Comparison of sensing strategies in SPR biosensor for rapid and sensitive enumeration of bacteria

Rapid and sensitive detections of microorganisms are very important for biodefence, food safety, medical diagnosis and pharmaceutics. The present study aims to find out the most proper bioactive surface preparation method to develop rapid, sensitive and selective bacteria biosensor, based on surface plasmon resonance (SPR) spectroscopy. Escherichia coli (E. coli) was used as a model bacterium and four sensing strategies in SPR were tested. Three of these strategies are antibody immobilization methods that are non-specific adsorption, specific adsorption via the avidin-biotin interaction, and immobilization of antibodies via self-assembled monolayer formation. The fourth strategy is a novel method for bacteria enumeration based on the combination of the SPR spectroscopy and immunomagnetic separation with using gold-coated magnetic nanoparticles. According to results, the most efficient SPR method is the one based on gold-coated magnetic nanoparticles. This method allows to specifically separate E. coli from the environment and to quantify rapidly without any labeling procedure. The developed method has a linear range between 30 and 3.0 × 10(4)cfu/ml, and a detection limit of 3 cfu/ml. The selectivity of the method was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The usefulness of the method to detect E. coli in real water samples was also investigated, and the results were compared with the results from plate-counting method. There was no significant difference between the methods (p>0.05).     

A mixed self-assembled monolayer-based surface plasmon immunosensor for detection of E. coli O157:H7

The sensitivity and specificity of a polyethylene glycol terminated alkanethiol mixed self-assembled monolayers (SAM) on surface plasmon resonance (SPR) immunosensor to detect Escherichia coli O157:H7 is demonstrated. Purified monoclonal (Mabs) or polyclonal antibodies (PAbs) against E. coli O157:H7 were immobilized on an activated sensor chip and direct and sandwich assays were carried to detect E. coli O157:H7. Effect of Protein G based detection and effect of concentrations of primary and secondary antibodies in sandwich assay were investigated. The sensor surface was observed under an optical microscope at various stages of the detection process. The sensor could detect as low as 10(3)CFU/ml of E. coli O157:H7 in a sandwich assay, with high specificity against Salmonella Enteritidis. The detection limit using direct assay and Protein G were 10(6)CFU/ml and 10(4)CFU/ml, respectively. Results indicate that an alkanethiol SAM based SPR biosensor has the potential for rapid and specific detection of E. coli O157:H7, using a sandwich assay.     

Long period grating based biosensor for the detection of Escherichia coli bacteria

In this paper we report a stable, label-free, bacteriophage-based detection of Escherichia coli (E. coli) using ultra sensitive long-period fiber gratings (LPFGs). Bacteriophage T4 was covalently immobilized on optical fiber surface and the E. coli binding was investigated using the highly accurate spectral interrogation mechanism. In contrast to the widely used surface plasmon resonance (SPR) based sensors, no moving part or metal deposition is required in our sensor, making the present sensor extremely accurate, very compact and cost effective. We demonstrated that our detection mechanism is capable of reliable detection of E. coli concentrations as low as 10(3)cfu/ml with an experimental accuracy greater than 99%.     

Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor

We report the quantitative and simultaneous detection of four species of bacteria, Escherichia coli O157:H7, Salmonella choleraesuis serotype typhimurium, Listeria monocytogenes, and Campylobacter jejuni, using an eight-channel surface plasmon resonance (SPR) sensor based on wavelength division multiplexing. Detection curves showing SPR response versus analyte concentration were established for each species of bacteria in buffer at pH 7.4, apple juice at native pH 3.7, and apple juice at an adjusted pH of 7.4, as well as for a mixture containing all four species of bacteria in buffer. Control experiments were performed to show the non-fouling characteristics of the sensor surface as well as the specificity of the amplification antibodies used in this study. The limit of detection (LOD) for each of the four species of bacteria in the tested matrices ranges from 3.4 x 10(3) to 1.2 x 10(5) cfu/ml. Detection curves in buffer of an individual species of bacteria in a mixture of all four species of bacteria correlated well with detection curves of the individual species of bacteria alone. SPR responses were higher for bacteria in apple juice at pH 7.4 than in apple juice at pH 3.7. This difference in sensor response could be partly attributed to the pH dependence of antibody-antigen binding.     

Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium

An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium. A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface. The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR. The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively. The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. typhimurium using protein G was developed with a detection range of 10(2) to 10(9)CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. typhimurium could be applied to construct other immnosensors or protein chips.     

Parallel-scan based microarray imager capable of simultaneous surface plasmon resonance and hyperspectral fluorescence imaging

With the development of the microarray technology, demands for array detection techniques become higher and higher. For many microarrays, several biomolecular interactions occur simultaneously and the interplay of various factors that affect these interactions remains poorly understood. Detecting such interactions with a single technique can often be a difficult and complicated process. In this work we propose a combined technique which enables simultaneous angle-interrogation surface plasmon resonance (SPR) sensing and hyperspectral fluorescence imaging. This tandem technique offers two-dimensional imaging of the whole array plane. The refractive index information obtained from SPR sensing and the physicochemical properties obtained from fluorescence imaging provide a comprehensive analysis of biological events on the array-chip. In addition, SPR and fluorescence detection techniques confirm each other in experimental results to exclude false-positive or false-negative cases. In terms of SPR sensing performance, the refractive index resolution is 3.86 × 10⁻⁶ refractive index units (RIU), and the detection limit is 10⁴ cfu/ml of Escherichia coli bacteria. The resolving power and detection sensitivity of fluorescence imaging are approximately 20 μm and 0.61 fluors/μm², respectively. Finally, two model experiments, detecting the DNA hybridization and biotin–avidin interactions respectively, demonstrate the biomedical application of this system.     

A SPR-based immunosensor for the detection of isoproturon

The proof of principle of a reusable surface plasmon resonance (SPR)-based immunosensor for the monitoring of isoproturon (IPU), a selective and systemic herbicide, is presented. The detecting rat monoclonal anti-isoproturon antibody (mAb IOC 7E1) was reversibly immobilized through the use of a capture mouse anti-rat (kappa-chain) monoclonal antibody (mAb TIB 172), which was covalently immobilized on the sensor chip surface. Such strategy features a controlled binding of the captured detecting antibody as well as facilitates the surface regeneration. The capture of the anti-IPU mAb by the antibody (TIB 172) coated sensor surface could be carried out up to 120 times (immobilization/regeneration cycles) without any evidence of activity loss. With a high test midpoint and a low associated SPR signal, the direct detection format was shown to be unsuitable for the routine analysis of isoproturon. However, the limit of detection (LOD) could be easily enhanced by using a strategy based on a surface competition assay, which improved all immunosensor parameters. Moreover, the sensitivity and working range of the indirect format were found to be dependent on the surface density of the anti-IPU mAb IOC 7E1. As expected for competitive formats, the lowest surface coverage (0.5 ng/mm(2)) allowed a lower detection of the herbicide isoproturon with a calculated LOD of 0.1 microg/l, an IC(50) (50% inhibition) of 5.3+/-0.6 microg/l, and a working range (20-80% inhibition) of 1.3-16.3 microg/l.     

Disposable amperometric immunosensor for the detection of polycyclic aromatic hydrocarbons (PAHs) using screen-printed electrodes

An amperometric immunosensor for polycyclic aromatic hydrocarbons (PAHs) was developed. The immunosensor was based on disposable screen-printed carbon electrodes. The coating antigen used was phenanthrene-9-carboxaldehyde coupled to bovine serum albumin (BSA) via adipic acid dihydrazide. Antibodies were monoclonal mouse anti-phenanthrene. The enzyme alkaline phosphatase (AP) was used in combination with the substrate p-aminophenyl phosphate (pAPP) for detection at +300 mV (vs. Ag/AgCl). Various assay types were compared. Good results were achieved with an indirect co-exposure competition assay with a LOD of 0.8 ng/ml (800 ppt) and an IC(50) of 7.1 ng/ml (7.1 ppb) for phenanthrene. An indirect competition assay could detect phenanthrene with a LOD of 2 ng/ml (IC(50): 15 ng/ml) and an indirect displacement assay with a LOD of 2 ng/ml (IC(50): 11 ng/ml) at a 5 microl surface coating of 8.8 microg/ml phenanthrene-BSA conjugate. A coating concentration of 2.2 microg/ml allowed detection with a LOD of 0.25 ng/ml (250 ppt) with the indirect competition assay. The influence of the coating concentration on the sensor performance was investigated. Cross-reactivities were tested for 16 important PAHs. Anthracene and chrysene showed strong cross-reactivity, whereas benzo[g,h,i]perylene and dibenzo[a,h]anthracene showed no cross-reactivity.     

