Concentration Of Target Bacteria Research Articles

Paper-based colorimetric detection of pathogenic bacteria in food through magnetic separation and enzyme-mediated signal amplification on paper disc

Herein, we report a colorimetric sensing system for the detection of highly virulent bacteria, Escherichiacoli O157:H7, in sausage by utilizing magnetic separation and enzyme-mediated signal amplification on paper disc. For magnetic separation, Poly-l-lysine coated starch magnetic particles (PLL@SMPs) were synthesized and utilized for the separation and concentration of the bacteria in sample suspension. Horseradish peroxidase-conjugated antibody (HRP-Antibody) and 3,3′,5,5′- tetramethylbenzidine (TMB) were employed for the specific signal amplification in the presence of target bacteria. The synthesized PLL@SMPs showed an excellent capture efficiency (>90%) for the pathogenic bacteria in large volume sample suspension. The intrinsic problems associated with the non-specific binding of sensing components that lead to the high background signal and low sensitivity in colorimetric detection was successfully resolved by employing hyaluronic acid as a blocking agent. The effective separation and concentration of target bacteria by PLL@SMPs and target-specific signal amplification with exceptionally high signal to noise ratio enabled the detection of target bacteria with a detection limit in the single digit regime. The sensing system proposed in this study was successfully used for the detection of the target pathogenic bacteria, E. coli O157:H7, in sausage sample with the limit of detection (LOD) as low as 30.8 CFU/mL with 95% probability. The simple nature of paper-based detection system with a great sensitivity and specificity would provide an effective means of evaluating the safety of food and environmental samples.

Monitoring of methylene blue monomers and dimers to control the bacterialogical water quality including application to photocatalysis.

In this study, we propose the development of a rapid and reliable method to control and to monitor microbial water quality. The methylene blue (MB) decolorization assay was based on the analysis of spectral profiles of dye in interaction with a different bacterial concentration. The determination of dye decolorization rate (DDR) shows a correlation between the MB reduction rate and the bacterial density. Moreover, the kinetic of the monomer and dimer equilibrium of MB in water mainly, the monitoring of bounded MB species in relationship with a knowed concentration of target bacteria, was allowed to establish a relationship between MB decolorization rate and bacterial density. Furthermore, this method was applied to evaluate the water quality after photocatalysis. Based on this method, the photocatalytic effects on bacterial density was highlighted by the decrease in DDR after photocatalytic treatment with fractioned times (0 to 5 h); this increase was followed by a decrease of bounded MB species and, an increase in free MB forms miming the reduction of bacterial density due to the biocide effects of photocatalysis process. However, the analysis of spectra profiles shows a weak but a continuous decrease in bounded MB dimer and monomer forms in the treated water samples exempt of culturable bacteria. Moreover, the MB spectra profiles were tended toward a negative control spectrum without superposition. Thus, the possibility of the presence of viable but non-culturable bacteria was expected; therefore, to optimize this tertiary water treatment process, an extending on proceeding time was recommended to avoid the bacterial resuscitation after photocatalysis.

Detection of E. coli O157:H7 in Food Using Automated Immunomagnetic Separation Combined with Real-Time PCR

In this study, we describe the development of an automated immunomagnetic separation device combined with real-time polymerase chain reaction (PCR) for detecting foodborne bacteria. Immunomagnetic separation (IMS) is a well-known method for the separation and concentration of target bacteria from a large volume of food samples. Magnetic beads functionalized with an antibody provide selectivity for target bacteria such as Escherichia coli O157:H7. Moreover, compared to conventional methods, real-time PCR enables high-sensitivity detection of target bacteria. The method proposed in this study involves three steps: (1) pre-enrichment, (2) automated IMS and concentration of target bacteria, and (3) detection of target bacteria by real-time PCR. Using food samples with a working sample volume as large as 250 mL, the whole process only requires 3 h. As a result, target bacteria in the range of 101–102 colony-forming units per mg or g of sample can be detected in food samples, such as milk, ground beef, and cabbage, by using the proposed approach. We anticipate that the automated IMS system combined with real-time PCR will contribute to the development of a fully automated system for detecting foodborne bacteria and serve as a multi-tester for a variety of bacterial strains in the capacity of a sample-to-answer device in the near future.

Continuous-Flow Separation and Efficient Concentration of Foodborne Bacteria from Large Volume Using Nickel Nanowire Bridge in Microfluidic Chip.

Separation and concentration of target bacteria has become essential to sensitive and accurate detection of foodborne bacteria to ensure food safety. In this study, we developed a bacterial separation system for continuous-flow separation and efficient concentration of foodborne bacteria from large volume using a nickel nanowire (NiNW) bridge in the microfluidic chip. The synthesized NiNWs were first modified with the antibodies against the target bacteria and injected into the microfluidic channel to form the NiNW bridge in the presence of the external arc magnetic field. Then, the large volume of bacterial sample was continuous-flow injected to the channel, resulting in specific capture of the target bacteria by the antibodies on the NiNW bridge to form the NiNW–bacteria complexes. Finally, these complexes were flushed out of the channel and concentrated in a lower volume of buffer solution, after the magnetic field was removed. This bacterial separation system was able to separate up to 74% of target bacteria from 10 mL of bacterial sample at low concentrations of ≤102 CFU/mL in 3 h, and has the potential to separate other pathogenic bacteria from large volumes of food samples by changing the antibodies.

