Microfluidic based impedance biosensor for pathogens detection in food products.

A MEMS-based impedance biosensor was designed, fabricated, and tested to effectively detect the presence of bacterial cells including E. coli O157:H7 and Salmonella typhimurium in raw chicken products using detection region made of multiple interdigitated electrode arrays. A positive dielectrophoresis based focusing electrode was used in order to focus and concentrate the bacterial cells at the centerline of the fluidic microchannel and direct them toward the detection microchannel. The biosensor was fabricated using surface micromachining technology on a glass substrate. The results demonstrate that the device can detect Salmonella with concentrations as low as 10 cells/mL in less than 1 h. The device sensitivity was improved by the addition of the focusing electrodes, which increased the signal response by a factor between 6 and 18 times higher than without the use of the focusing electrodes. The biosensor is selective and can detect other types of pathogen by changing the type of the antibody immobilized on the detection electrodes. The device was able to differentiate live from dead bacteria.

Effects of conservation buffer systems on adsorption of fluorescent-labeled Escherichia coli.

The magnitude of bacterial transport through runoff into surface water or infiltration into groundwater is influenced by the adsorption processes in soil. The objective of this study was to evaluate fluorescent-labeled Escherichia coli (E. coli) adsorption by soil under agroforestry buffer (AB), grass buffer (GB), and row crop (RC) management. Adsorption experiments were conducted by inoculating three masses (0.5, 1, and 10 g) of each treatment (AB, GB, and RC) with E. coli O157:H7-GFP with concentration ranges of 105 -108 colony-forming units (cfu) ml-1 . Adsorption data were evaluated using Langmuir, Freundlich, and Temkin adsorption isotherm models. The Freundlich isotherm model described the observed data well for all treatments using the 10-g soil mass, with the R2 values closer to unity in all treatments. The Freundlich Kf parameter, an indicator of adsorption capacity, was higher for the AB treatment (9.93 cfu ml-1 ) compared with the GB and RC treatments (2.32 and 1.27 cfu ml-1 , respectively). The multiple pairwise comparisons test (Tukey test) of the Freundlich 1/nf parameter demonstrated a significant difference (p < .05) between the AB treatment and the RC and GB treatments. Similarly, the Kf values were significantly (p = .05) higher for the 10-g mass under the same test conditions, but no significant differences were observed in the 0.5- and 1-g masses. This study demonstrated that AB has a higher E. coli adsorption capacity and the potential for mitigating the effects of E. coli O157:H7 transport to surface or groundwater through the soil ecosystem.

Nanoparticle immuno-fluorescent probes as a method for detection of viable E. coli O157:H7.

Development of revolutionary sensitive biosensors for detecting the presence of harmful biological species in the environment is a necessity for countering disease outbreaks. This work examined the interaction of fluorescence-labeled antibody on amine functionalized gold nanoparticles (GNP) as a model system. The synthesized tetramethylrhodamine isothiocyanate (TRITC) labeled antibody-amine functionalized GNP interaction was characterized using UV-Vis spectroscopy and Fluorescent Microscopy imaging. Transmission Electron Microscopy (TEM) was also used to observe the morphology of the GNP. In contrast to TEM, the fluorescence microscopy imaging revealed the coating of the TRITC labeled antibody on the surface of the GNP. The signals were measured using a Photon Technology Inc. fluorometer at excitation of 541 nm and emission at 555 nm to 650 nm. Tests were conducted at near real-time with results obtained using the biosensor assay within 5 min. Results indicated that there was a shift of the wavelength from lower to higher wavelength (blue to red shift) when conjugated GNP (anti-E. coliO157:H7; IgY-TRITC-GNP) are compared to free GNP, a difference of about 28 nm. The GNP demonstrated a quenching capability when compared to the TRITC labeled antibody (degree of labeling of 15.41 mol dye per mole of IgY) using fluorometer. The lower and upper detection range of this method was found to be 103-105 CFU/mL with observed fluorescence of about 42,000 counts per seconds as against 24,000 counts per seconds that was observed when the specificity of the sensor was tested using Salmonella enterica.

A Review of SARS-CoV-2 Disease (COVID-19): Pandemic in Our Time.

