Surface IR applied to rapid and direct immunosensing of environmental pollutants

This paper describes the elaboration of a model immunosensor monitored by polarization modulation reflection absorption infrared spectroscopy (PM-RAIRS) and quartz crystal microbalance (QCM-D), as well as its reactivity using PM-RAIRS as transduction technique. To prove its relevance, this immunosensor was applied to the detection of benzo[a]pyrene (BaP), a carcinogenic polycylic aromatic hydrocarbon. Very few immunoassays, and even fewer immunosensors, have been described for the assay of BaP, making it an interesting target for analytical device development. The PM-RAIRS immunosensor was constructed on planar gold-coated sensors functionalized with cystamine then glutaraldehyde. Antibodies were immobilized through their affinity to protein G, covalently coupled to the aldehyde layer. In a first stage, a model mouse IgG was utilized to optimize the elaboration parameters; then, a monoclonal anti-PAH antibody was used and detection tests were performed, monitored by PM-RAIRS. The successive functionalization steps were monitored by PM-RAIRS and QCM-D. The binding of the proteins to gold surface, their saturation coverages and association constants, as well as their capture efficiencies were discussed. BaP capture by the antibody layer was evidenced by the appearance of a new ν(C-H) band at 3039 cm⁻¹ typical of aromatic C-H bonds. The integrated area of this band varied linearly with the BAP concentration within the range of tested concentrations, with a limit of detection close to 3 μM. This represents the first example of direct, label-free immunodetection of a low molecular weight molecule by PM-RAIRS transduction. The simplicity and the rapid response of this IR sensor make it already very worthwhile to preliminary on-site measurements.     

Piezoelectric immuno sensor for the detection of candida albicans microbes

The system consists of an AT-cut quartz piezoelectric crystal, oscillator and frequency counter. The surface of the palladium-plated electrodes is oxidized anodically, and anti-Candida antibody is immobilized onto the surface. The crystal sensor is dipped into Candida suspension and the surface mass increase, caused by immuno-adsorption of Candida, is measured by the decrease in the resonant frequency of the crystal. The frequency shift is correlated with C. albicans concentrations in the range 10⁶?5 × 10⁸ cells cm^?3. The crystal sensor showed no response to Saccharomyces cerevisiae.     

Aptamer-quantum dots and teicoplanin-gold nanoparticles constructed FRET sensor for sensitive detection of Staphylococcus aureus

The detection of bacterial pathogen such as Staphylococcus aureus(S.aureus) is essential for the regulation of food hygiene and disease diagnosis.Herein,we developed a simple one-step fluorescence resonance energy transfer(FRET)-based sensor for specific and sensitive detection of S.aureus in food and serum samples,in which aptamer-modified quantum dots(aptamer-QDs) was employed as the energy donor and antibiotic of teicoplanin functionalized-gold nanoparticles(Teico-AuNPs) was chosen as the energy acceptor.Within 1 h,the FRET-based sensor showed a linear range of from 10 cfu/mL to 5 × 10~8 cfu/mL,with the low limit of detection(LOD,2 cfu/mL) for S.aureus in buffer.When further applied to assay S.aureus in real samples,the FRET-based sensor showed good recoveries ranging from 84.5% to 110.0%,with relative standard derivations(RSDs) of 0.01%-0.44% and a LOD of 100 cfu/mL in milk,orange juice and human serum.     

Development of an immunosensor for the detection of Francisella tularensis antibodies

Tularemia, also known as rabbit fever, is a highly infectious zoonotic disease caused by a non-motile and non-spore-forming Gram-negative coccoid rod bacterium, Francisella tularensis. It occurs naturally in lagomorphs (rabbits and hares), but many animals have been reported to be susceptible. Transmission to humans is mostly caused by inhalation of aerosolised bacteria, handling of infected animals, arthropod stings, and ingestion of contaminated foods and water. At present, pathogenic isolation, molecular detection, and serology are the most commonly used methods to confirm the diagnosis of tularemia. In this work, an electrochemical immunosensor for the detection of anti-F. tularensis antibodies was developed, consisting of gold-based self-assembled monolayers of a carboxylic-group-terminated bipodal alkanethiol that is covalently linked to a lipopolysaccharide (LPS) that can be found in the outer membrane of the bacteria F. tularensis. The presence of anti-F. tularensis antibodies was measured using horseradish peroxidase-labelled protein A (HRP-protein A) from Staphylococcus aureus, and the developed immunosensor gave a stable quantitative response to different anti-F. tularensis FB11 antibody concentrations after 30 min with a limit of detection of 15 ng/mL, RSD of 9 %, n?=?3. The developed immunosensor was tested with serum from animals infected with tularemia and was compared to the results obtained using ELISA showing an excellent degree of correlation.     

