Rapid and sensitive biosensor for Salmonella

The rapid and sensitive detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated. The binding of bacteria to the surface changed the crystal resonance parameters; these were quantified by the output voltage of the sensor instrumentation. The sensor had a lower detection limit of a few hundred cells/ml, and a response time of < 100 s over the range of 10(2)-10(10) cells/ml. The sensor response was linear between bacterial concentrations of 10(2)-10(7) cells/ml, with a sensitivity of 18 mV/decade. The binding of bacteria was specific with two binding sites needed to bind a single cell. The sensors preserve approximately 75% of their sensitivity over a period of 32 days.     

Detection of Escherichia coli 0157:H7 using a surface plasmon resonance biosensor

The BIAcore biosensor was used to detect binding of Escherichia coli O157:H7 with specific antibodies. Immobilized Protein A or Protein G captured antibodies which in turn bound to the bacteria. Alternatively, immobilized antibody captured the E. coli O157:H7 and the bacteria were further probed by a second antibody which enhanced the signal. The regenerated sensor surfaces were used for at least 50 separate analyses. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with different antisera or other molecular species. © Rapid Science Ltd. 1998     

Multipurpose Love acoustic wave immunosensor for bacteria,virus or proteins detection

A multipurpose Love acoustic wave biosensor is described in this article. As mass loading is one of the main effect involved in acoustic wave sensors, a great range of biomolecules could be detected using such sensors. In this way, the antibody/antigen binding property was used to immobilise the target species. We first compared different coupling agents to link the antibodies sensitive layer to the SiO₂ sensor surface. Results showed that GPTS monolayer, allowing covalent attachment of antibodies bioreceptors, is better suited than DTSP and protein G. It permits to obtain a dense, stable and reproducible sensitive layer of antibodies. Then, different biological species with different size and shape like proteins, bacteriophages or bacteria were detected using such sensor. Different models have been chosen to validate the effective detection of a large species range: an anti-mouse antibody has been used to simulate small molecules (< 10 nm) like proteins or toxins, bacteriophage M13 for species lower than 1 μm like virus, and Escherichia coli for bacteria which are typically longer than 1 μm. Each kind of species were successfully quickly detected from few seconds for small proteins to one hour for bacteria, with detection threshold down to 4 ng/mm² for protein and 10⁶ cfu per milliliter for bacteria.     

THE USE OF STREPTAVIDIN COATED MAGNETIC BEADS FOR DETECTING PATHOGENIC BACTERIA BY LIGHT ADDRESSABLE POTENTIOMETRIC SENSOR (LAPS)

A modified procedure for magnetic capture of antibody-conjugated bacteria for light addressable potentiometric sensor (LAPS) detection using the Threshold System was developed. Streptavidin coated magnetic beads, partially labeled with biotinylated anti Escherichia coli O157 antibodies, were used to capture Escherichia coli O157:H7. Captured bacteria were further labeled with fluorescein-conjugated anti -E. coli O157:H7 antibodies and urease-labeled. anti-fluorescein antibody. Magnetically concentrated bacteria-containing complexes were then immobilized through streptavidin-biotin interactions on 0.45 μ biotinylated nitro-cellulose membranes assembled as sample sticks for the Threshold instrument. The rate of pH change associated with the production of NH₃ by the urease in urea-containing solution was measured by a LAPS incorporated in the Threshold instrument. This approach allowed us to detect 10³ to 10⁴ CPU of cultured E. coli O157:H7 in PBS solutions. Furthermore, detectable LAPS signals of the sample sticks remained relatively constant for at least 24 h at 4C. The developed approach was applied to detect the E. coli in beef hamburger spiked with the bacteria. After a 5 to 6-h enrichment at 37C, as low as 1 CFU/g of E. coli O157:H7 in beef hamburger could be detected.     

