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Abstract

Development and validation of a mammalian cell-based biosensor for application in food defense and food safety was investigated. Three prototypes of the biosensor capable of handling different sample types were developed and tested with food and beverages. The sensing element is a B lymphocyte Ped-2E9 cell-line, encapsulated in collagen matrix in 3D scaffold. The uniqueness of this biosensor is that it detects analyte interaction with mammalian cells and is able to distinguish pathogenic from non-pathogenic and active from inactive toxins, rendering accurate estimation of the risk associated with the agents. This sensor gave positive signal for a broad range of bacterial pathogens; Listeria monocytogenes, enterotoxigenic Bacillus, Vibrio, Micrococcus and Serratia, and toxins; α-hemolysin from Staphylococcus aureus, phospholipase C from Clostridium perfringens, cytolysin from sea anemone Stoichactis helianthus, listeriolysin O from L. monocytogenes, and enterotoxin from Bacillus. Detection limit for toxins was 10-40 ng in 2 h while for a model bacterial pathogen, L. monocytogenes, 10(3)-10(4) CFU/ml in 4-6 h, even in the presence of a mixture of higher concentrations of non-pathogenic species of the same genera or common background microflora. With inoculated food and beverage, the sensor detected L. monocytogenes and Bacillus cereus at a low initial concentration of 10(2)-10(4) CFU/g from ready-to-eat meat and rice, and only active toxins at nanogram quantities from rice, milk and water samples. Though all the three prototypes performed well with beverages, Devices II & III are most suitable for testing particulate foods. These data present promising evidence for possible application of this biosensor for rapid detection of multiple pathogens or toxins for food defense and food safety application.

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... A solution can be found in cell-based detection systems which are emerging biosensor technologies that detect the biological activity of pathogens or toxins. Those biosensors have mammalian cells as sensing elements and allow monitoring perturbations in cell physiological activities following exposure [80]. Cell-based biosensors are capable of detecting the presence of pathogens or active toxin in clinical, environmental and food samples [2] rendering accurate estimation of the risk associated with the agent identification. ...
... Cell-based biosensors are capable of detecting the presence of pathogens or active toxin in clinical, environmental and food samples [2] rendering accurate estimation of the risk associated with the agent identification. Different device designs are proposed from a 96-well plate to modified electrodes carrying mammalian cells [80]. ...
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Infectious animal diseases caused by pathogenic microorganisms such as bacteria and viruses threaten the health and well-being of wildlife, livestock, and human populations, limit productivity and increase significantly economic losses to each sector. The pathogen detection is an important step for the diagnostics, successful treatment of animal infection diseases and control management in farms and field conditions. Current techniques employed to diagnose pathogens in livestock and poultry include classical plate-based methods and conventional biochemical methods as enzyme-linked immunosorbent assays (ELISA). These methods are time-consuming and frequently incapable to distinguish between low and highly pathogenic strains. Molecular techniques such as polymerase chain reaction (PCR) and real time PCR (RT-PCR) have also been proposed to be used to diagnose and identify relevant infectious disease in animals. However these DNA-based methodologies need isolated genetic materials and sophisticated instruments, being not suitable for in field analysis. Consequently, there is strong interest for developing new swift point-of-care biosensing systems for early detection of animal diseases with high sensitivity and specificity. In this review, we provide an overview of the innovative biosensing systems that can be applied for livestock pathogen detection. Different sensing strategies based on DNA receptors, glycan, aptamers and antibodies are presented. Besides devices still at development level some are validated according to standards of the World Organization for Animal Health and are commercially available. Especially, paper-based platforms proposed as an affordable, rapid and easy to perform sensing systems for implementation in field condition are included in this review.
... However, the most effective way to exploit the potential of CBBS is to use appropriate cell types in an array format (Figure 3) that are most sensitive to a group of pathogens/toxins. The use of a mouse lymphocyte cell line was able to detect a broad range of toxins from Listeria monocytogenes, Bacillus spp., Staphylococcus aureus, Clostridium perfringens, and sea anemone or Stoichactis helianthus at nanogram quantities within 1-3 hr (Banerjee and Bhunia, 2010). ...
... Importantly, it produces more realistic in vivo type response. The use of a lymphocyte cell line in 3D scaffold on a 96-well micro plate (Figure 3) demonstrates that high-throughput screening of toxins at nanogram quantities and bacteria at 100-1,000 cells/g/ml from foods and beverages is possible (Banerjee and Bhunia, 2010). ...
Article
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Several innovative spectral-and mammalian cell-based techniques offer faster detection of multiple and unknown pathogens in pre-harvest and postharvest products. The cost-effective rapid high-throughput prescreening tools could be used to sort out products that are free of harmful agents and subsequently released for consumption, while the suspect products can be tested with more specific analytical kits. The application of a broadband pathogen screening tool is also sensible when the nature of the contaminants is not well defined, especially in pre-harvest food. The laser-based light-scattering technology, BARDOT, allows realtime identification of bacterial colonies as they grow on a Petri dish. BARDOT requires bacterial growth and the total assay time starting with the food sample may take 12-30 hr, depending on the growth rate. A fully automated BARDOT system with a built-in incubator could screen plates without user intervention and transfer data via the Internet for a decision to be rendered about a product release or hold.
... However, a CBS can generate electrochemical (33,43), colorimetric (95), or fluorescent (90) signals similar to those generated by other types of biosensors. Three-dimensional CBSs are in high demand because cells grow in a three-dimensional matrix that mimics the actual tissue configuration of the mammalian host; therefore, these CBSs can be highly sensitive and accurate (13,14,43,95). ...
... The commonly used approach to monitoring cellular responses is to measure cytotoxicity (12)(13)(14). To and Bhunia (95) proposed a three-dimensional Vero cell-based sensor to detect STEC cells or Shiga toxin. ...
Article
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Foodborne disease outbreaks continue to be a major public health and food safety concern. Testing products promptly can protect consumers from foodborne diseases by ensuring the safety of food before retail distribution. Fast, sensitive, and accurate detection tools are in great demand. Therefore, various approaches have been explored recently to find a more effective way to incorporate antibodies, oligonucleotides, phages, and mammalian cells as signal transducers and analyte recognition probes on biosensor platforms. The ultimate goal is to achieve high specificity and low detection limits (1 to 100 bacterial cells or piconanogram concentrations of toxins). Advancements in mammalian cell–based and bacteriophage-based sensors have produced sensors that detect low levels of pathogens and differentiate live from dead cells. Combinations of biotechnology platforms have increased the practical utility and application of biosensors for detection of foodborne pathogens. However, further rigorous testing of biosensors with complex food matrices is needed to ensure the utility of these sensors for point-of-care needs and outbreak investigations. HIGHLIGHTS
... Two classes of biosensors have been developed to detect bacteria: (i) those which require sample processing (e.g., bacterial lysis) to liberate bacterial components, and (ii) processing-free systems, which target whole bacteria. In the first category, bacterial components such as DNA [11], RNA [20], coagulation factors [12], and exotoxins [21] can be detected. Its main disadvantage is the requirement for sample processing, which increases the time and cost of the analysis. ...
... This is the case of human cells. Mammalian cells express a wide range of proteins and receptors to perceive their environment, which make them appealing bioreceptors to sense bacteria [21,26,27]. Bacteriophages may fall within this category as well. ...
Article
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Bacterial infections represent a serious threat in modern medicine. In particular, biofilm treatment in clinical settings is challenging, as biofilms are very resistant to conventional antibiotic therapy and may spread infecting other tissues. To address this problem, biosensing technologies are emerging as a powerful solution to detect and identify bacterial pathogens at the very early stages of the infection, thus allowing rapid and effective treatments before biofilms are formed. Biosensors typically consist of two main parts, a biorecognition moiety that interacts with the target (i.e., bacteria) and a platform that transduces such interaction into a measurable signal. This review will focus on the development of impedimetric biosensors using antimicrobial peptides (AMPs) as biorecognition elements. AMPs belong to the innate immune system of living organisms and are very effective in interacting with bacterial membranes. They offer unique advantages compared to other classical bioreceptor molecules such as enzymes or antibodies. Moreover, impedance-based sensors allow the development of label-free, rapid, sensitive, specific and cost-effective sensing platforms. In summary, AMPs and impedimetric transducers combine excellent properties to produce robust biosensors for the early detection of bacterial infections.