Development and deployment of biosensors for the rapid detection of the 2019 novel severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) are of utmost importance and urgency during this recent outbreak of coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 infection, which spread rapidly around the world. Cases now confirmed in February 2022 indicate that more than 170 countries worldwide are affected. Recent evidence indicates over 430 million confirmed cases with over 5.92 million deaths scattered across the globe, with the United States having more than 78 million confirmed cases and over 920,000 deaths. The US now has many more cases than in China where coronavirus cases were first reported in late December 2019. During the initial outbreak in China, many leaders did not anticipate it could reach the whole world, spreading to many countries and posing severe threats to global health. The objective of this review is to summarize the origin of COVID-19, its biological nature, comparison with other coronaviruses, symptoms, prevention, treatment, potential, available methods for SARS-CoV-2 detection, and post-COVID-19 symptoms.

A microfluidic biosensor for rapid simultaneous detection of waterborne pathogens.

A microfluidic based biosensor was investigated for rapid and simultaneous detection of Salmonella, Legionella, and Escherichia coli O157:H7 in tap water and wastewater. The biosensor consisted of two sets of focusing electrodes connected in parallel and three sets of interdigitated electrodes (IDE) arrays. The electrodes enabled the biosensor to concentrate and detect bacteria at both low and high concentrations. The focusing region was designed with vertical metal sidewall pairs and multiple tilted thin-film finger pairs to generate positive dielectrophoresis (p-DEP) to force the bacteria moving toward the microchannel centerline. As a result, the bacterial pathogens were highly concentrated when they reached the detection electrode arrays. The detection IDE arrays were coated with three different antibodies against the target bacterial pathogens and a cross-linker to enhance the binding of antibodies to the detection electrode. As the binding of bacterial pathogen to its specific antibodies took place, the impedance value changed. The results demonstrated that the biosensors were capable of detecting Salmonella, Legionella, and E. coli 0157:H7 simultaneously with a detection limit of 3 bacterial cells/ml in 30 - 40 min.

A microfluidic based biosensor for rapid detection of Salmonella in food products.

An impedance based microfluidic biosensor for simultaneous and rapid detection of Salmonella serotypes B and D in ready-to-eat (RTE) Turkey matrix has been presented. Detection of Salmonella at a concentration as low as 300 cells/ml with a total detection time of 1 hour has been achieved. The sensor has two sensing regions, with each formed from one interdigitated electrode array (IDE array) consisting of 50 finger pairs. First, Salmonella antibody type B and D were prepared and delivered to the sensor to functionalize each sensing region without causing any cross contamination. Then the RTE Turkey samples spiked with Salmonella types B and D were introduced into the biosensor via the antigen inlet. The response signal resulted from the binding between Salmonella and its specific antibody demonstrated the sensor's ability to detect a single type of pathogen, and multiple pathogens simultaneously. In addition, the biosensor's selectivity was tested using non-specific binding of E. coli O157 and E. coli DH5 Alpha while the IDE array was coated with the Salmonella antibody. The results also showed the sensor is capable to differentiate low concentration of live Salmonella cells from high concentration of dead Salmonella cells, and high concentration of E. coli cells. A detailed study on antibody immobilization that includes antibody concentration, antibody coating time (0.5-3 hours) and use of cross-linker has been performed. The study showed that Salmonella antibody to Salmonella antigen is not a factor of antibody concentration after electrodes were saturated with antibody, while the optimal coating time was found to be 1.5 hours, and the use of cross-linker has improved the signal response by 45-60%.

An impedance biosensor for simultaneous detection of low concentration of Salmonella serogroups in poultry and fresh produce samples.

This paper reports the design, fabrication and testing of a microfluidic based impedance biosensor for rapid and simultaneous detection of three Salmonella serogroups. The microfluidic device consists of three microchannels, each one includes a region for focusing the Salmonella cells into the centerline of the microchannel and direct them toward the sensing region to obtain highly concentrated samples using positive dielectrophoresis force. A region for bacteria sensing consists of interdigitated electrode (IDE) array with 10 pairs of fingers. Three types of Salmonella antibodies (type B, D and E) were mixed separately with the cross-linker (Sulfo-LC-SPDP) to enhance the immobalization of the antibodies to the detection electrodes. The electrode surfaces was then functionalized with the three mixtures, one for each channel. As target antigen binds to the antibody, it results in impedance change. The Salmonella samples were spiked with Salmonella type B, introduced into the biosensor via the sample inlet into the focusing region, and then toward the sensing region where they bind to the immobilized antibody, causing a change in the impedance. The performance of the devices was tested using single Salmonella serotype B and two Salmonella serotypes B, and D, with a limit of detection of 7 cells/ml. The biosensor was also able to differentiate live from dead bacteria eliminating the false positive results. Finally, the device was also able to detect Salmonella selectively when other type of pathogen was present.

