Impedance characterization of a piezoelectric immunosensor part II: Salmonella typhimurium detection using magnetic enhancement

This study investigated the usefulness and characteristics of a 5-MHz quartz crystal resonator as a sensor of biological pathogens such as Salmonella typhimurium. An impedance analyzer measured the impedance behavior of the oscillating quartz crystal exposed to various concentrations of Salmonella (10(2)-10(8) cells per ml). The Salmonella cells were captured by antibody-coated paramagnetic microspheres, and then these complexes were moved magnetically to the sensing quartz and were captured by antibodies immobilized on the crystal surface. The response of the crystal was expressed in terms of equivalent circuit parameters. The motional inductance and the motional resistance increased as a function of the concentration of Salmonella. The viscous damping was the main contributor to the resistance and the inductance in a liquid environment. The load resistance was the most effective and sensitive circuit parameter. A magnetic force was a useful method to collect the complexes of Salmonella-microspheres on the crystal surface and enhance the response of the sensor. In this system, the detection limit, based on resistance monitoring, was about 10(3) cells per ml.     

Development of liposome immunoassay for salmonella spp. using immunomagnetic separation and immunoliposome

The ability to detect Salmonella spp. is essential in the prevention of foodborne illness. This study examined a Salmonella spp. detection method involving the application of immunomagnetic separation and immunoliposomes (IMS/IL) encapsulating sulforhodamine B (SRB), a fluorescent dye. A quantitative assay was conducted by measuring the fluorescence intensity of SRB that was produced from an immunomagnetic bead-Salmonella spp.-immunoliposome complex. The results indicated detection limits of 2.7x10(5) and 5.2x10(3) CFU/ml for Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), respectivley. The signal/noise ratio was improved by using 4% skim milk as a wash solution rather than 2% BSA. In addition, higher fluorescence intensity was obtained by increasing the liposome size. Compared with the conventional plating method, which takes 3-4 days for the isolation and identification of Salmonella spp., the total assay time of 10 h only including 6 h of culture enrichment was necessary for the Salmonella detection by IMS/IL. These results indicate that the IMS/ IL has great potential as an alternative rapid method for Salmonella detection.     

Concentration dependence of IgG-protein A affinity studied by wireless-electrodeless QCM

The binding affinity between human immunoglobulin G (IgG) and protein A was studied by the homebuilt wireless-electrodeless quartz crystal microbalance (QCM). Protein A was immobilized on the electrodeless AT-cut quartz plate of 0.05 mm thick and its fundamental resonance frequency near 34 MHz was measured by a noncontacting manner using a line antenna. The vibrational analysis was performed to ensure higher sensitivity of the electrodeless QCM. A flow-cell system was fabricated to continuously measure the resonance frequency during the injection sequence of the IgG solutions with concentrations of 1-20,000 ng/mL. The exponential frequency changes were recorded to determine the affinity based on the Langmuir kinetics. The equilibrium constant K(A) significantly varied between 6 x 10(6) and 6 x 10(10) M(-1), depending on the IgG concentration, which is attributed to various formations of IgG-protein A complexes.     

QCM-FIA with PGMA coating for dynamic interaction study of heparin and antithrombin III

In this work, we describe a method of constructing a film of linear poly(glycidyl methacrylate) (PGMA) polymer onto the surface of quartz crystal microbalance (QCM) electrode as a coating material that allows easy coupling of heparin molecules onto the electrode and facilitates the determination of the interaction between heparin and antithrombin III (AT III). The PGMA film was characterized with atomic force microscopy (AFM) and infra-red spectroscopy. The coupling of heparin was accomplished in one step solution reaction. A home-made quartz crystal microbalance-flow injection analysis (QCM-FIA) system with data analysis software developed in our laboratory was used to determine the interaction. The interactions between immobilized heparin and AT III were studied with various concentrations under various conditions. The obtained constants are kass=(1.49+/-0.12)x10(3)mol-1ls-1, kdiss=(3.94+/-0.63)x10(-2)s-1, KA=(3.82+/-0.33)x10(4)mol-1l.     

Sensitivity enhancement of DNA sensors by nanogold surface modification

A novel amplified microgravimetric gene sensing system was developed using quartz crystal microbalance modified by gold nanoparticles anchored on its 1,6-hexanedithiol modified gold electrode surface, and ultrasensitive detection of DNA hybridization was accomplished at the level of at least 2 x 10(-16) M.     