Surface plasmon resonance immunosensor for histamine based on an indirect competitive immunoreaction

The use of a surface plasmon resonance immunosensor for the analysis of histamine (β-imidazole ethylamine) is described. The method is based on an indirect competitive reaction of an anti-histamine antibody in a sample solution with histamine immobilized on a sensor chip and with histamine in the sample solution. A sensor chip immobilized with histamine was prepared using a self-assembly monolayer of 11-mercaptoundecanoic acid (11-MUA) as an anchor membrane, followed by an amino-coupling reaction with histamine after activation of the 11-MUA layer on the sensor chip by treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide. The sensor chip can be reused, after regeneration with a 10 mM HCl solution, which dissociates the anti-histamine antibody complex from histamine on the sensor chip. The affinity constants for the immunocomplex of the anti-histamine antibody with histamine in the solution and for that of the anti-histamine antibody with histamine immobilized on the sensor chip were calculated to be 1.5 × 10⁷ and 7.2 × 10⁵ M⁻¹, respectively, by assuming a Langmuir-type adsorption of the anti-histamine antibody to histamine immobilized on the sensor chip. The detection limit of the method was determined to be 3 ppb.     

In-depth investigation of protein adsorption on gold surfaces: correlating the structure and density to the efficiency of the sensing layer

Protein A (PrA), mouse monoclonal anti-IgG antibody (SAb) and deglycosylated avidin (NAV) were adsorbed on gold surfaces to capture the model rabbit IgG and build three immunosensing platforms. The assembling of immunosensors, their specificity, and the receptor accessibility were monitored by polarization modulation reflection-absorption infrared spectroscopy (PM-RAIRS) and quartz crystal microbalance with dissipation measurement (QCM-D) at each step. Combining these two techniques allows us to compare both chemical and structural properties of the sensing layers with the former bringing chemical and semiquantitative information on the grafted protein layers, whereas the latter, in addition to the mass uptake, enables us to take the layer rigidity into account. Grafting of the three capture proteins to the transducer surfaces, covered with appropriate self-assembled monolayers, yielded protein layers with variable properties. NAV formed a dense and rigid molecular layer, likely containing protein aggregates, whereas the amount of PrA was below one monolayer resulting in a flexible layer. The amount of immobilized rabbit IgG was different for the three systems with the densest capture protein layer exhibiting the lowest binding capacity. The accessibility of antibodies on the resulting immunosensors measured by interaction with a secondary antirabbit IgG antibody was found to be closely dependent on their coverage as well as on the rigidity of the protein layer. The overall study provides in-depth information on three of the most common immunosensor recognition interfaces and demonstrates the crucial influence of both structure and density of the protein layer on the efficiency of the molecular recognition phenomena.     

Evaluation of antibody immobilization technqiues for fiber optic-based fluoroimmunosensing

Fiber optic chemical sensors have been developed that employ immunochemicals to perform fluoroimmunoassays. A regenerable fiber optic sensor was developed with a capillary delivery system to perform repetitive assays using antibodies immobilized to beads, “immunobeads”. The sensitivity of these sensors is directly proportional to the amount of antibody present. For the immunobeads, the amount immobilized (loaded) and the ability of the immobilized antibody to maintain antibody recognition (activity) are two criteria which will affect the sensitivity of the sensors. Four reagents, [1,1′-carbonyldiimidazole (CDI), Protein A, glycidoxypropyltrimethoxysilane (GOPS) and 2-fluoro-1-methylpyridium toluene-4-sulfonate (FMP)], were evaluated using these two criteria. Two antibody—antigen systems were employed to investigate the four procedures. The first combination is a polyclonal rabbit anti-human IgG with a F(ab)^′₂ fragment human IgG as the antigen. The second combination is a monoclonal mouse anti-benzo[a]pyrene tetraol (BPT) IgG with BPT as the hapten. In the case of the BPT, CDI demonstrated superior performance, combining high loading and high retention of antibody activity. In the case of the large antigen, CDI again immobilized the most antibody but suffered activity losses of about 40%. The large amount of inactive antibody may make this procedure less attractive than the Protein A beads, which maintained the activity of the anti-human IgG but exhibited less loading than the CDI. However, in terms of active antibody there are similar amounts. FMP yielded similar results to CDI for the large antigen case but the sample preparation is more labor intensive. GOPS yielded losses of up to 70% of the antibody activity, which makes it unattractive as a reagent. The two systems tested give an indication of antibody—antigen interactions but may not be representative of all cases. The ideal immobilization reagent and conditions will probably change from system to system.     