Quantification of the effect of glycocalyx condition on membrane receptor interactions using an acoustic wave sensor

The effect of the cell glycocalyx on the binding of a membrane receptor, class I major histocompatibility complex (MHC) human leukocyte antigen (HLA)-A2, to an immobilized anti-HLA antibody was investigated using an acoustic sensor based on a Love wave geometry. The enzyme neuraminidase was used to remove sialic acid residues from the cell glycocalyx. Real-time measurements of the amplitude of the acoustic wave showed that treatment with neuraminidase facilitates HLA/anti-HLA-mediated cell attachment via a 3.6-fold increase of the two-dimensional (2D) binding constant of the interaction. This could be attributed to better approach of binding partners due to favorable condition of the desialylated glycocalyx. The results underline the importance of microtopological factors in membrane receptor binding and reveal the potential of the Love wave sensor and 2D binding parameters for studying cell–substrate binding events.     

Detection of Escherichia coli O157:H7 with langasite pure shear horizontal surface acoustic wave sensors

The toxigenic Escherichia coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure and death. On average, O157:H7 causes 73,000 illnesses, 2100 hospitalizations and 60 deaths annually in the United States alone. There is the need for sensors capable of rapidly detecting dangerous microbes in food and water supplies to limit the exposure of human and animal populations. Previous work by the authors used shear horizontal surface acoustic wave (SH SAW) devices fabricated on langasite (LGS) Euler angles (0°, 22°, 90°) to successfully detect macromolecular protein assemblies. The devices also demonstrated favorable temperature stability, biocompatibility and low attenuation in liquid environments, suggesting their applicability to bacterial detection. In this paper, a biosensor test setup utilizing a small volume fluid injection system, stable temperature control and high frequency phase measurement was applied to validate LGS SH SAW biosensors for bacterial detection. The LGS SH SAW delay lines were fabricated and derivatized with a rabbit polyclonal IgG antibody, which selectively binds to E. coli O157:H7, in this case a non-toxigenic test strain. To quantify the effect of non-specific binding (negative control), an antibody directed against the trinitrophenyl hapten (TNP) was used as a binding layer. Test E. coli bacteria were cultured, fixed with formaldehyde, stained with cell-permeant nucleic acid stain, suspended in phosphate buffered saline and applied to the antibody-coated sensing surfaces. The biosensor transmission coefficient phase was monitored using a network analyzer. Phase responses of about 14° were measured for the E. coli detection, as compared to 2° due to non-specific anti-TNP binding. A 30:1 preference for E. coli binding to the anti-O157:H7 layer when compared to the anti-TNP layer was observed with fluorescence microscopy, thus confirming the selectivity of the antibody surface to E. coli.     

Fluorescent homogeneous immunosensors for detecting pathogenic bacteria

We developed a straightforward antibody-based assay for rapid homogeneous detection of bacteria. Our sensors utilize antibody recognizing cell-surface epitopes of the target cell. Two samples of the antibody are prepared, each labeled via nanometer size flexible linkers with short complementary oligonucleotides that are modified with fluorochromes that could participate in fluorescence resonance energy transfer (FRET). The length of the complementary oligonucleotide sequences was designed such that very little annealing occurred in the absence of the target cells. In the presence of the target cells the two labeled antibodies bind to the surface of the cell resulting in a large local concentration of the complementary oligonucleotides that are attached to the antibody. This in turn drives the annealing of the complementary oligonucleotides which brings the fluorescence probes to close proximity producing large FRET signals proportional to the amount of target cells. Long flexible linkers used to attach the oligonucleotides to the antibody enable target-induced oligonucleotide annealing even if the density of surface antigens is only modest. We used Escherichia coli 0157:H7 and Salmonella typhimurium to demonstrate that this design produced sensors exhibiting rapid response time, high specificity, and sensitivity in detecting the target bacteria.     