... Alternative detection methods that are faster, user-friendly, and accurate are in high demand (Bhunia, 2014). Therefore, CBBs have been proposed to serve as a reliable tool for the rapid screening of viable pathogens or active toxins in foods (Ngamwongsatit et al., 2008;Banerjee and Bhunia, 2009;Bhunia, 2011;Ye et al., 2019;To et al., 2020). However, maintaining the viability of mammalian cells outside the laboratory environment is a major challenge thus limits CBB's utility in routine foodborne pathogen testing (Bhunia et al., 1995;Banerjee et al., 2007;Banerjee and Bhunia, 2009;Ye et al., 2019). ...
... Mammalian cell-based assays are highly attractive functional screening tools to assess the presence of viable pathogens or active toxins in near-real-time (Bhunia, 2011(Bhunia, , 2014To et al., 2020). CBB monitors host-hazard interaction (Banerjee and Bhunia, 2009); therefore, non-pathogenic, non-hazardous, dead, or non-toxic agents do not yield false results. ...
Article
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Rapid detection of live pathogens is of paramount importance to ensure food safety. At present, nucleic acid-based polymerase chain reaction and antibody-based lateral flow assays are the primary methods of choice for rapid detection, but these are prone to interference from inhibitors, and resident microbes. Moreover, the positive results may neither assure virulence potential nor viability of the analyte. In contrast, the mammalian cell-based assay detects pathogen interaction with the host cells and is responsive to only live pathogens, but the short shelf-life of the mammalian cells is the major impediment for its widespread application. An innovative approach to prolong the shelf-life of mammalian cells by using formalin was undertaken. Formalin (4% formaldehyde)-fixed human ileocecal adenocarcinoma cell line, HCT-8 on 24-well tissue culture plates was used for the capture of viable pathogens while an antibody was used for specific detection. The specificity of the Mammalian Cell-based ImmunoAssay (MaCIA) was validated with Salmonella enterica serovar Enteritidis and Typhimurium as model pathogens and further confirmed against a panel of 15 S. Enteritidis strains, 8 S. Typhimurium, 11 other Salmonella serovars, and 14 non-Salmonella spp. The total detection time (sample-to-result) of MaCIA with artificially inoculated ground chicken, eggs, milk, and cake mix at 1–10 CFU/25 g was 16–21 h using a traditional enrichment set up but the detection time was shortened to 10–12 h using direct on-cell (MaCIA) enrichment. Formalin-fixed stable cell monolayers in MaCIA provide longer shelf-life (at least 14 weeks) for possible point-of-need deployment and multi-sample testing on a single plate.
... Spores are then produced along with CPE, which is released after lysis of the sporulating cells (Grass et al., 2013). The α, β, ε, and ι toxinencoding gene (cpa or plc, cpb, cpe, and iA) are the key characters used for alternative methods and approaches for C. perfringens determination (Tansuphasiri, 2001;Shimizu et al., 2009;Banerjee and Bhunia, 2010;Yang et al., 2010;Kaneko et al., 2011;Chon et al., 2012;Guran and Oksuztepe, 2013). The potential sources of C. perfringens are meat products, stew, casseroles, and gravy. ...
Chapter
Food regulatory agencies have established strict control programs to avoid pathogen contamination through the food chain. Thus, a large number of samples need to be rapidly and efficiently analyzed with a simple operating procedure. According to these requirements, the detection methods with rapidity, efficiency, high sample throughput, and on-site analytical strategies have been widely developed. Especially, a nanotechnology-based method, called nanosensor, is one of the effective methods to be used as an “efficient alarm” to promptly detect the risk of contamination by pathogens in various foods. Additionally, nanosensors, such as enzyme-, immuno-, microbial-, and DNA-sensors have higher potential to further develop and apply as real-time monitoring devices to improve the food safety control system. This chapter provides a comprehensive review of current developments in application of nanotechnology on food contaminants, focusing specifically on pathogen detection and monitoring. The review includes (1) a brief overview of significant foodborne pathogens and their key properties in relation to detection, (2) discussion of nanosensors/assays developed for detection or monitoring of pathogens which include details of types, method efficiency, and applications, as well as current and future status of the methods.
... Biosensors are rapid, specific, sensitive, reproducible and most of the time user friendly methods for the detection of their targets including pathogens or toxins and other biomolecules [17,18]. Currently available biosensors for fast detection of S. aureus, are usually based on the indirect detection of the organism, based on the amplification of specific nucleic-acid sequences [19,20] or recognition of toxins and other biomolecules excreted by the pathogen under certain conditions [21,22]. Application of these methods are limited due to the fact that, sometimes, there is no correlation between indirect methods and the presence of the active viable pathogen microorganism in the analyzed samples [23]. ...
Article
Staphylococcus aureus (S. aureus) is most common causes of hospital-acquired infections and food-associated disease, also staphylococcal food poisoning (SFP) are known as the one of the most important foodborne illnesses worldwide. Many different methods have been developed for the detection and quantification of S. aureus and Staphylococcus aureus enterotoxins (SEs). But most of these methods are usually time-consuming and need complicated sample preparation steps. Therefore, it is of great interest to develop a rapid, simple, and sensitive detection method. In the last years, aptamers, are used as a new biosensor platform for detection of S. aureus in different samples. In this review, recent advances and applications of optical and electrochemical aptamer-based biosensors for the detection and quantitative of S. aureus and enterotoxins in details have been discussed.
... These biological sensing elements are either integrated within or associated with transducers, which transform physicochemical interaction into a decipherable form with the help of a transduction and electro- mechanical interpretation, depending upon target groups of analytes. A conventional biosensor consists of three components: bioreceptor, transducer, and detector [24][25][26]. The first component, bioreceptor, acts as a prototype for the detection of the analyte. ...
... Molecular imprinting, plastic membrane fabrication, and genetic engineering are used to design biomimetic-based bioreceptors [59]. An entire cell/microorganism of biorecognition, or a specific cellular component, is capable of creating a specific binding, which is used in cellular based bioreceptors [60], and nucleic acid/DNA based bioreceptor forms of the complementarity of adenine:thymine (A:T) and cytosine:guanosine (C:G) pairings inside the DNA, which ensures the specificity of the biorecognition [61,62]. Figure 3 shows schematic of antigen and nucleic-acid based bioreceptor. ...
Article
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Pathogen and toxin-contaminated foods and beverages are a major source of illnesses, even death, and have a significant economic impact worldwide. Human health is always under a potential threat, including from biological warfare, due to these dangerous pathogens. The agricultural and food production chain consists of many steps such as harvesting, handling, processing, packaging, storage, distribution, preparation, and consumption. Each step is susceptible to threats of environmental contamination or failure to safeguard the processes. The production process can be controlled in the food and agricultural sector, where smart sensors can play a major role, ensuring greater food quality and safety by low cost, fast, reliable, and profitable methods of detection. Techniques for the detection of pathogens and toxins may vary in cost, size, and specificity, speed of response, sensitivity, and precision. Smart sensors can detect, analyse and quantify at molecular levels contents of different biological origin and ensure quality of foods against spiking with pesticides, fertilizers, dioxin, modified organisms, anti-nutrients, allergens, drugs and so on. This paper reviews different methodologies to detect pathogens and toxins in foods and beverages.
... The advantages of living cells as recognition element are: sensitivity, they present functional analysis for biochemical agents, very low detection limit (Velusamy et al., 2010) [49] . Banerjee and Bhunia, 2010 [6] developed cell based biosensor for rapid detection of multiple pathogenic bacteria (Listeria monocytogenes, enterotoxigenic Bacillus, Vibrio, Micrococcus and Serratia) and toxin (from Staphylococcus aureus, Clostridium perfringens, Stoichactis helianthus, L. monocytogenes, and Bacillus) in food safety application. ...