Piezoelectric affinity sensors for cocaine and cholinesterase inhibitors

We report here the development of piezoelectric affinity sensors for cocaine and cholinesterase inhibitors based on the formation of affinity complexes between an immobilized cocaine derivative and an anti-cocaine antibody or cholinesterase. For both binding reactions benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on the surface of the sensor. For immobilization, pre-conjugated BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU) allowed the formation of a chemisorbed monolayer on the piezosensor surface.The detection of cocaine was based on a competitive assay. The change of frequency measured after 300 s of the binding reaction was used as the signal. The maximum binding of the antibody resulted in a frequency decrease of 35 Hz (with an imprecision 3%, n = 3) while the presence of 100 pmol l⁻¹ cocaine decreased the binding by 11%. The limit of detection was consequently below 100 pmol l⁻¹ for cocaine. The total time of one analysis was 15 min.This BZE-DADOO-modified sensor was adapted for the detection of organophosphates. BZE-DADOO - a competitive inhibitor - served as binding element for cholinesterase in a competitive assay.     

Immunomagnetic nanoparticle based quantitative PCR for rapid detection of Salmonella

We have developed a rapid and sensitive method for immunomagnetic separation (IMS) of Salmonella along with their real time detection via PCR. Silica-coated magnetic nanoparticles were functionalized with carboxy groups to which anti-Salmonella antibody raised against heat-inactivated whole cells of Salmonella were covalently attached. The immuno-captured target cells were detected in beverages like milk and lemon juice by multiplex PCR and real time PCR with a detection limit of 10⁴ cfu.mL^?1 and 10³ cfu.mL^?1, respectively. We demonstrate that IMS can be used for selective concentration of target bacteria from beverages for subsequent use in PCR detection. PCR also enables differentiation of Salmonella typhi and Salmonella paratyphi A using a set of four specific primers. In addition, IMS—PCR can be used as a screening tool in the food and beverage industry for the detection of Salmonella within 3–4 h which compares favorably to the time of several days that is needed in case of conventional detection based on culture and biochemical methods.

Development of an Amperometric Immunosensor for the Quantification of Staphylococcus aureus Using Self‐Assembled Monolayer‐Modified Electrodes as Immobilization Platforms

Amperometric immunosensors for the detection and quantification of S. aureus using MPA self‐assembled monolayer modified electrodes for the immobilization of the immunoreagents are reported. Two different immunosensor configurations were compared. A competitive mode, in which protein A‐bearing S. aureus cells and antiRbIgG labeled with horseradish peroxidase (HRP) compete for the binding sites of RbIgG immobilized onto the 3‐mercaptopropionic acid (MPA) modified electrode, was evaluated. Moreover, a sandwich configuration in which S. aureus cells were immobilized onto the MPA SAM, and RbIgG and antiRbIgG labeled with HRP were further linked to the electrode surface, was also tested. In both cases, TTF was used as the redox mediator of the HRP reaction with H₂O₂, and it was co‐immobilized onto the MPA‐modified gold electrode. After optimization of the working variables for both configurations, the analytical performance of the amperometric measurements carried out at 0.00 V (vs. Ag/AgCl) showed that the competitive immunosensor exhibited a lower limit of detection (1.6×10⁵ S. aureus cells mL^?1), as well as a better repeatability and reproducibility of the measurements.     

A versatile approach for the immobilization of amines via copper-free “click” reaction between azido self-assembled monolayer and alkynyl Fischer carbene complex. Application to the detection of staphylococcal enterotoxin A antibody

A copper-free “click” reaction between an azido-terminated self-assembled monolayer (SAM) chemisorbed on planar gold-coated glass sensors and an alkynyl Fischer carbene complex yielded functionalized surfaces onto which facile and swift grafting of amine-containing molecules was achieved via aminolysis of the Fischer carbene moieties. The course of this process was conveniently monitored by Polarization-Modulation Infrared Reflection-Absorption Spectroscopy (PM-IRRAS), contact angle measurements and X-ray photoelectron spectroscopy (XPS). A study of the parameters involved in the covalent grafting of the model protein bovine serum albumin (BSA) to the Fischer carbene modified SAM was carried out. As an application, the bacterial toxin staphylococcal enterotoxin A (SEA) produced by some pathogenic strains of Staphylococcus aureus was immobilized on the gold chips and immunocapture of a polyclonal antibody raised against SEA was detected by PM-IRRAS so as to ultimately construct an optical immunosensor for the detection of this toxin in food samples.     