A microfluidic signal-off biosensor for rapid and sensitive detection of Salmonella using magnetic separation and enzymatic catalysis

The key to prevent and control the spread of foodborne diseases is rapid screening and early warning of pathogenic bacteria in foods. In this study, a microfluidic biosensor using magnetic nanoparticles (MNPs) and catalases was developed for rapid and sensitive detection of Salmonella typhimurium. First, the MNPs modified with anti-Salmonella monoclonal antibodies were used to separate and enrich target bacteria from sample background to form magnetic bacteria, which were reacted with polystyrene microspheres (PSs) modified with anti-Salmonella polyclonal antibodies and catalases to form enzymatic bacteria. Then, the enzymatic bacteria were injected into the capillary in microfluidic chip and captured by high gradient magnetic field. After washing to remove unbound PSs, hydrogen peroxide was finally injected and catalyzed by the catalases on enzymatic bacteria to produce oxygen gap in the capillary, leading to electrical signal-off. The change of electrical voltage was found to have a good linear relationship with the concentration of target bacteria from 3.7 × 101 to 3.7 × 106 CFU/mL. The proposed biosensor was able to detect Salmonella as low as 33 CFU/mL within 2 h, and could be further combined with microfluidics to develop a lab-on-a-chip system for in-field detection of foodborne bacteria to ensure food safety.

Self-assembly kinetics of debranched short-chain glucans from waxy maize starch to form spherical microparticles and its applications.

Starch microparticles (SMPs) of well-defined size and morphology were synthesized through pullulanase-mediated debranching of waxy maize starch followed by spontaneous re-assembly of the resulting short-chain glucan molecules in aqueous solution. Enzymatic debranching of amylopectins from native starch generated two major fractions corresponding to a smaller glucan and partially digested larger amylopectin molecules. The ratio of short-chain glucan (SCG) over partially digested amylopectin (PDAp) turned out to be the deterministic factors for the size and crystallinity of SMPs, of which the ratio could be controlled by the concentration of debranching enzyme. The PDAp fraction was closely associated with the creation of nuclei, determining the growth kinetics of SMPs which led to the formation of SMPs with a diameter ranging from 0.52.5 μm. In addition, we demonstrated that iron oxide nanoparticles (IONPs) were successfully incorporated into the starch microstructure by introducing them during the self-assembly reaction, conferring desired functionality onto the final SMPs. The incorporated IONPs rendered the SMPs an excellent magnetic sensitivity, which were successfully applied for the separation and concentration of target bacteria upon conjugation of specific antibody on the surface of SMPs. The simple processes and biocompatible nature of starch would make this approach attractive for many applications in the area of food, medicine and other related materials sciences.

Large-volume immunomagnetic separation combined with multiplex PCR assay for simultaneous detection of Listeria monocytogenes and Listeria ivanovii in lettuce

Multiplex polymerase chain reaction (mPCR) has been widely used for the simultaneous detection of various target bacteria in vegetables. However, an enrichment period is necessary to improve the sensitivity of the mPCR method. In this paper, large-volume (10 mL) immunomagnetic separation (IMS) combined with mPCR for the rapid detection of Listeria monocytogenes and Listeria ivanovii in lettuce without further enrichment process is reported for the first time. Various parameters that affected the capture efficiency (CE) of IMS, including the amounts of streptavidin and biotinylated anti-Listeria monoclonal antibodies on the surface of magnetic nanobeads, the amount of immunomagnetic beads, immunoreaction time, and magnetic separation time, were systematically investigated. Moreover, the concentrations of primers, PCR conditions, and genomic DNA isolation for mPCR assay were optimized. Under optimum conditions, the CE of large-volume IMS for L. monocytogenes and L. ivanovii was greater than 90% when the concentration of target bacteria was less than 106 CFU/mL in pure culture, and was more than 80% when the concentration was below 105 CFU/mL in lettuce samples. The limit of detection of IMS combined with mPCR assay reached as low as 1.0 CFU/mL in pure culture and 10 CFU/g in lettuce. The overall assay time, including sample preparation, large-volume IMS, and mPCR assay, took less than 7 h. In summary, the developed large-volume IMS-based mPCR system exhibits great potential for routine screening detection of foodborne pathogenic bacteria for safety monitoring.

A label-free, microfluidics and interdigitated array microelectrode-based impedance biosensor in combination with nanoparticles immunoseparation for detection of Escherichia coli O157:H7 in food samples

Abstract A microfluidic flow cell with embedded gold interdigitated array microelectrode (IDAM) was developed and integrated with magnetic nanoparticle-antibody conjugates (MNAC) into an impedance biosensor to rapidly detect pathogenic bacteria in ground beef samples. The flow cell consisting of a detection microchamber and inlet and outlet microchannels was fabricated by bonding an IDAM chip to a poly(dimethylsiloxane) (PDMS) microchannel. The detection microchamber with a dimension of 6 mm × 0.5 mm × 0.02 mm and a volume of 60 nL was used to collect bacterial cells in the active layer above the microelectrode for sensitive impedance change. MNAC were prepared by conjugating streptavidin-coated magnetic nanoparticles with biotin-labeled polyclonal goat anti-E. coli antibodies and were used in the separation and concentration of target bacteria. The cells of E. coli O157:H7 inoculated in a food sample were first captured by the MNAC, separated, and concentrated by applying a magnetic field, washed, and then suspended in mannitol solution and finally injected through the microfluidic flow cell for impedance measurement. This impedance biosensor was able to detect as low as 1.6 × 102 and 1.2 × 103 cells of E. coli O157:H7 cells present in pure culture and ground beef sample, respectively. The total detection time from sampling to measurement was 35 min. Equivalent circuit analysis indicated that the bulk medium resistance, double layer capacitance, and dielectric capacitance were responsible for the impedance change due to the presence of E. coli O157:H7 cells on the surface of IDAM. Sample pre-enrichment, secondary antibodies on the microelectrode surface, and redox probes were not required in this impedance biosensor.