Evaluation of antibody immobilization methods for piezoelectric biosensor application

The immobilization of anti-Salmonella antibodies by two methods were studied and evaluated for their potential use in a piezoelectric biosensor. The optimum temperature-time combinations for the highest immobilization yields were determined for both methods. Protein A binding was found to be 67.4+/-3.8% on the gold surface which then allowed an immobilization of 42.1+/-2.09% antibody. The degree of antibody immobilization via surface aldehyde groups of glutaraldehyde (GA) on a precoated quartz crystal with polyethylenimine (PEI) was 31.6+/-0.3%. A piezoelectric probe was designed and used in dry assays to observe the frequency change due to addition of mass by the immobilization layers. The frequency changes recorded showed a better reproducibility and less added mass for the Protein A method. The frequency decrease due to microg of added antibodies was compared to frequency decrease calculated by the Sauerbrey equation. The experimental data was found to be only approximately 8% of theoretical data. The functionality of the immobilized antibodies with the Protein A method was tested with S. typhimurium in a wet chamber and the frequency decrease was compared to results of a similar system activated with PEI-GA immobilization. The frequency decreases with S. typhimurium concentration of approximately 1.5 x 10(9) CFU/ml were 50+/-2 Hz and 44+/-3 Hz for the Protein A method and PEI-GA method, respectively. It was concluded that although both methods resulted in comparable activities in terms of % immobilized protein and frequency decreases due to Salmonella binding, the Protein A method was favorable due to stability and better reproducibility of the immobilization layers.     

Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater.

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10(3) bacteria per ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10(5) to 3.3 x 10(7) salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.     

Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10(3) bacteria per ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10(5) to 3.3 x 10(7) salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.     

Monitoring and kinetic parameter estimation for the binding process of berberine hydrochloride to bovine serum albumin with piezoelectric quartz crystal impedance analysis

A new method for monitoring, in real time, the drug-binding process to protein with piezoelectric quartz crystal impedance (PQCI) is proposed. The method was used to monitor the binding process of berberine hydrochloride to bovine serum albumin (BSA). BSA was immobilized on the silver electrode surface of a piezoelectric quartz crystal and the optimized experimental conditions were established. The BSA-coated piezoelectric sensor was in contact with berberine solution. The time courses of the resonant frequency and equivalent circuit parameters of the sensor during the protein-drug binding were simultaneously obtained. On the basis of the analysis of the multidimensional information provided by PQCI, it was concluded that the observed frequency decrease was mainly ascribed to the mass increase of the sensor surface resulting from the binding. According to the frequency decrease with time, the kinetics of the binding process were quantitatively studied. A piezoelectric response model for the binding was theoretically derived. Fitting the experimental data to the model, the kinetic parameters, such as the binding and dissociation rate constants (k(1) and k(-1)) and the binding equilibrium constant (K(a)), were determined. The k(1), k(-1), and K(a) values obtained at 25 degrees C were 67.5 (+/-0.1) (mol liter(-1))(-1) s(-1), 1.7 (+/- 0.1) x 10(-3) s(-1), and 3.97 (+/- 0.06) x10(4) (mol liter(-1))(-1), respectively.     

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.     

High-sensitivity detection for model organophosphorus and carbamate pesticide with quartz crystal microbalance-precipitation sensor

The effect of acetylcholinesterase (AChE) immobilization over the surface of a quartz crystal microbalance (QCM), by chemisorption of the AChE thiolated with a heterobifunctional cross-linker, sulfo-succinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate, and carboxyl-amine coupling of AChE to 3-mercaptopropionic acid self-assembled monolayer, on the responses of a batch-type QCM-precipitation sensor was compared, resulting in a better sensitivity and binding efficiency in the former method. When an inhibition study with the developed sensor was undertaken at the optimized AChE immobilization with varying concentrations of a model organophosphorus pesticide EPN and carbamate one carbofuran, a sensitive detection for them was possible with the limit of detection corresponding to 1.55 x 10(-8) and 1.30 x 10(-9)M, respectively.     