Gas phase activity of anti-FITC antibodies immobilized on a surface acoustic wave resonator device

In this paper we present the results of a series of experiments on the activity of antibodies in a vapor phase sensor. For these experiments the sensor component was a ST-Quartz resonator with a center frequency of approximately 250 MHz. Anti-FITC antibodies were attached to the electrodes on the device surface via a protein-A crosslinker. Surface acoustic wave (SAW) resonator devices with various coatings were mounted in TO-8 packages, inserted into our sensor head module and subjected to various fluorescent analyte gases. Numerous controls were performed including the use of coated and uncoated devices along with devices coated with antibodies which were not specific for the target analyte. The SAW immunosensor response was monitored and a baseline frequency shift was observed when the analyte being presented was the antigen for the immobilized antibody. To provide an independent measure of antibody/antigen binding, the devices were removed from the sensor head, washed with a buffer solution to remove any unbound analyte, and then inspected using a confocal laser scanning microscope (CLSM). Since all the analytes being used in these experiments were fluorescent this afforded us the opportunity to visualize the attachment of the analyte to the antibody film. Given the high resolution of the CLSM, we were able to identify the location of the attachment of the fluorescent analytes relative to the 1.5 microm wide electrodes of the SAW device. We believe that these experiments demonstrate that we have achieved real time molecular recognition of these small molecules in the vapor phase.     

A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7

A quartz crystal microbalance (QCM) DNA sensor, based on the nanoparticle amplification method, was developed for detection of Escherichia coli O157:H7. A thiolated single-stranded DNA (ssDNA) probe specific to E. coli O157:H7 eaeA gene was immobilized onto the QCM sensor surface through self-assembly. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA, and resulted in the mass change and therefore frequency change of the QCM. Streptavidin conjugated Fe(3)O(4) nanoparticles (average diameter=145 nm) were used as "mass enhancers" to amplify the frequency change. Synthesized biotinylated oligonucleotides as well as E. coli O157:H7 eaeA gene fragments (151 bases) amplified using asymmetric PCR with biotin labeled primers were tested. As low as 10(-12)M synthesized oligonucleotides and 2.67 x 10(2) colony forming unit (CFU)/ml E. coli O157:H7 cells can be detected by the sensor. Linear correlation between frequency change and logarithmic number of bacterial cell concentration was found for E. coli O157:H7 from 2.67 x 10(2) to 2.67 x 10(6)CFU/ml.     

Detection of pathogen Escherichia coli O157:H7 using self-excited PZT-glass microcantilevers

Composite self-excited PZT-glass cantilevers (5 and 3 mm in length, 1.8 and 2.0 mm wide) were fabricated and their resonance characteristics were determined in air and at 1 mm liquid immersion. In air, resonance occurred at 65.8 and 63.4 kHz for the two cantilevers used in this paper. Monoclonal antibody (MAb) specific to the pathogen Escherichia coli (E. coli) O157:H7 was immobilized at the cantilever glass tip, and then exposed to pathogen in the concentration range of 7x10(2) to 7x10(7)bacteria/mL. Resonance of the second mode decreased due to pathogen attachment in accordance with a proposed kinetic model. The specific attachment rate constant was found to be 3x10(-9) to 5x10(-9) min-1 (cell/mL)-1. Exposure to a mixed population containing both a pathogenic and non-pathogenic strain showed that the antibody-immobilized cantilever is highly selective, thus demonstrating its usefulness for detecting water-borne pathogens.     

Real-time analysis of antibody binding interactions with immobilized E. coli O157:H7 cells using the BIAcore

The kinetic properties of antibody-antigen reaction and other interacting macromolecules can be analyzed in real-time using the surface plasmon resonance biosensor (BIAcore). The interactions of an antibody against Escherischia coli O157:H7 were studied using immobilized whole cells. The bacterial sensor surface was evaluated with anti-E. coli in a continuous flow system. Regeneration of the sensor surface with guanidine-HCl was more effective than with phosphoric acid and resulted in better binding reproducibility. The determined kinetic values, association and dissociation rate constants, can be used in the development of rapid immuno-techniques. This study also provides the basis to evaluate real-time interactions of macromolecules with immobilized cells.     