Low concentration E. coli O157:H7 bacteria sensing using microfluidic MEMS biosensor.

This paper reports the design, fabrication, and testing of a microfluidic MEMS biosensor for rapid sensing of low concentration Escherichia coli O157:H7. It consists of a specially designed focusing and sensing region, which enables the biosensor to detect low concentration of bacterial cells. The focusing region consists of a ramped vertical electrode pair made of electroplated gold along with tilted thin film finger pairs (45°) embedded inside a microchannel. The focusing region generates positive dielectrophoresis force, which moves the cells towards the edges of the tilted thin film electrode fingers, located at the center of the microchannel. The fluidic drag force then carries the focused cells to the sensing region, where three interdigitated electrode arrays (IDEAs) with 30, 20, and 10 pairs, respectively, are embedded inside the microchannel. This technique resulted in highly concentrated samples in the sensing region. The sensing IDEAs are functionalized with the anti-E. coli antibody for specific sensing of E. coli 0157:H7. As E. coli binds to the antibody, it results in an impedance change, which is measured across a wide frequency range of 100 Hz-10 MHz. The biosensor was fabricated on a glass substrate using the SU8 epoxy resist to form the microchannel, gold electroplating to form the vertical focusing electrode pair, a thin gold film to form the sensing electrode, the finger electrodes, traces and bonding pads, and polydimethylsiloxane to seal the device. The microfluidic impedance biosensor was tested with various low concentration bacterial samples and was able to detect bacterial concentration, as low as 39 CFU/ml with a total sensing time of 2 h.

An integrated impedance biosensor platform for detection of pathogens in poultry products.

This paper presents an impedance-based biosensor for rapid and simultaneous detection of Salmonella serotypes B, D, and E with very low concentration. The biosensor consists of a focusing region, and three detection regions. The cells focusing was achieved using a ramp down electroplated vertical electrode pair along with tilted thin film finger pairs that generate p-DEP forces to focus and concentrate the bacterial cells into the center of the microchannel, and direct them toward the detection region. The detection regions consist of three interdigitated electrode arrays (IDEA), each with 20 pairs of finger coated with a mixture of anti-Salmonella antibody and crosslinker to enhance the adhesion to IDEA. The impedance changes as the target Salmonella binds to the antibody. The biosensor has showed excellent performance as proven by the detection of a single Salmonella serotype B, and simultaneous detection of two Salmonella serotypes B and D with a limit of detection (LOD) of 8 Cells/ml in ready-to-eat turkey samples, the addition of focusing capability improved the measured signal by a factor of between 4-4.5, the total detection time of 45 minutes, selectivity of the sensor on different types of bacterial cells, and the ability to distinguish between dead and live cells.

A rapid and highly specific immunofluorescence method to detect Escherichia coli O157:H7 in infected meat samples.

Developing rapid and sensitive methods for the detection of pathogenic Escherichia coli O157:H7 remains a major challenge in food safety. The present study attempts to develop an immunofluorescence technique that uses Protein-A-coated, magnetic beads as the platform. The immunofluorescence technique described here is a direct detection method in which E. coli O157:H7 cells are labeled with tetramethylrhodamine (TRITC) fluorescent dye. TRITC-labeled bacteria are captured by the desired antibody (Ab), which is immobilized on the Protein-A magnetic beads. Fluorescence of the captured cells is recorded in a fluorescence spectrophotometer, where the fluorescence values are shown to be directly proportional to the number of bacteria captured on the immunobead. The formation of an immunocomplex is evidenced by the fluorescence of the beads under microscopy. The Ab immobilization procedure is also evidenced by microscopy using fluorescein isothiocyanate (FITC)-labeled Ab. The total experimental time, including preparation of the sample, is just 1h. The minimum bacterial concentration detected by this method is 1.2±0.06×10(3)CFUml(-1). The high specificity of this method was proved by using the specific monoclonal Ab (MAb) in the test. The proposed protocol was successfully validated with E. coli O157:H7-infected meat samples. This approach also opens the door for the detection of other bacterial pathogens using Protein-A magnetic beads as a detection platform.

