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.     

Fluorescence Polarization Based Nucleic Acid Testing for Rapid and Cost‐Effective Diagnosis of Infectious Disease

A new nucleic acid detection method was developed for a rapid and cost‐effective diagnosis of infectious disease. This approach relies on the three unique elements: 1) detection probes that regulate DNA polymerase activity in response to the complementary target DNA; 2) universal reporters conjugated with a single fluorophore; and 3) fluorescence polarization (FP) detection. As a proof‐of‐concept, the assay was used to detect and sub‐type Salmonella bacteria with sensitivities down to a single bacterium in less than three hours.     

Rapid Colorimetric Identification and Targeted Photothermal Lysis of Salmonella Bacteria by Using Bioconjugated Oval‐Shaped Gold Nanoparticles

Salmonella bacteria are the major cause for the infection of 16 million people worldwide with typhoid fever each year. Antibiotic‐resistant Salmonella strains have been isolated from various food products. As a result, the development of ultrasensitive sensing technology for detection and new approaches for the treatment of infectious bacterial pathogens that do not rely on traditional therapeutic regimes is very urgent for public health, food safety, and the world economy. Driven by this need, we report herein a nanotechnology‐driven approach that uses antibody‐conjugated oval‐shaped gold nanoparticles to selectively target and destroy pathogenic bacteria. Our experiments have shown the use of a simple colorimetric assay, with an anti‐salmonella antibody conjugated to oval‐shaped gold nanoparticles, for the label‐free detection of S. typhimurium with an excellent detection limit (10⁴ bacteria per mL) and high selectivity over other pathogens. When bacteria conjugated to oval‐shaped gold nanoparticles were exposed to near‐infrared radiation, a highly significant reduction in bacterial cell viability was observed due to photothermal lysis. Ideally, this nanotechnology‐based assay would have enormous potential for rapid, on‐site pathogen detection to avoid the distribution of contaminated foods.     

A sensitive liquid chromatography/mass spectrometry‐based assay for quantitation of amino‐containing moieties in lipid A

A novel sensitive liquid chromatography/mass spectrometry‐based assay was developed for the quantitation of aminosugars, including 2‐amino‐2‐deoxyglucose (glucosamine, GlcN), 2‐amino‐2‐deoxygalactose (galactosamine, GalN), and 4‐amino‐4‐deoxyarabinose (aminoarabinose, AraN), and for ethanolamine (EtN), present in lipid A. This assay enables the identification and quantitation of all amino‐containing moieties present in lipopolysaccharide or lipid A from a single sample. The method was applied to the analysis of lipid A (endotoxin) isolated from a variety of biosynthetic and regulatory mutants of Salmonella enterica serovar Typhimurium and Francisella tularensis subspecies novicida. Lipid A is treated with trifluoroacetic acid to liberate and deacetylate individual aminosugars and mass tagged with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate, which reacts with primary and secondary amines. The derivatives are separated using reversed‐phase chromatography and analyzed using a single quadrupole mass spectrometer to detect quantities as small as 20 fmol. GalN was detected only in Francisella and AraN only in Salmonella, while GlcN was detected in lipid A samples from both species of bacteria. Additionally, we found an approximately 10‐fold increase in the level of AraN in lipid A isolated from Salmonella grown in magnesium‐limited versus magnesium‐replete conditions. Salmonella with defined mutations in lipid A synthesis and regulatory genes were used to further validate the assay. Salmonella with null mutations in the phoP, pmrE, and prmF genes were unable to add AraN to their lipid A, while Salmonella with constitutively active phoP and pmrA exhibited AraN modification of lipid A even in the normally repressive magnesium‐replete growth condition. The described assay produces excellent repeatability and reproducibility for the detection of amino‐containing moieties in lipid A from a variety of bacterial sources. Copyright © 2009 John Wiley & Sons, Ltd.     