Detect of Escherichia coli O157:H7 in ground beef samples using piezoelectric excited millimeter-sized cantilever (PEMC) sensors

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 4 mm2 were fabricated. An antibody specific to Escherichia coli (anti-E. coli) O157:H7 was immobilized on PEMC sensors, and exposed to samples containing E. coli O157:H7 (EC) prepared in various matrices: (1) broth, broth plus raw ground beef, and broth plus sterile ground beef without inoculation of E. coli O157:H7 served as controls, (2) 100 mL of broth inoculated with 25 EC cells, (3) 100 mL of broth containing 25 g of raw ground beef and (4) 100 mL of broth with 25 g of sterile ground beef inoculated with 25 EC cells. The total resonant frequency change obtained for the broth plus EC samples were 16+/-2 Hz (n=2), 30 Hz (n=1), and 54+/-2 Hz (n=2) corresponding to 2, 4, and 6h growth at 37 degrees C, respectively. The response to the broth plus 25 g of sterile ground beef plus EC cells were 21+/-2 Hz (n=2), 37 Hz (n=1), and 70+/-2 Hz (n=2) corresponding to 2, 4, and 6 h, respectively. In all cases, the three different control samples yielded a frequency change of 0+/-2 Hz (n=6). The E. coli O157:H7 concentration in each broth and beef samples was determined by both plating and by pathogen modeling program. The results indicate that the PEMC sensor detects E. coli O157:H7 reliably at 50-100 cells/mL with a 3 mL sample.     

Recombinant single-chain variable fragment and single domain antibody piezoimmunosensors for detection of HIV1 virion infectivity factor

In this paper recombinant single-chain fragments (scFv-4BL), and single domain antibodies (4BL-V(H)) and (4BL-V(H)D) generated against HIV1 virion infectivity factor (Vif) are used to develop piezoimmunosensors for HIV1 recognition. Mixed self assembled monolayers were generated at the surface of gold coated crystal sensors to which scFv-4BL, 4BL-V(H), or 4BL-V(H)D were immobilized. Impedance analysis was used to discriminate interfering signals from frequency variation data and to increase the sensor sensitivity. The elimination of interfering signals enabled the quantification of the amount of immobilized protein and gave some indication on the viscoelasticity of immobilized biofilms. All the modified sensors were able to specifically recognize HIV1 Vif in liquid samples. The results indicate that lower sensitivities are obtained with 4BL-V(H) single domain antibodies, possibly due to its higher hydrophobic character. The sensitivity obtained when using scFv-4BL was reestablished when using the more hydrophilic 4BL-V(H)D single domain. 4BL-V(H)D piezoimunosensors were effective in recognizing HIV1 Vif from protein mixtures and from cell extracts of human embryonic kidney cells expressing HIV1 Vif. The results presented in this paper demonstrate the potential applicability of the developed piezoimmunosensors to monitor HIV1 infection evolution.     

Evaluation of a microwire sensor functionalized to detect Escherichia coli bacterial cells

A functionalized microwire sensor based on dielectrophoresis (DEP) and antigen-antibody reaction was initially developed for sensitive and selective detection of E. coli O157:H7. The dynamics of gold-tungsten microwires were manipulated using an automated X-Y-Z stage and the sensing process included antibody immobilization and bacterial detection, and cell quantification. Antibodies were first immobilized on surface of the microwire to improve sensing specificity, and then coupled with DEP for capture of E. coli cells in a mixture of E. coli cells and non-conductive polystyrene beads. Afterward, fluorescein-conjugated secondary antibodies were applied to the wire for quantification of captured bacteria. Field Emission Scanning Electron Microscope (FESEM) figures and fluorescence intensities of bacteria on the wire validated the sensing mechanism. The entire immobilization and detection procedure could be completed within 30 min with simple operations. Performance of the microwire sensor was not significantly affected when conducted in orange juice. In addition, the detection limit of this sensor was about 5 bacterial cells per microwire in 1000 CFU/mL bacterial suspensions when the electric field generated at 3 MHz and 20 peak to peak voltage (V(pp)), and only targeted E. coli cells were concentrated and captured.     