Highly specific and rapid immuno-fluorescent visualization and detection of E. coli O104:H4 with protein-A coated magnetic beads based LST-MUG assay.

A method combining immunomagnetic separation and fluorescent sensing was developed to detect Escherichia coli (E. coli) O104:H4. The antibody specific to E. coli O104:H4 was immobilized on protein A-coated magnetic beads. This protein-A-anti E. coli O104:H4 complex was used to bind Fluorescein IsoThioCyanate (FITC) labeled E. coli O104:H4 antigen (whole cell) on it. The goal was to achieve a fluorescently detectable protein-A-anti E. coli O104:H4-E. coli O104:H4 complex on the magnetic beads. Fluorescent microscopy was used to image the magnetic beads. The resulting fluorescence on the beads was due to the FITC labeled antigen binding on the protein-A-anti E. coli O104:H4 immobilized magnetic beads. This visually proves the antigen-antibody binding. The fluorescent imaging results were obtained in 2 h if the minimum available bacteria in the sample were at least 10(5) CFU/ml. If no fluorescence was observed on the magnetic beads during fluorescent imaging, it indicates the bacterial concentration in the sample to be too low for it to have bound to the magnetic beads and hence no detection was possible. To detect bacterial concentration less than 10(5) CFU/ml in the sample, an additional step was required for detection. The magnetic bead complex was added to the LST-MUG (lauryl sulfate tryptose-4-methylumbelliferyl-ß-D-glucuronide), a signaling reporter. The E. coli O104:H4 grows in LST-MUG and releases ß-glucuronidase enzyme. This enzyme cleaves the MUG substrate that produces 4-methylumbelliferone, a highly fluorescent species. This fluorescence was detected using a spectrofluorometer. The emission peak in the fluorescent spectrum was found to be at 450 nm. The lower and upper detection range for this LST-MUG assay was found to be 2.05×10(5)-4.09×10(8) CFU/ml. The results for the LST-MUG assay for concentrations below 10(5) CFU/ml were ascertained in 8h. The advantages of this technique include the specific detection of bacteria without an enrichment step and allowing the procedure to be completed in hours rather than days.

Postpartum multivessel coronary artery dissection treated with coronary artery bypass grafting.

Spontaneous coronary artery dissection is a rare but potentially life-threatening event associated with the peripartum period. We present a case of acute anterolateral ST elevation myocardial infarction in the postpartum period in a young woman. Thrombolytic therapy was successful, but later the patient required emergent coronary artery bypass grafting due to another acute inferolateral myocardial infarction that developed 4 h after cardiac catheterization which showed multivessel spontaneous coronary artery dissection.

Specific and targeted detection of viable Escherichia coli O157:H7 using a sensitive and reusable impedance biosensor with dose and time response studies.

A gold interdigitated microelectrode (IME) impedance biosensor was fabricated for the detection of viable Escherichia coli O157:H7. This sensor was fabricated using lithography techniques. The surface of the electrode was immobilized with anti-E. coli IgG antibodies. This approach is different from other studies where the change in impedance is measured in terms of growth of bacteria on the electrode, rather then the antibody/antigen bonding. The impedance values were recorded for frequency ranges between 100 Hz and 10 MHz. The working range of the dose response for this device was found to be between 2.5×10(4) CFU ml(-1) and 2.5×10(7) CFU ml(-1). The time response studies indicated that antibody/antigen binding is not a function of time, but can decrease if excess times are allowed for binding. It was observed that the impedance values for 60 min antibody/antigen binding were higher than the impedance values for 120 min binding time. The main advantages of the reported device are that, it provides for both qualitative and quantitative detection in 3h while other impedance sensors reported earlier may take up to 24h for detection. If enrichment steps are required then it may take 3-4 days to infer the results. This sensor can be used to detect different types of bacteria by immobilizing the antigen specific antibody. Most of the sensors are not reusable since they either use enzymes or enrichment steps for detection but this device can be reused, following a cleaning protocol which is easy to follow. Each device was used at least five times. The simplicity of this sensor and the ease of fabrication make this sensor a useful alternate to the microfluidics and enzyme based impedance sensors, which are relatively more difficult to fabricate, need programmable fluidic injection pumps to push the sample through the channel, suffer from limitation of coagulation and are difficult to clean.