

A comparative study of qualitative immunochemical screening assays for the combined measurement of T-2/HT-2 in cereals and cereal-based products

Many different immunochemical platforms exist for the screening of naturally occurring contaminants in food from the low cost enzyme linked immunosorbent assays (ELISA) to the expensive instruments such as optical biosensors based on the phenomenon of surface plasmon resonance (SPR). The primary aim of this study was to evaluate and compare a number of these platforms to assess their accuracy and precision when applied to naturally contaminated samples containing HT-2/T-2 mycotoxins. Other important factors considered were the speed of analysis, ease of use (sample preparation techniques and use of the equipment) and ultimately the cost implications. The three screening procedures compared included an SPR biosensor assay, a commercially available ELISA and an enzyme-linked immunomagnetic electrochemical array (ELIME array). The qualitative data for all methods demonstrated very good overall agreements with each other, however on comparison with mass spectrometry confirmatory results, the ELISA and SPR assay performed slightly better than the ELIME array, exhibiting an overall agreement of 95.8% compared to 91.7%. Currently, SPR is more costly than the other two platforms and can only be used in the laboratory whereas in theory both the ELISA and ELIME array are portable and can be used in the field, but ultimately this is dependent on the sample preparation techniques employed. Sample preparative techniques varied for all methods evaluated, the ELISA was the most simple to perform followed by that of the SPR method. The ELIME array involved an additional clean-up step thereby increasing both the time and cost of analysis. Therefore in the current format, field use would not be an option for the ELIME array. In relation to speed of analysis, the ELISA outperformed the other methods.

Detection of streptomycin residues in whole milk using an optical immunobiosensor

The development of an assay for the detection of streptomycin residues in pasteurized whole milk using an optical biosensor (Biacore) is reported. Streptomycin-adipic hydrazide coupled to bovine thyroglobulin was used to produce a sheep polyclonal antibody. The antibody displayed excellent cross-reactivity with dihydrostreptomycin (106%). There was no significant cross-reaction with other aminoglycosides or common antibiotics. Streptomycin was also immobilized onto a CM5 sensor chip to provide a stable, reusable surface. The developed assay permitted the direct analysis of whole milk samples (similar to3.5% fat) without prior centrifugation and defatting. Results were available in 5 min. The limit of detection of the assay was determined as 4.1 ng/mL, well below the European maximum residue limit (MRL) of 200 ng/mL. Repeatability (or coefficient of variation) between runs was determined as 3.5% (100 ng/mL; 0.5 x MRL), 5.7% (200 ng/mL; MRL), and 7.6% (400 ng/mL; 2 x MRL).

The development of a rapid immunobiosensor screening method for the detection of residues of sulphadiazine

A rapid imununoassay using an optical biosensor (BIAcore(TM)) for determining the presence of sulphadiazine (SDZ) residues in pig bile was developed. SDZ,cas immobilised onto the surface of a dextran-coated silicon chip and a solution containing SDZ antibody, sample and buffer was injected over the chip surface. The level of antibody binding to the chip was determined after 20 s and the surface of the chip was then regenerated over a 1-min period prior to another sample injection taking place. Standard curves were constructed to allow quantification of SDZ presence in sample. Concentrations ranging from 0 to 10.64 mu g ml(-1) SDZ were detected in bile samples taken from experimentally fed pigs and randomly selected pigs taken from a local slaughterhouse. These results were compared to the concentrations of SDZ detected by high-performance liquid chromatography: in associated tissues. When concentrations in bile exceeded 0.6 mu g ml(-1) SDZ, the corresponding edible tissue was above the maximum residue level (MRL), i.e. 0.1 mu g g(-1) in 13 out of 14 cases. Wizen the bile concentration was less than 0.6 mu ml(-1) the associated tissue concentrations never exceeded rite MRL. This experiment has indicated that biosensor analysis of bile is a highly effective method for detecting violative SDZ residues in meat.

A novel toxicity fingerprinting method for pollutant identification with lux-marked biosensors

A novel technique is described for the identification and quantification of environmental pollutants based on toxicity fingerprinting with a metabolic lux-marked bacterial biosensor. This method involved characterizing the toxicity-based responses of the biosensor to seven calibration pollutants as acute temporal-dose response fingerprints. An algorithm is described to allow comparisons of responses of an unknown pollutant to be made against the calibration data. This is based on predicting pollutant concentration at each of six different time points over the course of a 5-min assay. If the prediction is consistent between the unknown pollutant and a calibration pollutant at the 95% test level, this is considered to be a positive identification. All seven calibration pollutants could be successfully distinguished from each other with this technique. Environmental samples, individually spiked with single concentrations of pollutants, were compared in this way against the calibration pollutants. An 83% identification success was achieved, with no false positives at the 95% test level. This is a simple and rapid technique that potentially can be applied to monitoring of industrial wastewater or as a screening tool for regulators.