Label‐Free and Ultra‐Low Level Detection of Salmonella enterica Serovar Typhimurium Using Electrochemical Impedance Spectroscopy

An immunosensor for rapid and low level detection of the bacterial pathogen Salmonella enterica Serovar Typhimurium was designed and developed based upon label‐free electrochemical impedance spectroscopy and correlated to viable cell counts. The immunosensor was fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode by immobilizing monoclonal antibody (MAb) specific against Salmonella typhimurium cell surface lipopolysaccharide (LPS) onto the surface of the electrode. Use of mass‐fabricated and electroplated PCB electrodes allowed for disposable, highly sensitive, and rapid detection of Salmonella in an aqueous environment. Results demonstrate that in purified solution, Salmonella can be detected as low as 10 CFU in a 100 μL volume and label‐free and rapid manner in fewer than 90 s. The cost effective approach described here can be used for detection of pathogens with relevance for healthcare, food, and environmental applications.     

Engineering the Stereochemistry of Cephalosporin for Specific Detection of Pathogenic Carbapenemase‐Expressing Bacteria

Reported herein is the design of fluorogenic probes specific for carbapenem‐resistant Enterobacteriaceae (CRE) and they were designed based on stereochemically modified cephalosporin having a 6,7‐trans configuration. Through experiments using recombinant β‐lactamase enzymes and live bacterial species, these probes demonstrate the potential for use in the specific detection of carbapenemases, including metallo‐β‐lactamases in active bacterial pathogens.     

Development of a filtration-based SERS mapping platform for specific screening of Salmonella enterica serovar Enteritidis

The presence of Salmonella in natural freshwater and drinking water is a leading cause of intestinal illness all over the world; thus, the detection of Salmonella in water is of great importance to public health. The objective of this study is to develop a rapid screening method for the detection of Salmonella enterica serovar Enteritidis in water involving surface-enhanced Raman spectroscopy (SERS), aptamers, and filtration. SERS offers a great alternative to traditional methods of pathogen detection, with a simplified detection assay and shortened detection time. The specific capturing and labeling of Salmonella Enteritidis are realized by a specific single-stranded DNA aptamer, which is modified with an additional chain of adenine and fluorescein (FAM) and used as presence/absence indicator of Salmonella Enteritidis. By incorporating a vacuum filtration system, bacterial cells recognized by the specific aptamer are concentrated onto a membrane. With additional filtration of gold nanoparticles, the aptamer signals were captured and used to construct a SERS mapping indicating the presence and absence of target bacterial strains with potential quantitative capability. The specificity of the method was validated by using other strains of bacteria such as Escherichia coli and Listeria monocytogenes. The sensitivity of the method goes down to 10³ CFU/mL for 1 mL of sample with a total detection and analyzing time within 3 h. This study demonstrates the capability of the filtration-based SERS platform for detecting Salmonella Enteritidis in various aqueous matrices such as distilled water and rinsing water from fresh produce with high selectivity and sensitivity.

Graphical abstract

     

A Highly Efficient Chemiluminescence Probe for the Detection of Singlet Oxygen in Living Cells

Singlet oxygen is among the reactive oxygen species (ROS) with the shortest life‐times in aqueous media because of its extremely high reactivity. Therefore, designing sensors for detection of ¹O₂ is perhaps one of the most challenging tasks in the field of molecular probes. Herein, we report a highly selective and sensitive chemiluminescence probe ( SOCL‐CPP ) for the detection of ¹O₂ in living cells. The probe reacts with ¹O₂ to form a dioxetane that spontaneously decomposes under physiological conditions through a chemiexcitation pathway to emit green light with extraordinary intensity. SOCL‐CPP demonstrated promising ability to detect and image intracellular ¹O₂ produced by a photosensitizer in HeLa cells during photodynamic therapy (PDT) mode of action. Our findings make SOCL‐CPP the most effective known chemiluminescence probe for the detection of ¹O₂. We anticipate that our chemiluminescence probe for ¹O₂ imaging would be useful in PDT‐related applications and for monitoring ¹O₂ endogenously generated by cells in response to different stimuli.     