Article
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Food safety is becoming increasingly an important public health issue, as food borne diseases present a widespread and growing public health problem in both developed and developing countries. The rapid and precise monitoring and detection of food borne pathogens are some of the most effective ways to control and prevent human food borne infections. Biosensors offer rapid and cost effective method of food borne pathogen detection. It utilizes the unique properties of biological and physical materials to recognize a target molecule and effect transduction of an electronic signal. Varieties of biosensors have been developed, viz., electrochemical (amperometric, potentiometric and impedance), optical (light scattering, fibre optics and SPR) and mass based (piezoelectric and surface acoustic). This article gives an overview of electrochemical biosensors for detection of pathogenic bacteria in the food industry. Electrochemical biosensors have some advantages over other analytical transducing systems, such as the possibility to operate in turbid media, comparable instrumental sensitivity, and possibility of miniaturisation. Basically ectrochemical biosensor can be based on potentiometric, amperometric or impedimetric/conductimetric transducers. In recent years, nanotechnology has emerged as a promising field for solving food safety issues in terms of detecting contaminants. The introduction of nanomaterials into electrochemical sensors makes them suitable to reach lower detection limit, higher sensitivity values and bring novel labelling opportunities including multi detection capabilities.
... A mammalian cell-based biosensor was developed, wherein, the B-lymphocyte Ped-2E9 cell line embedded in a collagen/gel matrix served as the sensing element. This was tested for the detection of L. monocytogenes and its toxin (listeriolysin O) from the artificially inoculated food samples (Banerjee et al. 2008;Banerjee and Bhunia 2010). Such a unique sensor differentiated between pathogens and non-pathogens based on their cytotoxicity. ...
Article
Full-text available
The early detection of Listeria monocytogenes (L. monocytogenes) and understanding the disease burden is of paramount interest. The failure to detect pathogenic bacteria in the food industry may have terrible consequences, and poses deleterious effects on human health. Therefore, integration of methods to detect and trace the route of pathogens along the entire food supply network might facilitate elucidation of the main contamination sources. Recent research interest has been oriented towards the development of rapid and affordable pathogen detection tools/techniques. An innovative and new approach like biosensors has been quite promising in revealing the foodborne pathogens. In spite of the existing knowledge, advanced research is still needed to substantiate the expeditious nature and sensitivity of biosensors for rapid and in situ analysis of foodborne pathogens. This review summarizes recent developments in optical, piezoelectric, cell-based, and electrochemical biosensors for Listeria sp. detection in clinical diagnostics, food analysis, and environmental monitoring, and also lists their drawbacks and advantages.
... Multi-well plates containing Ped-2E9 cells were encapsulated in a collagen matrix and the amount of alkaline phosphatase released from infected cells was measured colorimetrically; the statuses of live or dead cells were confirmed using laser cytometry. This device can measure B. cereus having the concentration of 10 2 -10 4 CFU per g in meat and rice samples P (Banerjee and Bhunia, 2010). ...
Article
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Nanotechnology is one of the key fields for electrochemical, optical, and mass sensitive biosensors. The nanomaterials are used as an efficient catalytic tool for electron transfer, immobilization platforms as well as electroactive and optical labels to improve the biosensing performance in terms of high sensitivity, specificity and stability. Nanomaterials or nanoparticles such as carbon nanotubes, metal nanoparticles (Au, Pt, and Pd etc.), nanowires, quantum dots and hybrid nanocomposites are playing a crucial role during the design and development of various biosensing systems for the foodborne pathogen detection. We will discuss throughout this review on the bacterial pathogens associated with foodborne illness such as toxin-producing Escherichia coli, Staphylococcal enterotoxin B, Botulinum toxins, Salmonella species, Vibrio cholerae, etc.We will also focus on electrochemical (amperometry, impedance, voltammetry), optical (surface plasmon resonance and fluorescence approach) and mass sensitive (quartz crystal microbalance, microcantilever) biosensing techniques for the detection and characterization of foodborne pathogens. In addition, recent advances in the biosensor development based on various bio-probes such as enzymes, nucleic acids, antibodies, aptamers and phage-display peptides shall be discussed. Herein, next-generation approaches explored for the multiplexed detection of food borne pathogens shall also be enlightened.
... Cell-based biosensors have been utilized for cellular physiological analysis, pharmaceutical evaluation, environmental monitoring and medical diagnosis ( Banerjee and Bhunia, 2010;DeBusschere and Kovacs, 2001;Johnstone et al., 2010;Pan et al., 2019;Pancrazio and Borkholder, 1999;Ye et al., 2019). Electrogenic cells or tissues are cultured on top of microelectrode arrays known as MEAS ( Hierlemann et al., 2011). ...
Article
Herein, we describe an electrophysiological based sensor that reproducibly monitors and quantifies in real-time collective migration and the formation of cell-cell junctions by C6 glioma cells seeded on top of electrodes. The signal amplitude and frequency generated by the migrating cells changed over time and these parameters were used to accurately calculate the migration speed. Electrophysiological measurements could also distinguish individual from collective cell migration. The migration of densely packed cells generated strong signals, while dispersed cells showed weak bioelectrical activity. We propose this electrophysiological technique as a cell-based biosensor to gain insight into the mechanisms of cooperative migration of cancer cells. Possible applications include screening for anti-migratory compounds, which may lead to the development of novel strategies for antineoplastic chemotherapy.
... Lactate production in cell culture [3] Cell-based stress [4] Identification of optimal bioprocess parameters [24] Food safety Pathogens [37] Allergens [38] Functional biomaterials Proof-of-concept biomaterial to detect chemical inducers such as IPTG and DAPG [25] Figure 2. Bacterial two-component systems for biosensing: a) The sensor module of the TCS sensor kinase binds to the target resulting in activation of the kinase domain, the response regulator is then phosphorylated by the activated kinase domain to activate the response regulator. The response regulator will then bind to the output promoter (P R ) to generate the response. ...
Article
Full-text available
Cell‐based biosensors offer cheap, portable and simple methods of detecting molecules of interest but have yet to be truly adopted commercially. Issues with their performance and specificity initially slowed the development of cell‐based biosensors. With the development of rational approaches to tune response curves, the performance of biosensors has rapidly improved and there are now many biosensors capable of sensing with the required performance. This has stimulated an increased interest in biosensors and their commercial potential. However the reliability, long term stability and biosecurity of these sensors are still barriers to commercial application and public acceptance. Research into overcoming these issues remains active. Here we present the state‐of‐the‐art tools offered by synthetic biology to allow construction of cell‐based biosensors with customisable performance to meet the real world requirements in terms of sensitivity and dynamic range and discuss the research progress to overcome the challenges in terms of the sensor stability and biosecurity fears.
... [154] Writing uncovered that, cell based sensor have turned into a rising devices for restorative diagnostics (i.e. for example, sickness location), ecological examination, sustenance quality control, synthetic pharmaceutical industry and medications recognition. [155][156][157][158][159][160][161] ...
Article
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Recent advances in developing low-cost and highly efficient biosensors devices which are highly sensitive and specific have opened new scope for detection of sample e.g. drugs, contaminated bio agents ,metabolites, pollutants in atmosphere, and microbial load etc. by converting biochemical signals into measurable physiochemical signals which lead to quantify the amount of sample. Recently nano-biosensors, implanted biosensors and integrated biosensors are playing a critical role in current research area and development techniques. The various types of biosensors such as enzyme-based, tissue-based, immunosensors, and DNA biosensors, thermal and piezoelectric biosensors have been studied here to highlight their consequential applications in countless fields. Basic research is still required to refine the sensing strategies as well as analytical instrumentations and procedures to get new applications in numerous fields. Biosensors and their role in medical science including early stage detection of various diseases include human interleukin-10 causing heart diseases, rapid detection of human papilloma virus, etc. are critical aspects. Fluorescent biosensors play an important role in drug discovery and in cancer. DNA biosensors, based on nucleic acid recognition methods, are being developed towards the assay of rapid, simple and economical testing of genetic and infectious diseases. Moreover, the detection of specific DNA sequence is of importance in various areas of interest including clinical, environmental and food analysis. Biosensor applications are prevalent in the plant biology sector to find out the missing links required in metabolic processes. In this article, we present the basics of biosensing devices which can serve as an introductory part for those who are new to this field.
... In the absence of antibiotics, Clostridium infection incidents increase dramatically which emphasizes on the need for improvement of diagnostic kits and continual surveillance of pathogen occurrence. The main challenge in development of diagnostic kits is that it should be able to differentiate between virulent and non-virulent strains to account for the endemic non-pathogenic strains of bird gut (Banerjee and Bhunia 2010). This led to the emergence of a cell-based detection biosensor (Yoo and Lee 2016) which uses mammalian cell line as the sensing element to monitor perturbations in 146 S. Kaul et al. ...