An ecotoxicological approach to assessing the impact of tanning industry effluent on river health

A study was conducted to investigate the sediment health and water quality of the River Sagana, Kenya, as impacted by the local tanning industry. Chemical analysis identified the main chemical pollutants (pentachlorophenols and chromium) while a bioassay addressed pollutant bioavailability. The bioassay, exploiting the luminescence response of a lux marked bacterial biosensor, was coupled to a dehydrogenase and Dapnia magna test to determine toxicity effects on sediments. Results highlighted the toxicity of the tannery effluent to the sediments at the point of discharge (64% of control bioluminescence) with gradual improvement downstream. There was a significant increase in dehydrogenase downstream, with the enzyme activity attaining a peak at 600 m, also indicating a gradual reduction of toxicity. Biological oxygen demand (19.56 mg L(-1)) dissolved oxygen (3.97 mg L(-1)) and high lethal dose value (85%) of D. magna also confirmed an initial stress at the point of discharge and recovery downstream. Optical density of surface water demonstrated an increase in suspended particulates and colour after the discharge point, eventually decreasing beyond 400 m. In conclusion, the study highlighted the importance of understanding the biogeochemistry of river systems impacted by industries discharging effluent into them and the invaluable role of a biosensor-based ecotoxicological approach to address effluent hazards, particularly in relation to river sediments.

Biosensors for the analysis of microbiological and chemical contaminants in food

Increases in food production and the ever-present threat of food contamination from microbiological and chemical sources have led the food industry and regulators to pursue rapid, inexpensive methods of analysis to safeguard the health and safety of the consumer. Although sophisticated techniques such as chromatography and spectrometry provide more accurate and conclusive results, screening tests allow a much higher throughput of samples at a lower cost and with less operator training, so larger numbers of samples can be analysed. Biosensors combine a biological recognition element (enzyme, antibody, receptor) with a transducer to produce a measurable signal proportional to the extent of interaction between the recognition element and the analyte. The different uses of the biosensing instrumentation available today are extremely varied, with food analysis as an emerging and growing application. The advantages offered by biosensors over other screening methods such as radioimmunoassay, enzyme-linked immunosorbent assay, fluorescence immunoassay and luminescence immunoassay, with respect to food analysis, include automation, improved reproducibility, speed of analysis and real-time analysis. This article will provide a brief footing in history before reviewing the latest developments in biosensor applications for analysis of food contaminants (January 2007 to December 2010), focusing on the detection of pathogens, toxins, pesticides and veterinary drug residues by biosensors, with emphasis on articles showing data in food matrices. The main areas of development common to these groups of contaminants include multiplexing, the ability to simultaneously analyse a sample for more than one contaminant and portability. Biosensors currently have an important role in food safety; further advances in the technology, reagents and sample handling will surely reinforce this position.

Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: A femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG(3)-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and Fr-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG(6)-COOH and HS-OEG(3)-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Au Nanoparticles for Applications in Analysis of Cellular and Biomolecular Recognitions

Au nanoparticles (AuNPs) have attracted a great interest in fabrication of various biosensor systems for analysis of cellular and biomolecular recognitions. In conjunction with vast conjugation chemistry available, the materials are easily coupled with biomolecules such as nucleic acids, antigens or antibodies in order to achieve their many potential applications as ligand carriers or transducing platforms for preparation, detection and quantification purposes. Furthermore, the nanoparticles possess easily tuned and unique optical/ physical/ chemical characteristics, and high surface areas, making them ideal candidates to this end. In this topic, sensing mechanisms based on localized surface plasmon resonance (LSPR), particle aggregation, catalytic property, and Fluorescence Resonance Energy Transfer (FRET) of AuNPs as well as barcoding technologies including DNA biobarcodes will be discussed.

Effects of Gold Nanoparticle and Enzyme Precipitation on Enhancement of Surface Plasmon Resonance Immunoassay: A Super Sensitive Measurement of Anti- Glutamic Acid Decarboxylase Antibody

Introduction: In this study, colloidal gold nanoparticle and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance biosensor.