Development and evaluation of a novel nucleic acid sequence-based amplification method using one specific primer and one degenerate primer for simultaneous detection of Salmonella Enteritidis and Salmonella Typhimurium

Salmonella Enteritidis and Salmonella Typhimurium are the most widespread causes of salmonellosis and gastrointestinal diseases worldwide. Thus, their simple and sensitive detection is significantly important in biosafety and point-of-care diagnostics. In that regard, although present nucleic acid-based attempts are mainly focused on the detection methods encompassing all Salmonella enterica members in a single reaction, serotypes other than S. Enteritidis and S. Typhimurium are clinically and epidemiologically rare to humans. Therefore, regarding high ribosomal RNA (rRNA) copy numbers in a cell, isothermal nucleic acid sequence-based amplification (NASBA) technique was employed for simple, sensitive and simultaneous detection of the bacteria. However, due to high sequence homology among 16S rRNA genes and consequently, very few specific regions, we developed a novel NASBA method called “single specific primer-NASBA or SSP-NASBA” in which the specificity of the antisense primer is sufficient to perform a specific NASBA reaction. Accordingly, we designed highly specific NASBA antisense and degenerate sense primers for a segment of 16S rRNA variable region by universal sequence alignment to simultaneously detect S. Enteritidis and S. Typhimurium. Meanwhile, the approach was successfully evaluated for various Salmonella as well as closely related non-Salmonella serovars. Specific and simultaneous detection of both bacteria was achieved with the designed primer set in a single reaction environment with a detection limit of less than 10 CFUs mL⁻¹. The developed NASBA assay should facilitate the overall process and provide a simple, fast, specific and sensitive approach for molecular diagnostics of pathogens under various circumstances, e.g. outbreaks.     

Popcorn‐Shaped Magnetic Core–Plasmonic Shell Multifunctional Nanoparticles for the Targeted Magnetic Separation and Enrichment,Label‐Free SERS Imaging,and Photothermal Destruction of Multidrug‐Resistant Bacteria

Over the last few years, one of the most important and complex problems facing our society is treating infectious diseases caused by multidrug‐resistant bacteria (MDRB), by using current market‐existing antibiotics. Driven by this need, we report for the first time the development of the multifunctional popcorn‐shaped iron magnetic core–gold plasmonic shell nanotechnology‐driven approach for targeted magnetic separation and enrichment, label‐free surface‐enhanced Raman spectroscopy (SERS) detection, and the selective photothermal destruction of MDR Salmonella DT104. Due to the presence of the “lightning‐rod effect”, the core–shell popcorn‐shaped gold‐nanoparticle tips provided a huge field of SERS enhancement. The experimental data show that the M3038 antibody‐conjugated nanoparticles can be used for targeted separation and SERS imaging of MDR Salmonella DT104. A targeted photothermal‐lysis experiment, by using 670 nm light at 1.5 W cm^?2 for 10 min, results in selective and irreparable cellular‐damage to MDR Salmonella. We discuss the possible mechanism and operating principle for the targeted separation, label‐free SERS imaging, and photothermal destruction of MDRB by using the popcorn‐shaped magnetic/plasmonic nanotechnology.     

Stopped-flow microarray immunoassay for detection of viable <Emphasis Type="Italic">E. coli</Emphasis> by use of chemiluminescence flow-through microarrays