Chapter
Advancement in lifestyle and exponential population growth have evoked competitiveness and struggle for survival, resulting in the elevated levels of physiological stress that notably shows correlation with the rising health disparities within the population. Sustained level of stress based on environmental factors, gender inequalities, competitiveness and post-traumatic stress disorders (PTSDs) triggers the hypothalamic–pituitary–adrenal axis (HPA) for signalling an abnormal release of cortisol from cortex region of the adrenal gland. Although several biomolecules and hormones are known to be influenced by physiological stress, examining cortisol (a steroid hormone) is observed to be one of the potential clinical strategies to assess the levels of the stress. Cortisol level varies regularly during day–night cycles that eventually regulates circadian rhythm. Free form of cortisol can provide accurate and precise determination of stress and is a biomarker for early diagnosis of disorder; hence real time estimation of cortisol can be beneficial to overcome many health issues. Chromatographic techniques are the conventional technology used for cortisol determination; however they possess several limitations such as bulky and complex system, multi-step lengthy and expensive extraction and purification process as well as high limit of detection leading to superficial information. Nowadays, multiple detection techniques have been discovered which consist of high sensitivity, require less or no sample preparation, miniaturization, rapid quantification and easy to use with minimal limitations. Electrochemical immunosensors and bioelectronics integrated with microfluidic platforms started gaining attention recently due to their non-invasive, quick responsive, highly sensitive and portable nature with wearable features. Considering the testing devices either reported in the literature or available for clinical practices, there still remains some improvements and scope to develop miniaturized and wearable point-of-care diagnostics that may exhibit increased sensitivity performance, simple design and rapid fabrication. This book chapter attempts to highlight information regarding cortisol detection sources in the body, the available sensing techniques and the diagnostic devices. In addition, we focus on recent advancements in the biosensing strategies for cortisol detection in particular using microfluidic technology.
... Commonly used food additives are nitrite, benzoic acid, monosodium glutamate (MSG), propyl gallate, and food colorants, which are hazardous and undesirable if used beyond the maximum permissible limits. Batra et al. [96] developed an amperometric biosensor and enzyme biosensor (glutamate dehydrogenase and glutamate oxidase) to detect the presence of excessive amounts of MSG in food, which can pose a significant health risk to consumers. Wang et al. [97] discovered a biosensor for the determination of food colorants, especially Amaranth (E123) and Ponceau 4R (E124) in processed food products, based on the use of carbon nanotubes and a polypyrrole (ppy-) composite modified electrode. ...
Article
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The meat industry requires prompt and effective control measures to guarantee the quality and safety of its products and to avert the incidence of foodborne illnesses and disease outbreaks. Although standard microbiological methods and conventional analytical techniques are employed to monitor the quality and safety, these procedures are tedious and time-consuming, require skilled technicians, and sophisticated instruments. Therefore, there is an urgent need to develop simple, fast, and user-friendly hand-held devices for real-time monitoring of the quality of meat and meat products in the supply chain. Biosensors and chemical indicators, due to their high sensitivity, specificity, reproducibility, and stability, are emerging as promising tools and have the potential for monitoring and controlling the quality (freshness and sensory traits such as tenderness) and safety (metabolites, contaminants, pathogens, drug residues, etc.) of muscle foods. In this review, the application of biosensors in the meat industry and their emerging role in the quantification of key meat quality components are discussed. Furthermore, the role of different biosensors to identify and detect contaminants, adulterants, pathogens, antibiotics, and drug residues in meat and meat products is also summarized.
... In the absence of antibiotics, Clostridium infection incidents increase dramatically which emphasizes on the need for improvement of diagnostic kits and continual surveillance of pathogen occurrence. The main challenge in development of diagnostic kits is that it should be able to differentiate between virulent and non-virulent strains to account for the endemic non-pathogenic strains of bird gut (Banerjee and Bhunia 2010). This led to the emergence of a cell-based detection biosensor (Yoo and Lee 2016) which uses mammalian cell line as the sensing element to monitor perturbations in 146 S. Kaul et al. ...
Chapter
The development of biosensors and bioassays has focused recently on small, easy to use in a decentralized and portable manner and low-cost analytical devices for sensing and quantitative determination of biomarkers. Biosensors provide numerous advantages over other analytical techniques, including high selectivity and sensitivity, portability, miniaturization and possibility of on-site monitoring. Immunosensors were heavily used during the last decades, since they combine the specificity of the immune reaction between antibody and its specific antigen with the sensitivity provided by the detection platform. More recently, aptamers started to be used for the design of biosensors due to their improved characteristic compared to the antibody-based sensors. In this chapter an overview of the latest trends in electrochemical sensors design for diagnosis purposes of several diseases, including cancer, cardiovascular and neurological disorders, is presented.
... Ces protéines pourraient tout simplement être utilisées pour le transport d'un produit chimique d'un endroit à un autre, comme porteuse ou canal sur une surface cellulaire. Dans la littérature de nombreux capteurs couplés à des cellules sont utilisés pour le diagnostic médical[21]. ...
Article
This work deals with the association of molecularly imprinted polymers technology and Love wave devices in order to develop a biosensor in the frame of ANR project, Tecsan. The first part of this work concerned the development and the validation of a protocol for localized deposition of imprinted (MIP) and non-imprinted (NIP) polymeric thin film compatible with the propagation of acoustic wave. The second part exhibited the cahracterization of the coated sensors, by scanning microscopy and deep gas characterization. The proposed characterization technique gives information on surface morphology and porosity of thin MIP films before extraction of the target molecule, then after extraction and after rebinding. It allowed hence, the validation of the sensor principle. These results constitute a god background for future application in medium liquid.
... The possibility of performing multiplex assays within a miniaturized chip could be particularly suitable for biological defense applications (Rider et al., 2003). Several regulatory systems have been exploited including those for heavy metal resistance (for developing heavy metal responsive biosensors), for organic compound degradation (to obtain organic compound sensors), and for cellular stress responses (to obtain general toxicity sensors) (Banerjee and Bhunia, 2010;Raut et al., 2012;Bereza-Malcolm et al., 2014;Cerminati et al., 2015). ...
Article
Biosensors are a very active research field. They have the potential to lead to low-cost, rapid, sensitive, reproducible, and miniaturized bioanalytical devices, which exploit the high binding avidity and selectivity of biospecific binding molecules together with highly sensitive detection principles. Of the optical biosensors, those based on chemical luminescence detection (including chemiluminescence, bioluminescence, electrogenerated chemiluminescence, and thermochemiluminescence) are particularly attractive, due to their high-to-signal ratio and the simplicity of the required measurement equipment. Several biosensors based on chemical luminescence have been described for quantitative, and in some cases multiplex, analysis of organic molecules (such as hormones, drugs, pollutants), proteins, and nucleic acids. These exploit a variety of miniaturized analytical formats, such as microfluidics, microarrays, paper-based analytical devices, and whole-cell biosensors. Nevertheless, despite the high analytical performances described in the literature, the field of chemical luminescence biosensors has yet to demonstrate commercial success. This review presents the main recent advances in the field and discusses the approaches, challenges, and open issues, with the aim of stimulating a broader interest in developing chemical luminescence biosensors and improving their commercial exploitation. Copyright © 2015 Elsevier B.V. All rights reserved.
... A portable evanescent-wave fiber-optic biosensor fabricated (DeMarco and Lim, 2002) to determine E. coli in seeded samples of ground beef. A portable optical biosensor with specific sensitivities for pathogens that can evaluate the cytotoxicity by calculating the color change was used for the determination of L. monocytogenes in meats at 10 3 −10 4 CFU/ml in mammalian cells (Ped-2E9) (Banerjee and Bhunia 2010). Fourier transform infrared (FTIR) spectroscopy technique was used to analyze biological or chemical changes within the meat substrate (Ellis et al., 2002). ...
... Probing pathogen interactions with cell cultures has become much less laborious with recent advances in automation, high-throughput imaging, and isolation of single bacterial organisms by microfluidic devices [45,46]. Therefore, the development of cell-based imaging assays are critical for functional screening and elimination of false-negative results from dead, non-hazardous, or non-pathogenic bacteria [47]. ...