Immunosensor for detection of Legionella pneumophila using surface plasmon resonance

Immunosensor using surface plasmon resonance (SPR) onto self-assembled protein G layer was developed for the detection of Legionella pneumophila. A self-assembled protein G layer on gold (Au) surface was fabricated by adsorbing a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2) and the activation process for chemical binding between free amine (-NH(2)) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of self-assembled protein G layer on Au substrate and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analyses of self-assembled protein G layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein G were performed by atomic force microscope (AFM). The immunosensor for detection of L. pneumophila using SPR was developed and its detection limit could find up to 10(5) cells/ml.     

Conducting polyamic acid membranes for sensing and site-directed immobilization of proteins

A biosensor platform based on polyamic acid (PAA) is reported for oriented immobilization of biomolecules. PAA, a functionalized conducting polymer substrate that provides electrochemical detection and control of biospecific binding, was used to covalently attach biomolecules, resulting in a significant improvement in the detection sensitivity. The biosensor sensing elements comprise a layer of PAA antibody (or antigen) composite self-assembled onto gold (Au) electrode via N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) linking. The modified PAA was characterized by Fourier transform infrared (FTIR), (1)H nuclear magnetic resonance (NMR), and electrochemical techniques. Cyclic voltammetry and impedance spectroscopy experiments conducted on electrodeposited PAA on Au electrode using ferricyanide produced a measurable decrease in the diffusion coefficient compared with the bare electrode, indicating some retardation of electron transfer within the bulk material of the PAA. Thereafter, the modified PAA surface was used to immobilize antibodies and then to detect inducible nitric oxide synthase and mouse immunoglobulin G (IgG) using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and amperometric techniques. ELISA results indicated a significant amplified signal by the modified PAA, whereas the SPR and amperometric biosensors produced significant responses as the concentration of the antigen was increased. Detection limits of 3.1×10(-3)ng/ml and 2.7×10(-1)ng/ml were obtained for SPR and amperometric biosensors, respectively.     

Detection of fungal spores using a generic surface plasmon resonance immunoassay

This paper describes a biosensor-based method for detection of fungal spores using surface plasmon resonance (SPR). The approach involves the use of a mouse monoclonal antibody (Pst mAb8) and a SPR sensor for label-free detection of urediniospores from the model organism Puccinia striiformis f.sp. tritici (Pst). In the subtractive inhibition assay, urediniospores and Pst mAb8 were mixed, urediniospore-bound Pst mAb8 removed by centrifugation and the remaining Pst mAb8 quantified using the SPR sensor. Assay conditions were optimised and a detection limit of 3.1 x 10(5)urediniospores/ml was achieved. Spiked Pst samples were further examined in a background of a related spore and it was found that Pst detection was possible in this mixture. This study represent the first use of SPR technology for fungal spore detection as well as the first report of a successful biosensor-based detection strategy for Pst.     

Microfluidic systems integrated with two-dimensional surface plasmon resonance phase imaging systems for microarray immunoassay