Rapid multiplexed analysis of microorganisms is important in water analysis to control bacterial contamination for health and safety reasons. Direct quantification of bacteria by means of flow-through microarray immunoassays requires new analysis strategies for optimising sensitivity and the analysis time. For bacteria and for particles, hydrodynamic forces and sedimentation are the dominating effects for binding on surfaces in a flow-through system, whereas diffusion is insignificant. Therefore, we have implemented a stop and flow technique for quantification of viable E. coli cells. The method, with alternation of resting volume elements and pumping the elements forward, was more effective than continuous-flow approaches for analysis of bacteria. For quantification of viable E. coli cells, a chemiluminescence sandwich immunoassay test format was performed by means of antibody microarrays and flow-injection-based microarray analysis. Antibodies, which served as selective capture molecules, were immobilised on polymer-modified glass surfaces serving as microarray substrate. For the bacteria recognition step, a second detection antibody was used, forming a sandwich immunoassay at each spot of the microarray. Detection was carried out with a horseradish peroxidase catalysed chemiluminescence reaction. All assay steps were conducted with an automated flow-through chemiluminescence microarray readout system. Living E. coli cells could be detected in 67 min with a detection limit of 4 × 10⁵ cells mL⁻¹. By introduction of the stopped-flow technique and optimisation of interaction time and interaction steps the achieved detection of E. coli was faster and two orders of magnitude more sensitive than with a conventional ELISA technique in microplates.     

Pathogen detection in complex samples by quartz crystal microbalance sensor coupled to aptamer functionalized core–shell type magnetic separation

A quartz crystal microbalance sensor (QCM) was developed for sensitive and specific detection of Salmonella enterica serovar typhimurium cells in food samples by integrating a magnetic bead purification system. Although many sensor formats based on bioaffinity agents have been developed for sensitive and specific detection of bacterial cells, the development of robust sensor applications for food samples remained a challenging issue. A viable strategy would be to integrate QCM to a pre-purification system. Here, we report a novel and sensitive high throughput strategy which combines an aptamer-based magnetic separation system for rapid enrichment of target pathogens and a QCM analysis for specific and real-time monitoring. As a proof-of-concept study, the integration of Salmonella binding aptamer immobilized magnetic beads to the aptamer-based QCM system was reported in order to develop a method for selective detection of Salmonella. Since our magnetic separation system can efficiently capture cells in a relatively short processing time (less than 10 min), feeding captured bacteria to a QCM flow cell system showed specific detection of Salmonella cells at 100 CFU mL⁻¹ from model food sample (i.e., milk). Subsequent treatment of the QCM crystal surface with NaOH solution regenerated the aptamer-sensor allowing each crystal to be used several times.     

Interaction of bacteria and ion‐exchange particles and its potential in separation for matrix‐assisted laser desorption/ionization mass spectrometric identification of bacteria in water

Identification of microbial contaminants in drinking water is a challenge to matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) due to low levels of microorganisms in fresh water. To avoid the time‐consuming culture step of obtaining enough microbial cells for subsequent MALDI‐MS analysis, a combination of membrane filtration and nanoparticles‐ or microparticles‐based magnetic separation is a fast and efficient approach. In this work, the interaction of bacteria and fluidMAG‐PAA, a cation‐exchange superparamagnetic nanomaterial, was investigated by MALDI‐MS analysis and transmission electron microscopy. FluidMAG‐PAA selectively captured cells of Salmonella, Bacillus, Enterococcus and Staphylococcus aureus. This capture was attributed to the aggregation of negatively charged nanoparticles on bacterial cell regional surfaces that bear positive charges. Three types of non‐porous silica‐encapsulated anion‐exchange magnetic microparticles (SiMAG‐Q, SiMAG‐PEI, SiMAG‐DEAE) were capable of concentrating a variety of bacteria, and were compared with silica‐free, smaller fluidMAG particles. Salmonella, Escherichia coli, Enterococcus and other bacteria spiked in aqueous solutions, tap water and reservoir water were separated and concentrated by membrane filtration and magnetic separation based on these ion‐exchange magnetic materials, and then characterized by whole cell MALDI‐MS. By comparing with the mass spectra of the isolates and pure cells, bacteria in fresh water can be rapidly detected at 1 × 10³ colony‐forming units (cfu)/mL. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid separation and immunoassay for low levels of Salmonella in foods using magnetosome-antibody complex and real-time fluorescence quantitative PCR