Somatic and flagellar immunofluorescence of Salmonella

Caldwell, W. J. (The Child Research Center of Michigan, Detroit, Mich.), C. S. Stulberg, and W. D. Peterson, Jr. Somatic and flagellar immunofluorescence of Salmonella. J. Bacteriol. 92:1177-1187. 1966.-Labeled globulin fractions of flagellar (H) antisera, prepared against 20 frequently occurring Salmonella serotypes belonging to five major somatic (O) groups, were characterized for O and H immunofluorescence and for O and H agglutinin titers against 32 serotypes. The feasibility of immunofluorescent identification of both somatic and flagellar antigens was enhanced by staining formaldehyde-treated organisms in suspension. Relationships between homologous, partial, and unrelated antigen-antibody systems were then analyzed, and a high degree of correlation was shown between the results obtained by the two serological procedures. Flagellar staining was highly specific, and was bright, faint, or inapparent, depending on the relationship between the antigen-antibody systems involved. Somatic staining was also specific, but somewhat more difficult to interpret, because cells in the same preparation might exhibit a mixture of bright, faint, or no fluorescent intensities. Correlation was shown between the percentage of brightly staining cells found in these preparations and the agglutination titers of the comparable antigen-antibody systems. The phenomenon of a "percentage" reaction was unexplained. Absorption studies further confirmed the specificity of reactions. The techniques developed were applied to surveillance of several mouse colonies for the presence of Salmonella. Broth cultures of fecal specimens were treated with formaldehyde and stained in suspension with "polyvalent" labeled antibody reagents. Agreement was found in 97.6% of the instances between results obtained by immunofluorescence and cultural methods. In addition, preliminary evidence indicated the feasibility of presumptive serotyping of Salmonella isolates by immunofluorescence.     

Evaluation of different micro/nanobeads used as amplifiers in QCM immunosensor for more sensitive detection of E. coli O157:H7

Micro/nanobeads with different materials (magnetic, silica and polymer) and different sizes (diameters from 30nm to 970nm) were investigated for their use as amplifiers in a quartz crystal microbalance (QCM) immunosensor for more sensitive detection of Escherichia coli O157:H7. The micro/nanobeads were conjugated with anti-E. coli antibodies. E. coli O157:H7 cells were first captured by the first antibody immobilized on the electrode surface, and then micro/nanobeads labeled secondary antibodies attached to the cells, and finally the complexes of antibody-E. coli-antibody modified beads were formed. The results showed that antibody-labeled beads lead to signal amplification in both the change in frequency (ΔF) and the change in resistance (ΔR). Since the penetration depth of the oscillation-induced shear-waves for a ～8MHz crystal is limited to 200nm, the interpretation of how the signal is amplified by the adsorbed particles was represented in terms of the coupled-oscillator theory. The amplification is not sensed in terms of increase in mass on the sensor surface. Amplification is sensed as a change in bacterial resonance frequency when the spheres adsorb to the bacteria. The change in the values of ΔF caused by different micro/nanobeads (amplifiers) attaching on target bacterial cells is indicative of the ratio between the resonance frequency of the absorbed bacterial-particle complex (ω(s)), and the resonance frequency of the crystal (ω).     

Detection of pathogen Escherichia coli O157:H7 AT 70 cells/mL using antibody-immobilized biconical tapered fiber sensors

Optical fibers (core diameter 8 microm, cladding diameter 125 microm) was tapered to a waist diameter in the range of 8-12 microm, and then a monoclonal antibody to the pathogen, Escherichia coli O157:H7 was covalently bonded to the surface of the tapered region. Using 470 nm light, the taper was exposed to various concentrations (7 x 10(7), 7 x 10(5), 7 x 10(3), and 70 cells/mL) of the pathogen, and the sensor showed changes in transmitted light as the antigen attached to the antibody on the taper surface. The response was equal and opposite when the pathogen was released from the surface using a low pH buffer. The magnitude of the change was inversely proportional to the concentration of the pathogen. The sensor showed good sensitivity at as low a concentration as 70 cells/mL. The antibody-immobilized taper sensor was also exposed to a mixture of the pathogen and a non-pathogenic variant (JM101) at 0%, 50% and 70% by concentration. The sensor showed good selectivity to the pathogenic antigen. A first order attachment kinetic model is proposed to quantify the rate of attachment of pathogen to the sensor surface. The kinetic rate constant (k) of E. coli O157:H7 to the fiber was found to vary in the range of (2.5-6.1) x 10(-9) min(-1) (cells/mL)(-1).     