This study reports a microfluidic chip integrated with an arrayed immunoassay for surface plasmon resonance (SPR) phase imaging of specific bio-samples. The SPR phase imaging system uses a surface-sensitive optical technique to detect two-dimensional (2D) spatial phase variation caused by rabbit immunoglobulin G (IgG) adsorbed on an anti-rabbit IgG film. The microfluidic chip was fabricated by using micro-electro-mechanical-systems (MEMS) technology on glass and polydimethylsiloxane (PDMS) substrates to facilitate well-controlled and reproducible sample delivery and detection. Since SPR detection is very sensitive to temperature variation, a micromachine-based temperature control module comprising micro-heaters and temperature sensors was used to maintain a uniform temperature distribution inside the arrayed detection area with a variation of less than 0.3 degrees C. A self-assembled monolayer (SAM) technique was used to pattern the surface chemistry on a gold layer to immobilize anti-rabbit IgG on the modified substrates. The microfluidic chip is capable of transporting a precise amount of IgG solution by using micropumps/valves to the arrayed detection area such that highly sensitive, highly specific bio-sensing can be achieved. The developed microfluidic chips, which employed SPR phase imaging for immunoassay analysis, could successfully detect the interaction of anti-rabbit IgG and IgG. The interactions between immobilized anti-rabbit IgG and IgG with various concentrations have been measured. The detection limit is experimentally found to be 1 x 10(-4)mg/ml (0.67 nM). The specificity of the arrayed immunoassay was also explored. Experimental data show that only the rabbit IgG can be detected and the porcine IgG cannot be adsorbed. The developed microfluidic system is promising for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.     

Liquid chromatographic-mass spectrometric quantitation of Delta9-tetrahydrocannabinol and two metabolites in pharmacokinetic study plasma samples

A sensitive method for the determination of Delta(9)-tetrahydrocannabinol and its metabolites, 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid and 11-hydroxy-Delta(9)-tetrahydrocannabinol, in rat and guinea pig plasma was developed using high-performance liquid chromatographic separation with electrospray ionization mass spectrometry detection and a simple liquid-liquid extraction technique. The mean recoveries for Delta(9)-tetrahydrocannabinol, 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid, and 11-hydroxy-Delta(9)-tetrahydrocannabinol were 96, 92, and 85%, respectively. The lower limit of quantification (LLOQ) for all three compounds was 5 ng/ml and the limit of detection (LOD) was 2 ng/ml. This assay method utilizes the increased sensitivity and selectivity of mass spectrometric (MS) detection and a simple extraction step for the determination of Delta(9)-tetrahydrocannabinol and its metabolites in plasma, and thus yields a more efficient pharmacokinetic analysis method than has previously been described.     

Rapid identification of biological warfare agents using an instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system

An instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system has been developed for the rapid and specific identification of biological warfare (BW) agents. The system has been designed to assay for up to eight agents simultaneously and provides an indication of the absence or presence of a threat within 15 min. Parameters affecting the mixing of the reagents within the instrument's fluidic lines were investigated and optimized. Measurements of blank samples and samples containing Bacillus subtilis spores in the concentration range of 10(4) to 10(6) cfu/ml indicate the limit of detection (LOD) is 3 x 10(3) cfu/ml for B. subtilus. Although the LOD is higher than that of several technologies currently under development, this instrument offers an immediate interim approach for addressing the need to rapidly detect biological warfare agents in the field.     

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.     

Real-time protein biosensor arrays based on surface plasmon resonance differential phase imaging

This paper reports the application of differential phase surface plasmon resonance (SPR) imaging in two-dimensional (2D) protein biosensor arrays. Our phase imaging approach offers a distinct advantage over the conventional angular SPR technique in terms of utilization efficiency of optical sensor elements in the imaging device. In the angular approach, each biosensor site in the biosensor array requires a linear array of optical detector elements to locate the SPR angular dip. The maximum biosensor density that a two-dimensional imaging device can offer is a one-dimensional SPR biosensor array. On the other hand, the phase-sensitive SPR approach captures data in the time domain instead of the spatial domain. It is possible that each pixel in the captured interferogram represents one sensor site, thus offering high-density two-dimensional biosensor arrays. In addition, our differential phase approach improves detection resolution through removing common-mode disturbances. Experimental results demonstrate a system resolution of 8.8 x 10(-7)RIU (refractive index unit). Real-time monitoring of bovine serum albumin (BSA)/anti-BSA binding interactions at various concentration levels was achieved using a biosensor array. The detection limit was 0.77 microg/ml. The reported two-dimensional SPR biosensor array offers a real-time and non-labeling detection tool for high-throughput protein array analysis. It may find promising applications in protein therapeutics, drug screening and clinical diagnostics.     

