A magnetoelastic resonance biosensor immobilized with polyclonal antibody for the detection of Salmonella typhimurium

Mass-sensitive, magnetoelastic resonance sensors have a characteristic resonant frequency that can be determined by monitoring the magnetic flux emitted by the sensor in response to an applied, time varying, magnetic field. This magnetostrictive platform has a unique advantage over conventional sensor platforms in that measurement is wireless and remote. A biosensor for the detection of Salmonella typhimurium was constructed by immobilizing a polyclonal antibody (the bio-molecular recognition element) onto the surface of a magnetostrictive platform. The biosensor was then exposed to solutions containing S. typhimurium bacteria. Binding between the antibody and antigen (bacteria) occurred and the additional mass of the bound bacteria caused a shift in the sensor's resonant frequency. Sensors with different physical dimensions were exposed to different concentrations of S. typhimurium ranging from 10(2) to 10(9)CFU/ml. Detection limits of 5x10(3) CFU/ml, 10(5) CFU/ml and 10(7) CFU/ml were obtained for sensors with the size of 2 mmx0.4 mmx15 microm, 5 mmx1 mmx15 microm and 25 mmx5 mmx15 microm, respectively. Good agreement between the measured number of bound bacterial cells (as measured by scanning electron microscopy (SEM)) and frequency shifts was obtained.     

Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium

In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.     

In-situ monitoring of breast cancer cell (MCF-7) growth and quantification of the cytotoxicity of anticancer drugs fluorouracil and cisplatin

A wireless sensing device was developed for the in-situ monitoring of the growth of human breast cancer cells (MCF-7) and evaluation of the cytotoxicity of the anticancer drugs fluorouracil and cisplatin. The sensor is fabricated by coating a magnetoelastic ribbon-like sensor with a layer of polyurethane that protects the iron-rich sensor from oxidation and provides a cell-compatible surface. In response to a time-varying magnetic field, the magnetoelastic sensor longitudinally vibrates, emitting magnetic flux that can be remotely detected by a pick-up coil. No physical connections between the sensor and the detection system are required. The wireless property facilitates aseptic biological operation, especially in cell culture as illustrated in this work. The adhesion of cells on the sensor surface results in a decrease in the resonance amplitude, which is proportional to the cell concentration. A linear response was obtained in cell concentrations of 5x10(4) to 1x10(6)cellsml(-1), with a detection limit of 1.2x10(4)cellsml(-1). The adhesion strength of cells on the sensor is qualitatively evaluated by increasing the amplitude of the magnetic excitation field. And the cytotoxicity of the anticancer drugs fluorouracil and cisplatin is evaluated by the magnetoelastic biosensor. The cytostatic curve is related with the quantity of cytostatic drug. The lethal concentration (LC50) for cells incubated in the presence of drugs for 20h is calculated to be 19.9muM for fluorouracil and 13.1muM for cisplatin.     

Monitoring blood coagulation with magnetoelastic sensors

The determination of blood coagulation time is an essential part of monitoring therapeutic anticoagulants. Standard methodologies for the measurement of blood clotting time require dedicated personnel and involve blood sampling procedures. A new method based on magnetoelastic sensors has been employed for the monitoring of blood coagulation. The ribbon-like magnetoelastic sensor oscillates at a fundamental frequency, which shifts linearly in response to applied mass loads or a fixed mass load of changing elasticity. The magnetoelastic sensors emit magnetic flux, which can be detected by a remotely located pick-up coil, so that no direct physical connections are required. During blood coagulation, the viscosity of blood changes due to the formation of a soft fibrin clot. In turn, this change in viscosity shifts the characteristic resonance frequency of the magnetoelastic sensor enabling real-time continuous monitoring of this biological event. By monitoring the signal output as a function of time, a distinct blood clotting profile can be seen. The relatively low cost of the magnetoelastic ribbons enables their use as disposable sensors. This, along with the reduced volume of blood required, make the magnetoelastic sensors well suited for at-home and point-of-care testing devices.     