Specific enrichment of prokaryotic DNA using a recombinant DNA-binding protein

Targeted enrichment of DNA is often necessary for its detection and characterization in complex samples. We describe the development and application of the novel molecular tool for the specific enrichment of prokaryotic DNA. A fused protein comprising the DNA-binding subunit of the bacterial topoisomerase II, gyrase, was expressed, purified, and immobilized on magnetic particles. We demonstrated the specific affinity of the immobilized protein towards bacterial DNA and investigated its efficiency in the samples with high background of eukaryotic DNA. The reported approach allowed for the selective isolation and further detection of as few as 5 pg Staphylococcus aureus DNA from the sample with 4?×?10⁶-fold surplus of human DNA. This method is a promising approach for the preparation of such type of samples, for example in molecular diagnostics of sepsis.     

Optimization and characterization of a biosensor assembly for detection of <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium

The performance of biosensors depends directly on the strategies adopted during their development. In this paper, a fast and sensitive biosensor for Salmonella Typhimurium detection was assembled by using optimization studies in separate stages. The pre-treatment assays, biomolecular immobilization (primary antibody and protein A concentrations), and analytical response (hydroquinone and hydrogen peroxide concentrations) were optimized via voltammetric methods. In the biosensor assembly, a gold surface was modified via the self-assembled monolayer technique (SAM) using cysteamine thiol and protein A for immobilization of anti-Salmonella antibody. The analytical response of the biosensor was obtained through the use of a secondary antibody labeled with a peroxidase enzyme, and the signal was evaluated by applying the chronoamperometry technique. The biosensor was characterized by infrared spectroscopy and cyclic voltammetry. Optimization of protein A and primary antibody concentrations enabled higher analytical signals of 7.5 and 75 mg mL^?1, respectively, to be achieved. The hydroquinone and H₂O₂ concentrations selected were 3 and 300 mM, respectively. The biosensor developed attained a very low detection limit of 10 CFU mL^?1 and a fast response with a final detection time of 125 min. These results indicate that this biosensor is very promising for the food safety and emergency response applications.     

Surface plasmon resonance immunosensor for bacteria detection

This work describes an approach for the development of two bacteria biosensors based on surface plasmon resonance (SPR) technique. The first biosensor was based on functionalized gold substrate and the second one on immobilized gold nanoparticles. For the first biosensor, the gold substrate was functionalized with acid-thiol using the self-assembled monolayer technique, while the second one was functionalized with gold nanoparticles immobilized on modified gold substrate. A polyclonal anti-Escherichia coli antibody was immobilized for specific (E. coli) and non-specific (Lactobacillus) bacteria detection. Detection limit with a good reproducibility of 10⁴ and 10³ cfu mL⁻¹ of E. coli bacteria has been obtained for the first biosensor and for the second one respectively. A refractive index variation below 5 × 10⁻³ due to bacteria adsorption is able to be detected. The refractive index of the multilayer structure and of the E. coli bacteria layer was estimated with a modeling software.     

A novel piezoelectric immunosensor for the detection of malarial Plasmodium falciparum histidine rich protein-2 antigen

A novel piezoelectric (PZ) immunosensor for the direct detection of malarial Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen was developed. The mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol were formed on gold surface of quartz crystal. Cyclic voltammetry, electrochemical impedance spectroscopy and surface Raman spectroscopy techniques were used to characterize the mixed SAMs. The rabbit anti-PfHRP-2 antibodies were coupled on mixed SAM modified gold surface of quartz crystal via NHS/EDC activation method. The PZ immunosensor was applied to detect PfHRP-2 in the linear range of 15-60 ng/ml with a detection limit of 12 ng/ml. It was also found that even after 14 days of storage, 50% of the activity still remained. Clinical human serum samples were tested with this method, and the results were in agreement with those obtained from commercially available ICT kit (NOW^® Malaria).     

Dual gold nanoparticle lateflow immunoassay for sensitive detection of Escherichia coli O157:H7

Two patterns of signal amplification lateral flow immunoassay (LFIA), which used anti-mouse secondary antibody-linked gold nanoparticle (AuNP) for dual AuNP-LFIA were developed. Escherichia coli O157:H7 was selected as the model analyte. In the signal amplification direct LFIA method, anti-mouse secondary antibody-linked AuNP (anti-mouse-Ab-AuNP) was mixed with sample solution in an ELISA well, after which it was added to LFIA, which already contained anti-E. coli O157:H7 monoclonal antibody-AuNP (anti-E. coli O157:H7-mAb-AuNP) dispersed in the conjugate pad. Polyclonal antibody was the test line, and anti-mouse secondary antibody was the control line in nitrocellulose (NC) membrane. In the signal amplification indirect LFIA method, anti-mouse-Ab-AuNP was mixed with sample solution and anti-E. coli O157:H7-mAb-AuNP complex in ELISA well, creating a dual AuNP complex. This complex was added to LFIA, which had a polyclonal antibody as the test line and secondary antibody as the control line in NC membrane. The detection sensitivity of both LFIAs improved 100-fold and reached 1.14 × 10³ CFU mL⁻¹. The 28 nm and 45 nm AuNPs were demonstrated to be the optimal dual AuNP pairs. Signal amplification LFIA was perfectly applied to the detection of milk samples with E. coli O157:H7 via naked eye observation.     