Rapid method for detection of Salmonella in milk by surface plasmon resonance (SPR)

The Plasmonic surface plasmon resonance (SPR) device was used to develop a rapid, simple and specific immunoassay for detection of Salmonella in milk. Rapid detection of Salmonella contamination is a major challenge for the food industry. Salmonella contamination is well known in all foods including pasteurised milk. The SPR assay was developed as a sandwich model using a polyclonal antibody against Salmonella as capture and detection antibody. Milk spiked with Salmonella typhimurium cells, killed by thimerosal (1%, w/w) treatment was used. Using the Plasmonic SPR assay it was possible to detect S. typhimurium down to a concentration of 1.25 x 10(5) cells ml(-1) in both milk and buffer system. The results obtained are comparable with existing, approved rapid Salmonella detection techniques. No negative effects on the sensitivity of the assay are encountered due to the milk matrix. Hence, no sample preparation or clean-up steps are required. The sample volume requirement for the assay is only 10 microl. Using the assay S. typhimurium was detected in milk within 1h, whereas the cultural techniques require 3-4 days for presumptive positive isolates and further time for confirmation. The rapid tests require at least 24h for the results. The Plasmonic SPR device operates on the Kretschmann configuration and is a cuvette-based system with the advantage of having eight channels on one single SPR chip.     

Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi

A surface plasmon resonance (SPR) based immunosensor using self-assembled protein G was developed for the detection of Salmonella paratyphi. In order to endow a solid substrate binding affinity to protein G, the free amine (-NH2) of protein G was substituted into thiol (-SH) using 2-iminothiolane. Thus, self-assembled protein G was fabricated on gold (Au) substrate. The formation of protein G layer on Au surface, and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analysis of the protein G layer on Au surface was performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. paratyphi using self-assembled protein G was developed with a detection range of 10(2)-10(7) CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. paratyphi could be applied to construct other immnosensors or protein chips.     

Signal amplification on planar and gel-type sensor surfaces in surface plasmon resonance-based detection of prostate-specific antigen

This article describes surface plasmon resonance (SPR)-based detection of prostate-specific antigen (PSA), comparing amplification with colloidal gold (10nm diameter) and latex microspheres (120 nm diameter) on planar- and gel-type sensor surfaces. As matrix, 3% BSA in PBS was used. Experimental data were compared with model calculations that predict the SPR signal that results from covering of the different sensor surfaces with each of the particles used. Amplification with latex particles gave a higher signal than did that with colloidal gold. However, the limit of detection (LOD) attained by latex amplification was not as good as that obtained after gold amplification, and this was unexpected. LOD and sensitivity of the amplified PSA assays when performed with the planar-type sensor disc were equally good or better compared with those when performed with the gel-type sensor disc. Indirect evidence indicates a restricted accessibility of the gel layer on the gel-type sensor toward the colloidal gold. Application of colloidal gold led to a sensitivity increase of approximately three orders of magnitude compared with nonamplified detection. The corresponding LOD was approximately 0.15 ng PSA/ml, which is sufficient for measuring enhanced, clinically relevant PSA levels (>4 ng/ml).     

Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum

An immunochromatographic strip test using gold nanoparticles was developed for the rapid detection of Salmonella typhi (S. typhi) in human serum. The strip test based on the principle of sandwich immunoassay by the specific binding of antigens from S. typhi O901 and antibody of S. typhi O901 on a nitrocellulose membrane. Antibody-gold nanoparticle conjugate was used as the label and was coated onto a glass fiber membrane, which was used as a conjugate pad. To create a test and control zone, antibody of S. typhi O901 and an anti-IgG were dotted on the nitrocellulose membrane, respectively. Positive samples were displayed as red dots at the test and control zones of the nitrocellulose membrane, while negative samples resulted in a red dot only in the control zone. The limit of detection (LOD) was found to be 1.14×10(5) cfu mL(-1), which could be visually detected by the naked eye within 15 min. This strip test provided a lower detection limit and analysis time than a dot blot immunoassay (8.88×10(6) cfu mL(-1) for LOD and 110 min for reaction time). In addition, our immunochromatographic strip test was employed to detect S. typhi in human serum effectively, with high accuracy. This strip test offers great promise for a rapid, simple and low-cost analysis of S. typhi.