John G. Quinn

Advances in biosensors for detection of pathogens in food and water

Abstract While most microbes play an important role in nature, certain potentially harmful microbes can contaminate food and water, and cause a plethora of infectious diseases in both animals and humans. Conventional methods for detecting microbial contamination have primarily relied on time-consuming enrichment steps, followed by biochemical identification, having a total assay time of up to 1 week in certain cases. Over the last decade, a great deal of research has focused on the development of biological sensors for the detection of micro-organisms, allowing rapid and “real-time” identification. This paper reviews some of the most commonly used biosensor systems based on their transducer properties, which include surface plasmon resonance (SPR), amperometric, potentiometric, and acoustic wave sensors and their applications for the detection of pathogens in food and water. It also highlights some of the limitations of applying biosensors for the detection of pathogens, such as sensitivity, cost and the need for sample pre-treatment.

Detection of blood group antigens utilising immobilised antibodies and surface plasmon resonance

Surface plasmon resonance (SPR) detection using the BIAcore biosensing system was employed for the detection of blood group-associated antigens (BGAA) on whole erythrocytes. The quantitative detection of erythrocytes was accomplished by covalently immobilising blood group-specific antibodies (IgM) to a dextran matrix and monitoring the cell binding response. Non-specific binding of erythrocytes to the IgM coated surface was not detected. Relatively mild regeneration conditions (20 mM NaOH) were employed to elute bound erythrocytes in order to preserve the activity of the immobilised antibody and allow the surface to be used repeatedly. Regeneration of the surface was particularly difficult when a high IgM immobilisation level was used and when the number of bound cells was high. Despite these considerations, a quantitative relationship between the cell binding response and erythrocyte concentration was confirmed. Erythrocyte preparations, diluted by a factor of ten as compared to physiological concentrations, were detectable. The occurrence of non-specific false positives appears to be minimal and allows the system to be used for blood typing. As a model study, the lectin concanavalin A (ConA) was covalently immobilised onto a hydrophilic dextran matrix and successfully used to support the capture of erythrocytes from suspension.

Development of a surface plasmon resonance-based immunoassay for Listeria monocytogenes

A polyclonal antibody was produced against Internalin B (InlB)-enriched extract and used to develop an inhibition assay to detect Listeria monocytogenes cells in solution using surface plasmon resonance. The gene sequence encoding for the InlB protein was cloned into a Qiagen pQE-60 vector, expressed in Escherichia coli, and purified by immobilized metal affinity chromatography. Protein G-purified anti-InlB-enriched extract polyclonal antibody was incubated with various concentrations of L. monocytogenes cells and subsequently injected over a purified-recombinant InlB (rInlB)-immobilized CM5 sensor chip surface. A decrease in antibody binding response was observed with increasing L. monocytogenes cell concentrations. Intraday and interday assay variability studies were carried out to evaluate precision and reproducibility. The assay had a limit of detection of less than 2 x 10(5) cells per ml and could be successfully reproduced with coefficients of variation of between 2.5 and 7.7%.

Evaluation of Taylor dispersion injections: determining kinetic/affinity interaction constants and diffusion coefficients in label-free biosensing.

In label-free biomolecular interaction analysis, a standard injection provides an injection of uniform analyte concentration. An alternative approach exploiting Taylor dispersion produces a continuous analyte titration allowing a full analyte dose response to be recorded in a single injection. The enhanced biophysical characterization that is possible with this new technique is demonstrated using a commercially available surface plasmon resonance-based biosensor. A kinetic interaction model was fitted locally to Taylor dispersion curves for estimation of the analyte diffusion coefficient in addition to affinity/kinetic constants. Statistical confidence in the measured parameters from a single Taylor dispersion injection was comparable to that obtained for global analysis of multiple standard injections. The affinity constants for multisite interactions were resolved with acceptable confidence limits. Importantly, a single analyte injection could be treated as a high-resolution real-time affinity isotherm and was demonstrated using the complex two-site interaction of warfarin with human serum albumin. In all three model interactions tested, the kinetic/affinity constants compared favorably with those obtained from standard kinetic analysis and the estimates of analyte diffusion coefficients were in good agreement with the expected values.

Applications and recent developments in the use of antibodies for analysis

ABSTRACT There are many compounds that require analysis ranging from pesticide levels in corn to disease markers in human patients. There are copious challenges to be met when measuring analytes such as the matrix in which they are to be determined, the amounts present, the cost and the rapidity of the result required. Enzyme immunoassays, immunoaffinity chromatography, immunomagnetic polymerase chain reaction, flow cytometry and immunobiological biosensors have all characteristics that can enhance analytical techniques. Antibody-based methods have found applications in a large number of diverse areas such as food and water analysis, clinical diagnosis and therapeutics The structure and modes of production of antibodies and antibody-based derivatives is described and their applications in analysis critically examined.