Rapid and sensitive magnetoelastic biosensors for the detection of Salmonella typhimurium in a mixed microbial population

In this article, we report the results of an investigation into the performance of a wireless, magnetoelastic biosensor designed to selectively detect Salmonella typhimurium in a mixed microbial population. The Langmuir-Blodgett (LB) monolayer technique was employed for antibody (specific to Salmonella sp.) immobilization on rectangular shaped strip magnetoelastic sensors (2 x 0.4 x 0.015 mm). Bacterial binding to the antibody on the sensor surface changes the resonance parameters, and these changes were quantified as a shift in the sensor's resonance frequency. Response of the sensors to increasing concentrations (5 x 10(1) to 5 x 10(8) cfu/ml) of S. typhimurium in a mixture of extraneous foodborne pathogens (Escherichia coli O157:H7 and Listeria monocytogenes) was studied. A detection limit of 5 x 10(3) cfu/ml and a sensitivity of 139 Hz/decade were observed for the 2 x 0.4 x 0.015 mm sensors. Binding kinetics studies have shown that the dissociation constant (K(d)) and the binding valencies for water samples spiked with S. typhimurium was 435 cfu/ml and 2.33 respectively. The presence of extraneous microorganisms in the mixture did not produce an appreciable change in the biosensor's dose response behavior.     

A staphylococcal enterotoxin B magnetoelastic immunosensor

A magnetoelastic immunosensor for detection of staphylococcal enterotoxin B (SEB) is described. The magnetoelastic sensor is a newly developed mass/elasticity-based transducer of high sensitivity having a material cost of approximately $0.001/sensor. Affinity-purified rabbit anti-SEB antibody was covalently immobilized on magnetoelastic sensors, of dimensions 6 mm x 2 mm x 28 microm. The affinity reaction of biotin-avidin and biocatalytic precipitation are used to amplify antigen-antibody binding events on the sensor surface. Horseradish peroxidase (HRP) and alkaline phosphatase were examined as the labeled enzymes to induce biocatalytic precipitation. The alkaline phosphatase substrate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) produces a dimer, which binds tightly to the sensor surface, inducing a change in sensor resonance frequency. The biosensor demonstrates a linear shift in resonance frequency with staphylococcal enterotoxin B concentration between 0.5 and 5 ng/ml, with a detection limit of 0.5 ng/ml.     

A remote-query sensor for predictive indication of milk spoilage

Described is application of the remote-query (wireless, passive) magnetoelastic sensor platform for direct detection and monitoring of bacterium contamination of milk within hermetically sealed containers. Specific application is made to the quantification of Staphylococcus aureus ssp. anaerobius (S. aureus) concentrations in milk. S. aureus growth changes milk viscosity, in turn changing the resonance frequency of the liquid immersed sensor allowing S. aureus concentrations of 10³ to 10⁷ cells ml⁻¹ to be directly quantified.     

Application of parylene-coated quartz crystal microbalance for on-line real-time detection of microbial populations

A novel technique of applying a quartz crystal microbalance (QCM) sensor to the on-line real-time detection of microbial populations is described. The pQCM sensor was fabricated by depositing di-para-xylene (parylene) over the entire surface of a QCM sensor through a chemical vapor deposition (CVD) process. An electrically insulated film of parylene on the QCM sensor enabled the operation of the sensor in the liquid environment, and the resonance frequency of the pQCM sensor set in the medium of a cultivation flask shifted in response to the microbial population. The effects of pH, conductivity, and viscosity of the medium on the frequency shift of the pQCM sensor were investigated. Ignorable responses (less than 1% at 10(3)cells) were obtained during an incubation cycle. The detection limit of the pQCM sensor was identified as 10(2) cells ml(-1) with a frequency shift of around 2 x 10(3)Hz. The cell numbers of Escherichia coli cultivated in both the YEM medium and whole milk were detected. A satisfactory correlation (r(2)=0.95) was obtained between the cell number and the response of the pQCM sensor. Experimental results suggest that the pQCM described here is applicable to the continuous long-term detection of microbial populations during a fermentation process.     

Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1mg/ml BSA (blocking agent) onto the magnetoelastic resonator's surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor's resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.     

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.     