Colonization of barley (Hordeum vulgare) with Salmonella enterica and Listeria spp

Colonization of barley plants by the food-borne pathogens Salmonella enterica serovar typhimurium and three Listeria spp. (L. monocytogenes, L. ivanovii, L. innocua) was investigated in a monoxenic system. Herbaspirillum sp. N3 was used as a positive control and Escherichia coli HB101 as a negative control for endophytic root colonization. Colonization of the plants was tested 1-4 weeks after inoculation by determination of CFU, specific PCR assays and fluorescence in situ hybridization (FISH) with fluorescently labelled oligonucleotide probes in combination with confocal laser scanning microscopy (CLSM). Both S. enterica strains were found as endophytic colonizers of barley roots and reached up to 2.3 x 10(6) CFU per g root fresh weight after surface sterilization. The three Listeria strains had 10-fold fewer cell numbers after surface sterilization on the roots and therefore were similar to the results of nonendophytic colonizers, such as E. coli HB101. The FISH/CSLM approach demonstrated not only high-density colonization of the root hairs and the root surface by S. enterica but also a spreading to subjacent rhizodermis layers and the inner root cortex. By contrast, the inoculated Listeria spp. colonized the root hair zone but did not colonize other parts of the root surface. Endophytic colonization of Listeria spp. was not observed. Finally, a systemic spreading of S. enterica to the plant shoot (stems and leaves) was demonstrated using a specific PCR analysis and plate count technique.     

Immunosensor for the differentiation and detection of Salmonella species based on a quartz crystal microbalance

Immunosensors based on the microgravimetric quartz crystal microbalance (QCM) technique have been developed for the detection of Salmonella species from serogroups A, B and D. Salmonella serogroup-specific murine monoclonal antibodies, respectively, raised against these serogroups were immobilized onto the silver electrodes of piezoelectric (PZ) crystals by cross-linkage via glutaraldehyde (GA) to the electrode surfaces pre-coated with thin polyethyleneimine (PEI) layer. The specific immunosensors developed gave responses in linear ranges from 10(5) to 5x10(8) cells per ml with no significant interference from other strains of Salmonella and Escherichia coli up to 10(8) cells per ml. They showed good repeatability and excellent linear range, achieving detection limits down to 10(4) cells per ml with ability to distinguish different strains of Salmonella. These biosensors exhibited an exquisite specificity evidenced by their ability to discriminate antigens, the structures of which differ only by the isomeric form of di-deoxyhexose. The antibody-modified crystals showed no loss in activity over 4 days under storage at 4 degrees C.     

Immunochemical detection of Salmonella group B,D and E using an optical surface plasmon resonance biosensor

A surface plasmon resonance biosensor (Biacore) was used to detect Salmonella through antibodies reacting with Salmonella group A, B, D and E (Kauffmann-White typing). In the assay designed, anti-Salmonella antibodies immobilized to the biosensor surface were allowed to bind injected bacteria followed by a pulse with soluble anti-Salmonella immunoglobulins to intensify the signal. No significant interference was found for (mixtures of) 30 non-Salmonella serovars at 10(9) CFU ml(-1). A total of 53 Salmonella serovars were successfully detected at 1 x 10(7) CFU ml(-1), except those of groups C, G, L and P, as expected. The cut-off point was determined with an equicellular mixture of Salmonella enteritidis and Salmonella typhimurium at a final amount of 1.7 x 10(3) CFU per test portion. Although further work is needed to cover the detection of all relevant Salmonella serovars in food-producing animals and food products, this work demonstrates the merits of this alternative biosensor approach in terms of automation, sensitivity, specificity, simple handling and limited hands-on time.     

Planar coil excitation of multifrequency shear wave transducers

A transducer format that replaces the electrode of an acoustic resonator with a planar spiral coil is used to extract multifrequency spectral information from adsorbed protein films. Both amorphous silica and crystalline piezoelectric resonators are driven to resonance by forces induced across an air gap by magnetic direct generation and piezoelectric excitation induced by the electromagnetic field of the coil. Inspection of the harmonic frequencies between 6 MHz and 0.6 GHz indicates that the response of these two resonator types is described by different families of shear acoustic standing waves, with similar acoustic features to the quartz crystal microbalance. Exposure of the devices to protein solutions results in reproducible shifts of their harmonic frequencies, up to a maximum of 15 kHz and increasing linearly with frequency and operating mode. The gradient, determined from the ratio of the frequency change to the operating frequency was determined as 21.5 x 10(-6) for the quartz device and 60.9 x 10(-6) for the silica device. Consistency with the Sauerbrey equation for the piezoelectric linear shear mode was comparable at a predicted value of 22.5 x 10(-6), but not for the radial shear mode of the silica device at 12.7 x 10(-6). Opportunities resulting from the wide bandwidth of the planar coil excitation and choice of acoustic mode are discussed with respect to acoustic fingerprinting of adsorbed proteins.     