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Cell culture systems have greatly expanded our understanding of how bacterial pathogens target signaling pathways to manipulate the host and cause infection. Advances in genetic engineering have allowed for the creation of fluorescent protein readouts within signaling pathways, but these techniques have been underutilized in pathogen biology. Here, we genetically engineered a lung cell line with fluorescent reporters for extracellular signal-related kinase (ERK) and the downstream transcription factor FOS-related antigen 1 (Fra1) and evaluated signaling after inoculation with pathogenic and non-pathogenic bacteria. Cells were inoculated with 100 colony-forming units of Acinetobacter baylyi, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus agalactiae, or Staphylococcus epidermidis and imaged in a multi-mode reader. The alamarBlue cell viability assay was used as a reference test and showed that pathogenic P. aeruginosa induced significant (p < 0.05) cell death after 8 h in both wild-type and engineered cell lines compared to non-pathogenic S. epidermidis. In engineered cells, we found that Fra1 signaling was disrupted in as little as 4 h after inoculation with bacterial pathogens compared to delayed disruption in signaling by non-pathogenic S. epidermidis. Overall, we demonstrate that low levels of pathogenic versus non-pathogenic bacteria can be rapidly and sensitively screened based on ERK-Fra1 signaling.
... In another approach, mast cells from mice have been engineered to express receptor proteins specific for E. coli [117]; subsequently, fluorescent dye introduced into the mast cells is expelled via cellular exocytosis upon binding with E. coli. One use for these biosensors is for the detection of pathogens in food samples [118] such as fried rice and hot dogs. This approach has successfully distinguished active toxins from the inactive ones, and similarly, severe pathogens from their more benign counterparts with detection limits as low as 10 2 -10 4 colony forming units per gram (CFU/g) and toxins in amounts as low as 10-40 ng. ...
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Membranes constitute the interface between the basic unit of life-a single cell-and the outside environment and thus in many ways comprise the ultimate "functional biomaterial". To perform the many and often conflicting functions required in this role, for example to partition intracellular contents from the outside environment while maintaining rapid intake of nutrients and efflux of waste products, biological membranes have evolved tremendous complexity and versatility. This article describes how membranes, mainly in the context of living cells, are increasingly being manipulated for practical purposes with drug discovery, biofuels, and biosensors providing specific, illustrative examples. Attention is also given to biology-inspired, but completely synthetic, membrane-based technologies that are being enabled by emerging methods such as bio-3D printers. The diverse set of applications covered in this article are intended to illustrate how these versatile technologies-as they rapidly mature-hold tremendous promise to benefit human health in numerous ways ranging from the development of new medicines to sensitive and cost-effective environmental monitoring for pathogens and pollutants to replacing hydrocarbon-based fossil fuels.
... Ces protéines pourraient tout simplement être utilisées pour le transport d'un produit chimique d'un endroit à un autre, comme porteuse ou canal sur une surface cellulaire. Dans la littérature de nombreux capteurs couplés à des cellules sont utilisés pour le diagnostic médical[21]. ...
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These research works aim at combining the technology of polymers with molecular imprints in acoustic devices with acoustic wave (Love wave) to realize a biosensor. The major objective was the development of a protocol for the localized deposit of not-printed polymers (NIP) and printed (MIP), compatible with the distribution of the elastic wave. A second part of the works concerned characterization of films and sensors so realized, by microscopy with scanning and measures of detection under gas. Elements relative to the mechanical properties (porosity, specific surface) of NIPs or MIPs before extraction of the target molecule, then after extraction and after recapture, allowed to validate the principle of the sensor, opening the way to the application in liquid environment within the framework of works begun at the same time.
... Biofilm-isolated L. monocytogenes were less cytotoxic to Ped-2E9 and Caco-2 cells than the planktonic bacteria To characterize pathogenic attributes of biofilm-isolated cells, Ped-2E9 (a hybrid murine B lymphocyte line)-based in vitro cytotoxicity assay was conducted 44,45 . Ped-2E9 has been established to be a sensitive model to respond to the apoptosis triggered by Lm 46-48 . ...
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Environmental cues promote microbial biofilm formation and physiological and genetic heterogeneity. In food production facilities, biofilms produced by pathogens are a major source for food contamination; however, the pathogenesis of biofilm-isolated sessile cells is not well understood. We investigated the pathogenesis of sessile Listeria monocytogenes ( Lm ) using cell culture and mouse models. Lm sessile cells express reduced levels of the lap , inlA, hly , prfA , and sigB and show reduced adhesion, invasion, translocation, and cytotoxicity in the cell culture model than the planktonic cells. Oral challenge of C57BL/6 mice with food, clinical, or murinized-InlA (InlA m ) strains reveals that at 12 and 24 h post-infection (hpi), Lm burdens are lower in tissues of mice infected with sessile cells than those infected with planktonic cells. However, these differences are negligible at 48 hpi. Besides, the expressions of inlA and lap mRNA in sessile Lm from intestinal content are about 6.0- and 280-fold higher than the sessle inoculum, respectively, suggesting sessile Lm can still upregulate virulence genes shortly after ingestion (12 h). Similarly, exposure to simulated gastric fluid (SGF, pH 3) and intestinal fluid (SIF, pH 7) for 13 h shows equal reduction in sessile and planktonic cell counts, but induces LAP and InlA expression and pathogenic phenotypes. Our data show that the virulence of biofilm-isolated Lm is temporarily attenuated and can be upregulated in mice during the early stage (12–24 hpi) but fully restored at a later stage (48 hpi) of infection. Our study further demonstrates that in vitro cell culture assay is unreliable; therefore, an animal model is essential for studying the pathogenesis of biofilm-isolated bacteria.
... Hence, recently, other biosensors have attracted scientific attentions that detect the bacteria like S. aureus in indirect process. These biosensors operates based on the amplification of specific nucleic-acid sequences [59,60] or detection of toxins and other biomolecules excreted by the pathogen under certain situations [61,62]. Thus, these biosensors do not require further oligonucleotides like aptamers that they are expensive and susceptible to digestion by nucleases. ...
Article
One of the most common and important pathogenic bacteria is Staphylococcus aureus (S. aureus) which is known as a foodborne illness all over the world. The detection of micrococcal nuclease (MNase) can act as a unique diagnostic biomarker for the identification of S. aureus. So far, various complex methods have been introduced for the evaluation of S. aureus bacterium. However, they have different limitations such as labor-intensive, inaccurate results and time-consuming procedures. Thus, it is of particular attention to develop fast, easy, simple and more approachable detection methods based on nanotechnology and MNase detection. In this review, recent advances and modern techniques of ultrasensitive biosensors based on quantum dots (QDs), noble metal and magnetic nanoparticles (NPs), graphene oxide (GO) nanosheets, and also transfer energy strategy have been discussed for the identification of MNase activity and S. aureus contamination. Besides, advantages and disadvantages of different types of fluorescent, phosphorescent and colorimetric biosensors have been discussed.
... Cell-based sensor developed fast in the past two decades [6][7][8]. This method is based on physiological reactions within living cells. ...
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Shellfish toxin can be easily found in seafood, and bring potential harm to human health. Nowadays some analytical methods are used for shellfish toxin quantitative detection. However, there are some disadvantages in these analytical methods such as time consuming, high cost, etc. Therefore, a more suitable method is in demand. In this study, a novel method using high frequency surface acoustic wave resonator (SAWR) based sensor combined with living cells was developed for shellfish toxin continuous monitoring. ECA9706 cell lines were used as the sensing elements to establish the sensor device for DSP toxin okadaic acid (OA) quantitative monitoring. The sensor device is in serial with SAWR. SAWR output frequency was recorded for detecting characterization. The OA concentration of prepared solutions could be characterized by using SAWR sensor, and good linear relationship between sensor responses and OA concentration is achieved. Liquid chromatography tandem mass spectrometry (LC–MS/MS) is used for characterization of OA content in shellfish extract. Good linear relationship between OA content and the concentration determination provided by SAWR sensor. HEK293 cells were fixed on electrode and utilized as negative control. The results showed that ECA9706 cell-based sensor had selective responses to OA. This method is promising in shellfish toxins rapid quantitative determination.
... Furthermore, their robustness, high selectivity, specificity, and evolvability have raised cell-based biosensors as a significant revolution in analytical science [13]. However, only a few studies have been performed on L. monocytogenes detection using cell-based biosensors [14][15][16]. ...