Modeling Taylor dispersion injections: determination of kinetic/affinity interaction constants and diffusion coefficients in label-free biosensing.

A new method based on Taylor dispersion has been developed that enables an analyte gradient to be titrated over a ligand-coated surface for kinetic/affinity analysis of interactions from a minimal number of injections. Taylor dispersion injections generate concentration ranges in excess of four orders of magnitude and enable the analyte diffusion coefficient to be reliably estimated as a fitted parameter when fitting binding interaction models. A numerical model based on finite element analysis, Monte Carlo simulations, and statistical profiling were used to compare the Taylor dispersion method with standard fixed concentration injections in terms of parameter correlation, linearity of parameter error space, and global versus local model fitting. A dramatic decrease in parameter correlations was observed for TDi curves relative to curves from standard fixed concentration injections when surface saturation was achieved. In FCI the binding progress is recorded with respect to injection time, whereas in TDi the second time dependency encoded in the analyte gradient increases resolving power. This greatly lowers the dependence of all parameters on each other and on experimental interferences. When model parameters were fitted locally, the performance of TDis remained comparable to global model fitting, whereas fixed concentration binding response curves yielded unreliable parameter estimates.

Immunoassay for the Determination of 7-Hydroxycoumarin in Serum Using 'Real-Time' Biosensor Analysis

ABSTRACT An inhibition immunoassay for the determination of total 7-hydroxycoumarin (7-OHC) in human serum samples using the BIACORE biosensor is described. 7-OHC-BSA was immobilised onto BIACORE sensor chips and 7-OHC was premixed with a polyclonal anti-7-OHC antibody and injected over this surface. Excess antibody bound to the immobilised conjugate, generating a binding response that was inversely proportional to the amount of 7-OHC in the sample. The sample buffer was formulated to minimise non-specific binding of serum components to the sensor surface. Regeneration of the sensor surface was optimised such that the conjugate-coated surface retained acceptable antibody-binding capacity over 64 binding-regeneration cycles. The recovery of free drug from samples spiked with 7-hydroxycoumarin-glucuronide (7-OHCG) approximated 100%, while the inter- and intra-day coefficients of variation for the assay were less than 8.76% for 5 replicates. The assay had a measuring range of 0.5 – 80 μg/ml. This BIACORE-bas...

Bispecific multivalent antibody studied by real-time interaction analysis for the development of an antigen-inhibition enzyme-linked immunosorbent assay

A bispecific antibody with specificities for both 7-hydroxycoumarin (7-OHC) and alkaline phosphatase (AP) was produced by chemically cross-linking two parental polyclonal antibodies. Real-time interaction analysis of the bispecific multivalent antibody (bsMAb) was performed using BIAcore, a surface plasmon resonance (SPR)-based biosensor, in order to confirm its bispecific nature. A 7-OHC-BSA conjugate was covalently immobilized to a dextran matrix to serve as the reaction surface and unconjugated bovine serum albumin (BSA) was immobilized on to a separate dextran matrix as a control surface. Immunoaffinity-purified bsMAb, parental anti-7-OHC antibody and AP were injected over both surfaces. The bsMAb was shown to bind both antigens, 7-OHC and AP, simultaneously. Comparison of the ratio of mass bound for bsMAb and AP (5:1) with the ratio of the molecular masses of bsMAb (approximately 300 kDa) and AP (85 kDa) (3.5:1) suggests that most of the bsMAb species possess both specificities. The bsMAb was employed in a one-step antigen-inhibition ELISA for the detection of 7-OHC. The assay was compared with a conventional ELISA approach employing an AP-labelled secondary antibody. The bispecific antibody approach proved to be faster and more sensitive, with a detection limit of 6 ng ml-1 as compared with approximately 50 ng ml-1 for the conventional approach. The assay was used for the quantification of free and total 7-OHC in urine samples from two healthy volunteers who had been administered coumarin. The accuracy and precision of the assay were assessed. The bispecific antibody-based assay gave similar results, accuracy and precision, but proved to be far more sensitive (limit of determination 6 ng ml-1 for total 7-OHC). It is concluded that real-time interaction analysis using BIAcor provides a rapid method for the evaluation of the bsMAb and it was verified that the bispecific product formed by chemical cross-linking of two parental antibodies offers a simple alternative for the development of a highly sensitive ELISA.