Development of a liposome-based immunochromatographic strip assay for the detection of Salmonella

Salmonella species are ubiquitous human pathogens which pose a dangerous threat to the elderly and children worldwide. In this study, to develop a more efficient assay procedure for the rapid detection of Salmonella Typhimurium, an immunochromatographic strip assay was developed using immunoliposome (anti-Salmonella IgG-tagged) encapsulated with sulforhodamine B (SRB). The detection sensitivity of the developed immunochromatographic assay was compared with a commercial immunochromatographic test strip using colloidal gold nanoparticles. The liposomes were prepared through a reverse-phase evaporation method by using a lipid and phospholipid mixture and SRB, a fluorescence dye, which was encapsulated in the phospholipid bilayer. Furthermore, the outer surface of the SRB-encapsulated liposome was conjugated with antibody (affinity-purified polyclonal goat anti-Salmonella IgG) to form an immunoliposome (size, 223 nm), used as the analytical reagent in the developed immunoassay. For this strip assay, a plastic-backed nitrocellulose strip was immobilized with two antibody zones. The lower zone of the strip referred to Salmonella antigen capture zone (test line), while the other zone served as a positive control (control line). The lower zone was coated with affinity-purified polyclonal goat anti-Salmonella IgG, while the upper zone was coated with rabbit anti-goat IgG. During capillary migration of the wicking solution (diluted liposome and Salmonella culture, each 50 μl), Salmonella was captured with surface-bound immunoliposomes at the antigen capture zone, while the unbound liposomes migrated upward and bound to another zone. The color density of the antigen capture zone was directly proportional to the amount of S. Typhimurium in the test sample. As a result, the detection limit of the immunochromatographic strip assay developed in this study against S. Typhimurium was found to be 10² CFU/ml, which was significantly higher than the detection limit (10⁷ CFU/ml) of the commercial immunochromatographic test strip assay.     

Light scattering sensing detection of pathogens based on the molecular recognition of immunoglobulin with cell wall-associated protein A

In this contribution, we report a rapid optical detection method of pathogens using Staphylococcus aureus (S. aureus) as the model analyte based on the molecular recognition of immunoglobulin with cell wall-associated Protein A (SpA). It was found that the molecular recognition of human immunoglobulin (IgG) with protein A on the cell wall of S. aureus on glass slide sensing area could result in strong surface enhanced light scattering (SELS) signals, and the SELS intensity (ΔI) increases proportionally with the concentration of S. aureus over the range of 2.5 × 10⁵-1.0 × 10⁸ CFU mL⁻¹ with right angle light scattering (RALS) signals detection mode. In order to identify the solid support based molecular recognition between IgG with SpA, we also employed water-soluble CdS quantum dots (CdS-QDs) as a fluorescent marker for IgG by immobilizing the IgG onto the surfaces of CdS-QDs through covalent binding in order to generate recognition probes for SpA on the cell wall of S. aureus. Consequently, the fluorescent method also showed that the detection for pathogens with solid supports is reliable based on the molecular recognition of IgG with SpA.     

Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor

A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-β-alanine-ovalbumin (TNP-β-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-β-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3 × 10⁻¹¹ to 3 × 10⁻⁷ g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1 × 10⁻¹¹ g/ml.     

Piezoelectric Crystal Biosensor System for Detection of Escherichia Coli

Abstract

A piezoelectric crystal biosensor system was applied to the detection of Escherichia coli. the system consists of an oscillator, a frequency counter, a flow cell and a modified piezoelectric crystal. Anti-E. coli antibody is immobilized on the surface of the crystal. It is used as an E. coli detection by measuring its resonant frequency shift due to a mass change caused by specific binding of the micro organisms to the surface. the frequency shift correlates with an E. coli concentration in the range of 10⁶?10⁸ cells·cm^?3. the resonant frequency shift is increased by further treatment to bind micro-particles modified with anti-E. coli antibody. This method allows us to improve the determination limit to 10⁵ cells · cm^?3.