Article
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Human food-borne diseases caused by pathogenic bacteria have been significantly increased in the last few decades causing numerous deaths worldwide. The standard analyses used for their detection have significant limitations regarding cost, special facilities and equipment, highly trained staff, and a long procedural time that can be crucial for foodborne pathogens with high hospitalization and mortality rates, such as Listeria monocytogenes. This study aimed to develop a biosensor that could detect L. monocytogenes rapidly and robustly. For this purpose, a cell-based biosensor technology based on the Bioelectric Recognition Assay (BERA) and a portable device developed by EMBIO Diagnostics, called B.EL.D (Bio Electric Diagnostics), were used. Membrane engineering was performed by electroinsertion of Listeria monocytogenes homologous antibodies into the membrane of African green monkey kidney (Vero) cells. The newly developed biosensor was able to detect the pathogen’s presence rapidly (3 min) at concentrations as low as 102 CFU mL−1, demonstrating a higher sensitivity than most existing biosensor-based methods. In addition, lack of cross-reactivity with other Listeria species, as well as with Escherichia coli, was shown, thus, indicating biosensor’s significant specificity against L. monocytogenes.
... Ohk et al. (2010) described an alternative, optical (fluorescence) based, fibre-optic approach in which they used a capture J Food Sci Technol antibody and a fluorescently labelled aptamer to sandwich Listeria monocytogenes with a LOD of 10 2 CFU/25 g in ready-to-eat meat samples. Banerjee and Bhunia (2010) used mammalian cells (Ped-2E9), with specific sensitivities for pathogens in a portable optical (absorbance) biosensor that assessed cytotoxicity by measuring the colour change and detected L. monocytogenes in ready-to-eat meats at 10 3 -10 4 CFU/ml. ...
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The meat industry associated with the health hazards like deadly pathogens, veterinary drugs, pesticide residues, toxins and heavy metals is in need of a tool to tackle the awful situation and ensure safer product to consumer. The growth in the industry, global trade scenario, stringent laws and consumer awareness has placed an extra onus on the meat industry to meet out the expectations and demands. Biosensors are the latest tool of detection in the fast growing industries including the food industry. Hence an attempt is envisaged here to review the possibility of harnessing biosensors as tool of safety to safe guard the consumer health and address safety issues in reference to the common threats of concern in the meat industry.
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The sections in this article are
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Contamination of food and water with pathogens can cause serious human illness and concern for public safety. Governments and food safety authorities now recognize the need to regulate and closely monitor foods for human and animal consumption. The establishment of food, water and regulatory authorities including the European Food Safety Authority and the National Antimicrobial Resistance Monitoring System for Enteric Bacteria has enabled investigation and monitoring of pathogenic outbreaks globally. This process is hampered by the conventional methods used for monitoring and detecting pathogens, which require expensive, laboratory-based, time-consuming analyses. This chapter focuses on the development of electrochemical and optical biosensors for rapid, cheap and simple detection of microorganisms, mycotoxin food and marine toxin contaminates at the point-of-need. The associated challenges of biosensor development and the future directions are also discussed.
Article
This paper describes fabrication of a DNA-based Au-nanoparticle modified pencil graphite electrode (PGE) biosensor for detection of Bacillus cereus, causative agent of two types of food-borne disease, i.e., emetic and diarrheal syndrome. The sensing element of the biosensor was comprised of gold nanoparticles (GNPs) self-assembled with single-stranded DNA (ssDNA) of nheA gene immobilized with thiol linker on the GNPs modified PGE. The size, shape and dispersion of the GNPs were characterized by field emission scanning electron microscope (FESEM). Detection of B. cereus was carried out based on an increase in the charge transfer resistance (Rct) of the biosensor due to hybridization of the ss-DNA with target DNA. An Atomic force microscope (AFM) was used to confirm the hybridization. The biosensor sensitivity in pure cultures of B. cereus was found to be 10° colony forming units per milliliter (CFU/mL) with a detection limit of 9.4×10−12 mol L−1. The biosensor could distinguish complementary from mismatch DNA sequence. The proposed biosensor exhibited a rapid detection, low cost, high sensitivity to bacterial contamination and could exclusively and specifically detect the target DNA sequence of B. cereus from other bacteria that can be found in dairy products. Moreover, the DNA biosensor exhibited high reproducibility and stability, thus it may be used as a suitable biosensor to detect B. cereus and to become a portable system for food quality control.
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Shiga-toxin producing Escherichia coli (STEC) is a serious public health concern. Current Vero cell assay, although sensitive, is lengthy and requires 48-72 h to assess STEC presence in a sample. In this study, we investigated if Vero cells in a three-dimensional (3D) platform would provide improved sensitivity for rapid screening of STEC. Vero cells (epithelial kidney cell line) were grown as a monolayer (2D) or in a collagen-matrix (3D) and exposed to Shiga-toxin (Stx) preparation or STEC cells that were pre-exposed to antibiotics (mitomycin C, ciprofloxacin, or polymyxin B) for toxin induction. Lactate dehydrogenase (LDH) release from Vero cells was used as a biomarker for cytotoxicity. Modified tryptic soy broth (mTSB) as enrichment broth containing mitomycin C (2 µg/ml) or ciprofloxacin (100 ng/ml) significantly induced Shiga-toxin production, which was further confirmed by the dot-immunoblot assay. The 3D Vero platform detected STEC after 6 h post-infection with cytotoxicity values ranging from 33%-79%, which is considerably faster than the traditional 2D platform, when tested with STEC. The cytotoxicity for non- Shiga-toxin producing bacteria, Salmonella, Listeria, Citrobacter, Serratia, and Hafnia was found to be below the cytotoxicity cutoff value of 15%. The detection limit for the 3D Vero cell assay was estimated to be 107 CFU/ml for bacteria and about 32 ng/ml for Stx in 6 h. STEC-inoculated ground beef samples (n=27) resulted in 38-46% cytotoxicity, and the bacterial isolates (n=42) from ground beef samples were further confirmed to be stx1 and stx2 positive in a multiplex PCR yielding a very low false-positive result. This 3D cell-based screening assay relies on mammalian cell pathogen interaction that can complement other molecular techniques for the detection of cell-free Stx or STEC cells from food samples for early detection and prevention.
Article
This work presents a disposable, self-aligned, and die-level socketed 3D integrated biosensing-interface module (BIM) for biosensing applications. The enabling technologies of the BIM include: 1) mechanically flexible interconnects (MFIs) that provide temporary electrical interconnections to enable disposability, 2) sapphire precision balls and positive self-alignment structures (PSAS), which in combination with KOH-etched pits provide the BIM self-alignment capabilities, and 3) a clamped socket that temporarily provides the necessary force for the MFIs to form and maintain electrical contact with the underlying test die during testing. Repeated alignment measurements show that the alignment of the BIM to the biosensor varies little between uses, hence demonstrating that the biosensor system can maintain accurate alignment. Additionally, four-point resistance measurements were taken after assembly indicating that temporary electrical connections were formed and maintained.
Article
The development and application of cell-based biosensors (CBBs) provides a convenient strategy for rapid detection of target analytes. The CBBs had been widely applied in the fields of food safety, environment monitoring, and medicine diagnosis due to their advantages of short response time, easy operation, low toxicity, and portability. However, the CBBs based on two-dimensional (2D) cultured cells in-vitro suffer from a lower cell viability and isolated physiology, which had blocked the accurate evaluations of these biosensors. With the development of nanotechnology and three-dimensional (3D) printing technology, cells fixed in a 3D biosensor or a 3D microenvironment have shown great improvement in the sensitivity and detection authenticity than conventional CBBs. To promote the further development of CBBs, in this paper, we reviewed the related technologies used to construct 3D bionic cell chips including organic/inorganic agents and operating approaches suitable for constructing 3D cell cultural microenvironment. Then, the applications of 3D bionic cell chip based on microbial and mammalian cell biosensors in food safety field were discussed during recent ten years. Finally, the current challenges and further directions were summarized and prospected.