Series quartz crystal sensor for remote bacteria population monitoring in raw milk via the Internet

A remote monitoring system based on a piezoelectric quartz crystal (SPQC) sensor was developed for the determination of the bacteria population in raw milk. The system employs the Windows XP server operating system, and its programs for data acquisition, display and transmission were developed using the LabVIEW 7.1 programming language. The circuit design consists of a circuit with a piezoelectric quartz crystal (SPQC) and a pair of electrodes. This system can provide dynamic data monitoring on a web-page via the Internet. Immersion of the electrodes in a cell culture with bacteria inoculums resulted in a change of frequency caused by the impedance change due to microbial metabolism and the adherence of bacteria on the surface of the electrodes. The calibration curve of detection times against density of bacteria showed a linear correlation coefficient (R(2) = 0.9165) over the range of 70-10(6) CFU ml(-1). The sensor could acquire sufficient data rapidly (within 4 h) and thus enabled real-time monitoring of bacteria growth via the Internet. This system has potential application in the detection of bacteria concentration of milk at dairy farms.     

A QCM immunosensor for Salmonella detection with simultaneous measurements of resonant frequency and motional resistance

A quartz crystal microbalance (QCM) immunosensor was described for the detection of Salmonella Typhimurium with simultaneous measurements of the resonant frequency and motional resistance. The immunosensor was fabricated using protein A for the antibody immobilization. High-frequency impedance analysis indicated that the changes in resonant frequency and motional resistance (DeltaF and DeltaR) of the QCM were significant while the changes in static capacitance, motional capacitance, and motional inductance were insignificant. In the direct detection of S. Typhimurium in chicken meat sample, DeltaF and DeltaR were proportional to the cell concentration in the range of 10(5) - 10(8) and 10(6) - 10(8) cells/ml, respectively. Using anti-Salmonella-magnetic beads as a separator/concentrator for sample pretreatment as well as a marker for signal amplification, the detection limit was lowered to 10(2) cells/ml based on the DeltaR measurement; however, DeltaF was not related to the cell concentration. No interference was observed from E. coli K12 or the sample matrix.     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.     

Application of a flow-type antibody sensor to the detection of Escherichia coli in various foods

A flow-type biosensor system which uses a broad-spectrum anti-Escherichia coli antibody and quartz crystal microbalance as biological component and transducer was developed. Biosensor responses were initiated by injecting viable E. coli suspensions through a flow cell and the sensor system was optimized for response time according to flow rate and injection time, followed by the measurement of responses for various E. coli strains. As expected, the sensor system showed a characteristic broad binding feature against E. coli strains. A linear sensor response in double-logarithmic scale was observed for the microbial suspensions ranging from 1.7 x 10(5) to 8.7 x 10(7) CFU/ml. Sample measurements could be done within 20-30 min after Stomacher treatment followed by spiking or enrichment.     

Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance

A biosensor based on bovine serum albumin (BSA) for the detection of lead (Pb(2+)) ion was developed and characterized. BSA was immobilized onto a colloidal Au-modified piezoelectric quartz crystal (PQC) as a biosensor for the detection of Pb(2+) ion by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb(2+) ion showed excellent linearity throughout the concentration range from 1.0 x 10(-7) to 3.0 x 10(-9)mol/L. The interaction between the Pb(2+) ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 x 10(-9)mol/L for Pb(2+) was obtained. Kinetic parameters of the Pb(2+)-BSA interactions were also determined by using this chip. The sensor chip could be regenerated for use by dipping in the ethylenediaminetetraacetic acid (EDTA) solution for approximately 2h, and the chip was used to detect Pb(2+) ion for eight times without obvious signal attenuation.