A rapid and economical method for detecting Salmonella was developed, based on a novel complex for immunomagnetic separation, which was composed of anti‐Salmonella polyclonal antibody (Ab) and magnetosome (bacterial magnetic particle, BMP) produced by the bacterium Magnetospirillum gryphiswaldense MSR‐1. BMP‐Ab complex was used to capture Salmonella from pure suspensions of S. dublin, S. enteritidis, S. aesch, S. agona, S. abony and S. bareily, from mixed suspensions of S. dublin and Vibrio parahaemolyticus, and from artificially contaminated food samples. Captured Salmonella were then detected by plate count, or real‐time fluorescence quantitative PCR. Capture efficiencies, calculated from plate count, were >80% for the pure Salmonella suspensions of all six strains, and >70% for the mixed suspension. Samples of six food products, with artificial contamination by 6000, 600, 60, or 0.6 cfu/mL S. dublin, were captured by complex and detected by real‐time fluorescence quantitative PCR. Threshold cycle values varied depending on type of food. The lower limit of detectability was 60 cfu/mL without pre‐enrichment, and <0.6 cfu/mL after 3‐h pre‐enrichment. The method described here, based on capture pathogens by BMP‐Ab complex, is sensitive, rapid, and considerably simpler than traditional methods for Salmonella detection. It can be extended to other pathogens by the use of appropriate antibodies.     

An AIEgen‐Peptide Conjugate as a Phototheranostic Agent for Phagosome‐Entrapped Bacteria

The detection and elimination of intracellular bacteria remain a major challenge. In this work, we report an aggregation‐induced emission (AIE) bioprobe that can detect bacterial infection and kill bacteria surviving inside macrophages through a dynamic process, notably specific molecular tailoring of the probe by caspase‐1 activation in infected macrophages and accumulation of the residue on phagosomes containing bacteria, leading to light‐up fluorescent signals. Moreover, the AIEgen can serve as a photosensitizer for generation of reactive oxygen species (ROS); and the average ROS indicator fluorescent signal intensity per unit area in the bacterial phagosomes is approximately 2.7‐fold higher than that in the cytoplasm. This, in turn, induces bacteria killing with high efficiency and minimal cytotoxicity towards macrophages. We envision that this specific light‐up bioprobe may provide a new approach for selective and sensitive detection and eradication of intracellular bacterial infections.     

Gold Nanoparticles as Dual Functional Sensor to Detect E.coliDH5α as a Model for Gram‐negative Bacteria

4‐aminothiophenol‐modified gold nanoparticles (PATP‐AuNPs) were used as colorimetric and Surface Enhanced Raman Scattering (SERS) probes for the sensitive detection of Escherichia coliDH5α, as a model for Gram‐negative bacteria. The nano‐probes were easy to prepare through Au‐S bonding. Under optimized conditions, the PATP‐AuNPs surface with positive charge can bind with negatively charged E.coliDH5α via electrostatic adhesion, resulting in a quick color change from red to blue, and also a dramatic SERS signal enhancement from thousands of AuNPs aggregated on the surface of bacteria, which was utilized for both colorimetric and SERS detection of E.coliDH5α. For colorimetric analysis, it is the first time that the classical partial least square (PLS) regression was utilized to deal with the relationship between adsorption and E.coliDH5α concentrations. Excellent linear relationship was observed from 1.1 x 10⁷ to 1.3 x 10⁸ cfu mL^‐1 with the average relative error (ARE) of 5.430, which was more accurate than the traditional extinction ration method. When coupled with confocal Raman microscope, this PATP‐AuNPs probes could be used to detection SERS signals produced from even one single bacterium. This bioassay is rapid, less expensive and convenient for bacteria detection and analysis. Therefore, PATP‐AuNPs system as a novel, versatile, on‐site and real‐time Gram‐negative bacteria sensor, would have a wide range of practical applications.     