Microscale biosensor for measurement of volatile fatty acids in anoxic environments

A microscale biosensor for acetate, propionate, isobutyrate, and lactate is described. The sensor is based on the bacterial respiration of low-molecular-weight, negatively charged species with a concomitant reduction of NO(-)(3) to N(2)O. A culture of denitrifying bacteria deficient in N(2)O reductase was immobilized in front of the tip of an electrochemical N(2)O microsensor. The bacteria were separated from the outside environment by an ion-permeable membrane and supplied with nutrients (except for electron donors) from a medium reservoir behind the N(2)O sensor. The signal of the sensor, which corresponded to the rate of N(2)O production, was proportional to the supply of the electron donor to the bacterial mass. The selectivity for volatile fatty acids compared to other organic compounds was increased by selectively enhancing the transport of negatively charged compounds into the sensor by electrophoretic migration (electrophoretic sensitivity control). The sensor was susceptible to interference from O(2), N(2)O, NO(2)(-), H(2)S, and NO(-)(3). Interference from NO(-)(3) was low and could be quantified and accounted for. The detection limit was equivalent to about 1 microM acetate, and the 90% response time was 30 to 90 s. The response of the sensor was not affected by changes in pH between 5.5 and 9 and was also unaffected by changes in salinity in the range of 2 to 32 per thousand. The functioning of the sensor over a temperature span of 7 to 30 degrees C was investigated. The concentration range for a linear response was increased five times by increasing the temperature from 7 to 19.5 degrees C. The life span of the biosensor varied between 1 and 3 weeks after manufacturing.     

Designing a Non-invasive Surface Acoustic Resonator for Ultra-high Sensitive Ethanol Detection for an On-the-spot Health Monitoring System

Surface acoustic wave (SAW) sensors–based on piezoelectric crystal resonators–are extremely sensitive to even very small perturbations in the external atmosphere, because the energy associated with the acoustic waves is confined to the crystal surface. In this study, we present a critical review of the recent researches and developments predominantly used for SAW-based organic vapor sensors, especially ethanol. Besides highlighting their potential to realize real-time ethanol sensing, their drawbacks such as indirect sensing, invasive, time initializing, and low reliability, are properly discussed. The study investigates a proposed YZ-lithium niobate piezoelectric substrate with interdigital transducers patterned on the surface. Design of the resonator plays an important role in improving mass sensitivity, particularly the sensing area. Accordingly, a tin dioxide (SnO₂) layer with a specific thickness is generated on the surface of the sensor because of its high affinity to ethanol molecules. To determine the values of sensor configuration without facing the practical problems and the long theoretical calculation time, it is shown that the mass sensitivity of SAW sensors can be calculated by a simple three-dimensional (3-D) finite element analysis (FEA) using a commercial finite-element platform. In design validation step, different concentrations of ethanol are applied to investigate the acoustic wave properties of the sensor. The FEA data are used to obtain the surface and bulk total displacements of the sensor and fast Fourier transform (FFT) on output spectrum. The sensor could develop into highly sensitive and fast responsive structure so that a positive intensity shift of 0.18e-2 RIU is observed when the sensor is exposed to 15 ppm ethanol. It is capable of continuously monitoring the ethanol gas whether as an ultra-high sensitive sensor or switching applications for medical and industrial purposes.     

Peptide biosensor for recognition of cross-species cell surface markers

Biosensors based on phage display-derived peptides as biorecognition molecules were used for the detection of cell surface cross-species markers in tissue homogenates. The peptide selected for murine myofibers was immobilized onto the surface of an acoustic wave sensor by biotin-streptavidin coupling. To detect peptide-receptor interaction, the sensors were exposed to muscle and control (kidney, liver, brain) tissue homogenates. The sensor showed a strong response to murine muscle. The amplitudes of the responses to the feline muscle homogenates were lower compared to those of the murine muscle, while the same K(d) indicated that the peptide has cross-species affinity. In contrast, murine kidney, liver and brain homogenates produced insignificant responses. Specificity of the sensor was shown in a blocking experiment, as reduced signal was detected when muscle preparations were preincubated with free peptide. Additionally, when muscle-specific peptide was replaced with two different random control peptides, the sensors produced no response to murine muscle. Suitability of peptide ligands for a variety of species can be evaluated using this technology.