A magnetoelastic bioaffinity-based sensor for avidin

A magnetoelastic bioaffinity sensor coupled with biocatalytic precipitation is described for avidin detection. The non-specific adsorption characteristics of streptavidin on different functionalized sensor surfaces are examined. It is found that a biotinylated poly(ethylene glycol) (PEG) interface can effectively block non-specific adsorption of proteins. Coupled with the PEG immobilized sensor surface, alkaline phosphatase (AP) labeled streptavidin is used to track specific binding on the sensor. This mass-change-based signal is amplified by the accumulation on the sensor of insoluble products of 5-bromo-4-chloro-3-indolyl phosphate catalyzed by AP. The resulting mass loading on the sensor surface in turn shifts the resonance frequency of the magnetoelastic sensors, with an avidin detection limit of approximately 200 ng/ml.     

A rapid highly-sensitive endotoxin detection system

This paper presents a rapid, highly-sensitive, and low-cost method of endotoxin quantification based on the use of stress-responsive magnetoelastic sensors, that monitor the gel formation (viscosity change) of the Limulus Amoebocyte Lysate (LAL) assay in response to endotoxin. Ribbon-like magnetoelastic sensors, 12.7 mm × 6 mm × 28 μm, were immersed in a LAL assay after mixing with test samples of variable endotoxin concentration, and the decrease in resonance amplitude of the sensor was recorded as a function of time. Experimental results show excellent correlation between endotoxin concentration and the maximum clot rate, determined by taking the minimum point of the first derivative of the amplitude–time curve, as well as the clotting-time, defined as the time that corresponds to the maximum clot rate. Using a LAL gel–clot assay with a sensitivity of 0.06 EU/ml (EU: endotoxin unit), the magnetoelastic sensor based technology can detect the presence of endotoxin at 0.0105 EU/ml in test requiring approximately 20 min. Unlike optical methods used for determining endotoxin concentration, the color of the test solution does not impact the magnetoelastic sensor measurement. Due to the small size of the sensor reader electronics and low cost, the magnetoelastic sensor based endotoxin detection system is ideally suited for wide-spread use in endotoxin screening for sepsis prevention.     

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.     

The effect of salt and phage concentrations on the binding sensitivity of magnetoelastic biosensors for Bacillus anthracis detection

This article presents an investigation of the effect of salt and phage concentrations on the binding affinity of magnetoelastic (ME) biosensors. The sensors were fabricated by immobilizing filamentous phage on the ME platform surface for the detection of Bacillus anthracis spores. In response to the binding of spores to the phage on the ME biosensor, a corresponding decrease occurs in resonance frequency. Transmission electron microscopy (TEM) was used to verify the structure of phage under different combinations of salt/phage concentration. The chemistry of the phage solution alters phage bundling characteristics and, hence, influences both the sensitivity and detection limit of the ME biosensors. The frequency responses of the sensors were measured to determine the effects of salt concentration on the sensors' performance. Scanning electron microscopy (SEM) was used to confirm and quantify the binding of spores to the sensor surface. This showed that 420 mM salt at a phage concentration of 1 x 10(11) vir/mL results in an optimal distribution of immobilized phages on the sensor surface, consequently promoting better binding of spores to the biosensor's surface. Additionally, the sensors immobilized with phage under this condition were exposed to B. anthracis spores in different concentrations ranging from 5 x 10(1) to 5 x 10(8) cfu/mL in a flowing system. The results showed that the sensitivity of this ME biosensor was 202 Hz/decade.     

Detection of Mycoplasma genitalium using a wireless magnetoelastic immunosensor

A wireless immunosensor for the detection of Mycoplasma genitalium was fabricated by immobilizing polyclonal antibody onto the surface of a magnetostrictive strip. In response to a time-varying magnetic field, the immunosensor longitudinally vibrates at a resonance frequency, emitting magnetic flux that can be remotely detected by a pickup coil. No physical connections between the immunosensor and the detection system are required, facilitating wireless aseptic operation. The binding of M. genitalium to the immunosensor surface resulted in a decrease in the resonance frequency of the immunosensor. When solutions with varying concentrations of the bacteria were tested, the shift of the resonance frequency was proportional to the concentration of M. genitalium. Under the optimized conditions, the linear range for the determination of M. genitalium was 2.0 × 10³ to 2.9 × 10⁴ color change units (ccu)/ml with a detection limit of 3.4 × 10² ccu/ml. The immunosensor was successfully applied to real samples containing M. genitalium with results similar to those previously obtained by the color change unit method.     