Article
Bacterial toxins are food safety hazards causing about 10% of all reported foodborne outbreaks in Europe. Pertinent to Gram‐positive pathogens, the most relevant toxins are emetic toxin and diarrheal enterotoxins of Bacillus cereus , neurotoxins of Clostridium botulinum , enterotoxin of Clostridium perfringens , and a family of enterotoxins produced by Staphylococcus aureus and some other staphylococci. These toxins are the most important virulence factors of respective foodborne pathogens and a primary cause of the related foodborne diseases. They are proteins or peptides that differ from each other in their size, structure, toxicity, toxicological end points, solubility, and stability, types of food matrix to which they are mostly related to. These differences influence the characteristics of required detection methods. Therefore, detection of these toxins in food samples, or detection of toxin production capacity in the bacterial isolate, remains one of the cornerstones of microbial food analysis and an essential tool in understanding the relevant properties of these toxins. Advanced research has led into new insights of the incidence of toxins, mechanisms of their production, their physicochemical properties, and their toxicological mode of action and dose‐response profile. This review focuses on biological, immunological, mass spectrometry, and molecular assays as the most commonly used detection and quantification methods for toxins of B. cereus, C. botulinum, C. perfringens , and S. aureus . Gathered and analyzed information provides a comprehensive blueprint of the existing knowledge on the principles of these assays, their application in food safety, limits of detection and quantification, matrices in which they are applicable, and type of information they provide to the user.
Article
Foodborne diseases caused by pathogens and toxins are a serious threat to food safety and human health; thus, they are major concern to society. Existing conventional foodborne pathogen or toxin detection methods, including microbiological assay, nucleic acid-based assays, immunological assays, and instrumental analytical method, are time-consuming, labor-intensive and expensive. Because of the fast response and high sensitivity, cell-based biosensors are promising novel tools for food safety risk assessment and monitoring. This review focuses on the properties of mammalian cell-based biosensors and applications in the detection of foodborne pathogens (bacteria and viruses) and toxins (bacterial toxins, mycotoxins and marine toxins). We discuss mammalian cell adhesion and how it is involved in the establishment of 3D cell culture models for mammalian cell-based biosensors, as well as evaluate their limitations for commercialization and further development prospects.
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Immunosensing has played a pivotal role in health management of humans and animals. In recent year, we have been witnessing a significant change in advancements made in the field of immunosensing in terms of enhanced sensitivity, ease of performing the assays, field applicability, and commercialization potential. Immunosensing technology or immunosensors (also called biosensors) are affinity-based devices capable of direct or indirect detection of an analyte of interest. This field of biotechnology possesses the capacity for invention of analytical tools and devices capable of powerful and precise detection of specific biotargets by the use of enzymes, aptamers, nanomaterials, antibodies, peptides, with a wide range of electrochemical, fluorescent-tagged, specific bioreceptor molecules. Animal production, perpetuation of selected traits, and maintenance of livestock health have long been a subject of extensive research. The biggest limitations of the diagnostic platforms developed are inability of its translation for real-time, on-site application owing to multiple factors. The most plausible solution proposed is the development of minimalistic device(s) capable of a rapid detection reaction with negligible sample processing, thus leading to surfacing of immunosensor devices. This review is aimed at emphasizing on the expansion of these devices which led to the advent of a concept popularized as “lab-on-a-chip” enabling the monitoring of physiological and reproductive health. The products’ mass-commercialization and applicability are the major challenges encountered in the process.
Article
Gold nanoparticles have emerged as a prominent tool in nanomedicine, particularly for applications in cancer diagnostic and treatment. One of the challenges for the successful implementation of gold nanoparticles in cancer therapy is their delivery to the specific cancer area within the tumor microenvironment. The presence of cancer enables a poorly organized vascularization system, increasing the pressure with the microenvironment, limiting the uptake of particles. The physicochemical properties of the gold nanoparticles (size, shape, and surface charge) also have a significant effect on diffusion to the tumor site and cellular uptake. In this work, we analyzed the transport of 10 nm gold nanoparticles with different surface charges (neutral, negative, and positive) through a hydrogel composite. Three-dimensional in vitro models composed of breast cancer cells loaded in the hydrogel composite were used for the qualitative and quantitative evaluation of cellular uptake of the gold nanoparticles. Surprisingly, an inverse correlation between the diffusion coefficients of the nanoparticles and cellular uptake was demonstrated. Positively charged gold nanoparticles displayed high cellular uptake, although their diffusion coefficient indicated slow transport through the hydrogel matrix. Neutral particles, on the other hand, displayed fast diffusion but the lowest cellular uptake. The results obtained indicate that nanoparticle diffusion and cellular uptake should be studied together in realistic in vitro models for a true evaluation of transport in tumor microenvironments.
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Animals play a vital role in our lives as they provide milk, meat, and other by-products for daily consumption. At the same time, their health directly/indirectly affects human beings as we both share a common environment. The One Health concept makes it imperative that the animals should remain healthy as it will have an impact on our health as well as economy. Animal disease diagnosis, their health monitoring, prevention, and control of diseases are important from this aspect. Tools for rapid diagnosis of animal diseases are crucial for early diagnosis and imposing control measures. Molecular techniques such as, genome sequencing, restriction fragment length polymorphism (RFLP), DNA microarray, PCR, and real-time PCR are some of the rapid techniques, but they need expertise and sophisticated labs. Conventional methods such as isolation of the pathogen, serological techniques, are laborious and time taking process. Therefore, to overcome the said limitations of conventional and molecular tools, biosensors are the better alternatives as they are rapid and can be used as pen-side diagnostic tests. They can provide test results in a few minutes under field conditions. In this chapter, we give an elaborative description of various types of biosensors and their utility in animal disease diagnostics and health monitoring.
Article
The design and fabrication of high sensitive and selective biosensing platforms areessential goals to precisely recognize biomaterials in biological assays. In particular, determination of adenosine triphosphate (ATP) as the main energy currency of the cells and one of the most important biomolecules in living organisms is a pressing need in advanced biological detection. Recently, aptamer-based biosensors are introduced as a new direct strategy in which the aptamers (Apts) directly bind to the different targets and detect them on the basis of conformational changes and physical interactions. They can also be conjugated to optical and electronic probes such as quantum dot (QD) nanomaterials and provide unique QD aptasensing platforms. Currently, these Apt-based biosensors with excellent recognition features have attracted extensive attention due to the high specificity, rapid response and facile construction. Therefore, in this review article, recent achievements and advances in aptasensing detection of ATP based on different detection methods and types of QDs are discussed. In this regard, the optical and electrochemical aptasensors have been categorized based on detection methods; fluorescence (FL), electrochemiluminescence (ECL) and photoelectrochemical (PEC) and they have been also divided to two main groups based on QDs; metal-based (M-based) and carbon-based (C-based) materials. Then, their advantages and limitations have been highlighted, compared and discussed in detail.
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Irresistible animal viruses brought about by pathogenic microorganisms, for example, microscopic organisms and viruses undermine the well-being and prosperity of untamed life, livestock, and limit efficiency and increment essentially monetary misfortunes to every division. The virus detection is a significant advance for the indications, effective cure of creature contamination illness in farms and field conditions. Thusly, there is solid enthusiasm for growing new quick purpose of care nanosensor frameworks for early identification of livestock infections with more affectability and particularity. In this chapter, we are presenting a layout of the nanosensing frameworks which can be applicable for animal virus identification. Diverse detecting procedures dependent on DNA receptors, glycan, aptamers and antigens are introduced.
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Rapid detection and identification of infectious pathogens in clinical samples is very challenging, and it is essential for proper treatment and predicts the outcome. To date, several notorious viruses had an impact on the health and social well-being across the globe in a short period. Bioanalytes like nucleic acids or proteins are usually the target for viral detection. Prospective biosensors for rapid, sensitive, specific, and cost-effective detection of viruses have been reported as an alternative to conventional viral detection methods such as polymerase chain reactions, enzyme-linked immunosorbent assays. With its higher advantages, nowadays the impact of biosensors to diagnose viruses is of great importance. Viruses are subject to mutations frequently and the genomes of different viruses can recombine to form novel progeny and therefore it is hard to detect by using a single biomarker. Therefore it is very important to detect multiple analytes concurrently using a smart designing multiplex biosensor platform that can improve the diagnostic efficacy. Nowadays humanity faces rising risks from life-threatening fast-spreading viral infectious diseases such as severe acute respiratory syndrome-coronavirus 2; coronavirus disease 2019 (SARS-CoV-2; COVID-19), Zika virus (ZIKV), Ebola virus (EBOV), and dengue virus (DENV) that eventually affects substantial global economic and social impacts. Considering the phenomenal advances in the field of biosensors over classical methods, herein we provide an overview of different types of biosensors, and the latest developments in the field of multiplex biosensors for detection of the deadliest viruses, like SARS-CoV-2, ZIKV, EBOV, and DENV along with the future outlook of biosensor technology.