Methods: The colloidal gold nanoparticle was synthesized as described by Turkevitch et al., and their surface was firstly functionalized with HS(CH2)11(OCH2CH2)3COOH (OEG3¬-COOH) by self assembling technique. Thereafter, those OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti-IgG antibody (specific to the Fc portion of all human IgG subclasses) to form an enzyme-immunogold complex. Characterization was performed by several methods: UV-Vis absorption, dynamic light scattering (DLS), transmission electron microscopy (TEM) and FTIR. The as-prepared enzyme-immunogold complex has been applied in enhancement of SPR immunoassay. A sensor chip used in the experiment was constructed by using 1:10 molar ratio of HS(CH2)11(OCH2CH2)6COOH and HS(CH2)11(OCH2CH2)3OH. The capture protein, GAD65 (autoantigen) which is recognized by anti-GAD antibody (autoantibody) in the sera of insulin-dependent diabetes mellitus patients, was immobilized onto the 1:10 surface via biotin-streptavidin interaction.

Results and conclusions: In the research, we reported the influences of gold nanoparticle and enzyme precipitation on the enhancement of SPR signal. Gold nanoparticle showed its enhancement as being consistent with other previous studies, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. As the results, anti-GAD antibody could be detected at pg/ml level which is far higher than that of commercial ELISA detection kit. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Development of M13 bacteriophage-based SPR detection using Salmonella detection as a case study

Surface plasmon resonance (SPR)-based biosensor is a popular platform for real-time monitoring and sensitive detection for a myriad of targets. However, only a few studies have reported the use of bacteriophages as specific binders for SPR-based detection. This study aimed to demonstrate how filamentous M13 bacteriophages expressing 12-mer peptides can be employed in an SPR-based assay, using a Salmonella-specific bacteriophage as a model binder to detect the foodborne bacterium Salmonella. Several important factors (immobilization buffers and methods, and interaction buffers) for a successful bacteriophage-based SPR assay were optimized. As a result, a Salmonella-specific bacteriophage-based SPR assay was achieved, with very low cross reactivity with other non-target foodborne pathogens and detection limits of 8.0 × 10⁷ and 1.3 × 10⁷ CFU/mL for one-time and five-time immobilized sensors, respectively. This proof-of-concept study demonstrates the feasibility of using M13 bacteriophages expressing target-specific peptides as a binder in a rapid and label-free SPR assay for pathogen detection.

Metamaterials-Based Label-Free Nanosensor for Conformation and Affinity Biosensing

Analysis of molecular interaction and conformational dynamics of biomolecules is of paramount importance in understanding of their vital functions in complex biological systems, disease detection, and new drug development. Plasmonic biosensors based upon surface plasmon resonance and localized surface plasmon resonance have become the predominant workhorse for detecting accumulated biomass caused by molecular binding events. However, unlike surface-enhanced Raman spectroscopy (SERS), the plasmonic biosensors indeed are not suitable tools to interrogate vibrational signatures of conformational transitions required for biomolecules to interact. Here, we show that highly tunable plasmonic metamaterials can offer two transducing channels for parallel acquisition of optical transmission and sensitive SERS spectra at the biointerface, simultaneously probing the conformational states and binding affinity of biomolecules, e.g. G-quadruplexes, in different environments. We further demonstrate the use of the metamaterials for fingerprinting and detection of arginine-glycine-glycine domain of nucleolin, a cancer biomarker which specifically binds to a G-quadruplex, with the picomolar sensitivity.

Nile blue-based nano-sized pH sensors for simultaneous far-red and near-infrared live bioimaging

Diblock copolymer vesicles are tagged with pH-responsive Nile Blue-based labels and used as a new type of pH-responsive colorimetric/fluorescent biosensor for far-red and near-infrared imaging of live cells. The diblock copolymer vesicles described herein are based on poly(2-(methacryloyloxy)ethyl phosphorylcholine-block-2-(diisopropylamino)ethyl methacrylate) [PMPC-PDPA]: the biomimetic PMPC block is known to facilitate rapid cell uptake for a wide range of cell lines, while the PDPA block constitutes the pH-responsive component that enables facile vesicle self-assembly in aqueous solution. These biocompatible vesicles can be utilized to detect interstitial hypoxic/acidic regions in a tumor model via a pH-dependent colorimetric shift. In addition, they are also useful for selective intracellular staining of lysosomes and early endosomes via subtle changes in fluorescence emission. Such nanoparticles combine efficient cellular uptake with a pH-responsive Nile Blue dye label to produce a highly versatile dual capability probe. This is in marked contrast to small molecule dyes, which are usually poorly uptaken by cells, frequently exhibit cytotoxicity, and are characterized by intracellular distributions invariably dictated by their hydrophilic/hydrophobic balance.