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.     

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.     

Isolation and antifungal and antioomycete activities of aerugine produced by Pseudomonas fluorescens strain MM-B16

The bacterial strain MM-B16, which showed strong antifungal and antioomycete activity against some plant pathogens, was isolated from a mountain forest soil in Korea. Based on the physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, the bacterial strain MM-B16 was identical to Pseudomonas fluorescens. An antibiotic active against Colletotrichum orbiculare and Phytophthora capsici in vitro and in vivo was isolated from the culture filtrates of P. fluorescens strain MM-B16 using various chromatographic procedures. The molecular formula of the antibiotic was deduced to be C(10)H(11)NO(2)S (M(+), m/z 209.0513) by analysis of electron impact mass spectral data. Based on the nuclear magnetic resonance and infrared spectral data, the antibiotic was confirmed to have the structure of a thiazoline derivative, aerugine [4-hydroxymethyl-2-(2-hydroxyphenyl)-2-thiazoline]. C. orbiculare, P. capsici, and Pythium ultimum were most sensitive to aerugine (MICs for these organisms were approximately 10 micro g ml(-1)). However, no antimicrobial activity was found against yeasts and bacteria even at concentrations of more than 100 micro g ml(-1). Treatment with aerugine exhibited a significantly high protective activity against development of phytophthora disease on pepper and anthracnose on cucumber. However, the control efficacy of aerugine against the diseases was in general somewhat less than that of the commercial fungicides metalaxyl and chlorothalonil. This is the first study to isolate aerugine from P. fluorescens and demonstrate its in vitro and in vivo antifungal and antioomycete activities against C. orbiculare and P. capsici.     

SPR biosensor for the detection of L. monocytogenes using phage-displayed antibody

Whole cells of Listeria monocytogenes were detected with a compact, surface plasmon resonance (SPR) sensor using a phage-displayed scFv antibody to the virulence factor actin polymerization protein (ActA) for biorecognition. Phage Lm P4:A8, expressing the scFv antibody fused to the pIII surface protein was immobilized to the sensor surface through physical adsorption. A locally constructed fluidics system was used to deliver solutions to the compact, two-channel SPREETA sensor. Specificity of the sensor was tested using common food-borne bacteria and a control phage, M13K07 lacking the scFv fusion on its coat protein. The detection limit for L. monocytogenes whole cells was estimated to be 2 x 10(6)cfu/ml. The sensor was also used to determine the dissociation constant (Kd) for the interaction of phage-displayed scFv and soluble ActA in solution as 4.5 nM.     

Improved bacteria detection by coupling magneto-immunocapture and amperometry at flow-channel microband electrodes

This paper describes the first immunosensing system reported for the detection of bacteria combining immunomagnetic capture and amperometric detection in a one-step sandwich format, and in a microfluidic environment. Detection is based on the electrochemical monitoring of the activity of horseradish peroxidase (HRP), an enzyme label, through its catalysis of hydrogen peroxide (H(2)O(2)) in the presence of the mediator hydroquinone (HQ). The enzymatic reaction takes place in an incubation micro-chamber where the magnetic particles (MPs) are confined, upstream from the working electrode. The enzyme product is then pumped along a microchannel, where it is amperometrically detected by a set of microelectrodes. This design avoids direct contact of the biocomponents with the electrode, which lowers the risk of electrode fouling. The whole assay can be completed in 1h. The experiments performed with Escherichia coli evidenced a linear response for concentrations ranging 10(2)-10(8) cell ml(-1), with a limit of detection of 55 cells ml(-1) in PBS, without pre-enrichment steps. Furthermore, 100 cells ml(-1) could be detected in milk, and with negligible interference by non-target bacteria such as Pseudomonas.