Fluorescent Triazole Urea Activity‐Based Probes for the Single‐Cell Phenotypic Characterization of Staphylococcus aureus

Phenotypically distinct cellular (sub)populations are clinically relevant for the virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Herein, we introduce fluorescent activity‐based probes as chemical tools for the single‐cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3‐triazole urea library to identify selective inhibitors of fluorophosphonate‐binding serine hydrolases and lipases in S. aureus and synthesized target‐selective activity‐based probes. Molecular imaging and activity‐based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single‐cell phenotypic heterogeneity. We propose the labeling of enzymatic activities by chemical probes as a generalizable method for the phenotyping of bacterial cells at the population and single‐cell level.     

Multiplex identification of sepsis‐causing Gram‐negative pathogens from the plasma of infected blood

Early and accurate detection of bacterial pathogens in the blood is the most crucial step for sepsis management. Gram‐negative bacteria are the most common organisms causing severe sepsis and responsible for high morbidity and mortality. We aimed to develop a method for rapid multiplex identification of clinically important Gram‐negative pathogens and also validated whether our system can identify Gram‐negative pathogens with the cell‐free plasm DNA from infected blood. We designed five MLPA probe sets targeting the genes specific to major Gram‐negative pathogens (uidA and lacY for E. coli, ompA for A. baumannii, phoE for K. pneumoniae, and ecfX for P. aeruginosa) and one set targeting the CTX‐M group 1 to identify the ESBL producing Gram‐negative pathogens. All six target‐specific peaks were clearly separated without any non‐specific peaks in a multiplex reaction condition. The minimum detection limit was 100 fg of pathogen DNA. When we tested 28 Gram‐negative clinical isolates, all of them were successfully identified without any non‐specific peaks. To evaluate the clinical applicability, we tested seven blood samples from febrile patients. Three blood culture positive cases showed E. coli specific peaks, while no peak was detected in the other four culture negative samples. This technology can be useful for detection of major sepsis‐causing, drug‐resistant Gram‐negative pathogens and also the major ESBL producing Gram‐negatives from the blood of sepsis patients in a clinical setting. This system can help early initiation of effective antimicrobial treatment against Gram‐negative pathogens for sepsis patients, which is very crucial for better treatment outcomes.     

Differential characterization of biogenic amine‐producing bacteria involved in food poisoning using MALDI‐TOF mass fingerprinting

Histamine poisoning is caused by the consumption of fish and other foods that harbor bacteria possessing histidine decarboxylase activity. With the aim of preventing histamine formation, highly specific mass spectral fingerprints were obtained from the 16 major biogenic amine‐producing enteric and marine bacteria by means of MALDI‐TOF MS analysis. All bacterial strains analyzed exhibited specific spectral fingerprints that enabled its unambiguous differentiation. This technique also identified peaks common to certain bacterial groups. Thus, two protein peaks at m/z 4182±1 and 8363±6 were found to be present in all Enterobacteriaceae species analyzed except for Morganella morganii. Peaks at m/z 3635±1 and 7267±2 were specific to both M. morganii and Proteus spp. Biogenic amine‐forming Proteus spp. exhibited three genus‐specific peaks at m/z 3980, 7960±1 and 9584±2. The genus Photobacterium also showed three genus‐specific peaks at m/z 2980±1, 4275±1 and 6578±1. The two histamine‐producing Gram‐positive bacteria Lactobacillus sp. 30A and Staphylococcus xylosus exhibited a few protein peaks in the 2000–7000 m/z range and could be easily distinguished from biogenic amine‐forming Gram‐negative bacteria. Clustering based on MALDI‐TOF MS also exhibited a good correlation with phylogenetic analysis based on the 16S rRNA gene sequence, validating the ability of the MALDI‐TOF technique to establish relationships between microbial strains and species. The approach described in this study leads the way toward the rapid and specific identification of major biogenic amine‐forming bacteria based on molecular protein markers with a goal to the timely prevention of histamine food poisoning.