Article
Abiotic stresses of various chemical contamination of physical, inorganic, organic and biotoxin origin and biotic stresses of bacterial, viral, parasitic and fungal origins are the significant constraints in achieving higher aquaculture production. Testing and rapid detection of these chemical and microbial contaminants are crucial in identifying and mitigating abiotic and biotic stresses, which has become one of the most challenging aspects in aquaculture and culture-based fisheries. The classical analytical techniques, including titrimetric methods, spectrophotometric, mass spectrometric, spectroscopic, and chromatographic techniques, are tedious and sometimes inaccessible when required. The development of novel and improved bioanalytical methods for rapid, selective and sensitive detection is a wide and dynamic field of research. Biosensors offer precise detection of biotic and abiotic stressors in aquaculture and culture-based fisheries within no time. This review article allows filling the knowledge gap for detection and monitoring of chemical and microbial contaminants of abiotic and biotic origin in aquaculture and culture-based fisheries using nano(bio-) analytical technologies, including nano(bio-)molecular and nano(bio-)sensing techniques.
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The chapter highlights new concepts for the design of biosensing ­material in order to develop sensors for different biological warfare agents. Biosensing technologies that use electrochemical, piezoelectric, optical, acoustic and thermal transducers for detection of pathogenic bacteria are highlighted. Special attention is paid to methods for improving the sensitivity and analysis time of biosensors. Recent developments in physical transducers in biosensors for bacterial detection are overviewed.
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Antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. A critical assessment of the implementation of such formats is provided, with reference to their principles, problems and potential for 'on-site' analysis. Particular emphasis is placed on the detection of foodborne bacterial pathogens, such as Escherichia coli and Listeria monocytogenes, and additional examples relating to the monitoring of fungal pathogens, viruses, mycotoxins, marine toxins and parasites are also provided.
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Dendritic cells (DCs) are a specialized family of antigen presenting cells. They play critical roles in sensing and processing microbial information through a series of pattern recognition receptors (PPRs), including the well-characterized toll-like receptors (TLRs). In this study, we demonstrated the utilization of a DC cell line, DC2.4, as a cell source for the detection and differentiation of microbes towards the development of cell-based biosensors. As a proof of principle, the gram-negative bacteria Escherichia coli K12 strain D21 and its lipopolysaccharide (LPS) mutants were used as model targets. The stimulation of DCs by bacterial strains was monitored by the production of nitric oxide (NO), and the colorimetric Greiss assay was used to quantify the level of NO produced. Our results demonstrated that DCs could detect and differentiate microbes with subtle differences in the composition of specific cell surface components, i.e. LPS, within minutes. Though the current colorimetric-based NO assay limited the detection sensitivity, we showed that DCs were able to detect as low as 2-3 bacteria per cell. Furthermore, compared to macrophages, DCs were superior in discriminating LPS mutants. Our study demonstrates that DCs possess great potential as cell sources for the development of novel cell-based biosensors for detecting microbes with high selectivity and sensitivity and rapid responsiveness. In addition, when DCs are coupled with other biosensor platforms, higher sensitivity can be expected.
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An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 10(8) cfu/ml into a suspension of Ped-2E9 (10(6)/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test. Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 microns filter.
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Murine hybridoma cells, designated Ped-2E9, when stored up to 60 days at -196 degrees C or up to 48 days at -80 degrees C, gave results equivalent to those for freshly grown murine hybridoma cells in an in vitro pathogenicity assay of Listeria species. Thus, laboratories do not need to have their own tissue culture facilities to maintain the hybridoma cells for the assay described.
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A simple cytotoxicity assay for Listeria species was developed by assaying alkaline phosphatase (AP) release from an infected hybrid B lymphocyte (Ped-2E9) line. Eight of eight L. monocytogenes and six of 11 L. ivanovii strains induced significantly high AP release from Ped-2E9 cells compared to five other L. ivanovii strains and other Listeria spp. In contrast, all L. monocytogenes and L. ivanovii test strains showed high release of lactate dehydrogenase (LDH) activity from Ped-2E9 cells. The molecular mass of AP was estimated to be about 128-165 kDa, suggesting severe membrane damage in Ped-2E9 cells due to Listeria infection. The data presented here indicate that AP assay could be used over LDH assay to detect Listeria-induced cell cytotoxicity.
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Serratia marcescens culture filtrates have been reported to be cytotoxic to mammalian cells. Using biochemical and genetic approaches, we have identified a major source of this cytotoxic activity. Both heat and protease treatments abrogated the cytotoxicity of S. marcescens culture filtrates towards HeLa cells, suggesting the involvement of one or more protein factors. A screen for in vitro cytotoxic activity revealed that S. marcescens mutant strains that are deficient in production of a 56-kDa metalloprotease are significantly less cytotoxic to mammalian cells. Cytotoxicity was significantly reduced when culture filtrates prepared from wild-type strains were pretreated with either EDTA or 1,10-phenanthroline, which are potent inhibitors of the 56-kDa metalloprotease. Furthermore, cytotoxic activity was restored when the same culture filtrates were incubated with zinc divalent cations, which are essential for enzymatic activity of the 56-kDa metalloprotease. Finally, recombinant expression of the S. marcescens 56-kDa metalloprotease conferred a cytotoxic phenotype on the culture filtrates of a nonpathogenic Escherichia coli strain. Collectively, these data suggest that the 56-kDa metalloprotease contributes significantly to the in vitro cytotoxic activity commonly observed in S. marcescens culture filtrates.
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Chapter
Introduction The Need for Protective Food Security Programs Vulnerability Assessment Emergency Response and Product Recovery Prevention as the First Line of Defense Development of a Food Security Plan Based on HACCP Principles Evaluating Security Risks and Identifying Hazards Managing Risk: Preventive Measures Security Strategies Appendix: An Example References
Chapter
Antibody, also known as immunoglobulin, is normally made in the body in defense of foreign antigen or invading pathogen. Highly specific biorecognition property of antibody with antigen has made antibody as one of the most indispensable molecules for broad application, not only in the diagnosis or detection but also in prevention or curing of diseases. Animals are routinely used for production of both polyclonal and monoclonal antibodies; however, recombinant and phage display technologies are being adopted to improve antibody specificity and to cut cost for antibody production. Available genome sequence of pathogens is also allowing researchers to find and select suitable target antigens for production of antibody with improved specificity. In recent years, however, demand for antibody is even greater as novel biosensor or nanotechnology-based methods continue to utilize antibody for analyte capture and interrogation. Conventional immunoassay methods such as lateral flow and enzyme-linked immunoassays, though lack sensitivity, are available commercially and are widely used. While biosensor-based methods such as time-resolved fluorescence immunoassay, chemiluminescence assay, electrochemical immunoassay, surface plasmon resonance sensor, fiber optic sensor, and microfluidic biochip have, in some cases, demonstrated improved sensitivity, they require further optimization with real-world samples. Furthermore, environmental stress and the growth media are known to affect the physiological state of microorganism and antigen expression, often rendering unsatisfactory signal response from immunoassays. Thus, one must understand the microorganisms’ response to these factors before designing an immunoassay to avoid false results. With the advent of microfluidics and nanotechnology, the adaptation of lab-on-chip concept in immunoassays will soon be a reality for near real-time detection of pathogens from food or clinical specimens.
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Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes and Campylobacter jejuni are considered important pathogens causing the most food-related human illnesses worldwide. Current methods for pathogen detection have limitations in the effectiveness of identifying multiple foodborne pathogens. In this study, a pathogen detection microarray was developed using various 70-mer oligonucleotides specifically targeting the above pathogens. To reduce the cost of detection, each microarray chip was designed and fabricated to accommodate 12 identical arrays which could be used for screening up to 12 different samples. To achieve high detection sensitivity and specificity, target-specific DNA amplification instead of whole genome random amplification was used prior to microarray analysis. Combined with 14-plex PCR amplification of target sequences, the microarray unambiguously distinguished all 4 pathogens with a detection sensitivity of 1 x 10(-4) ng (approximately 20 copies) of each genomic DNA. Applied the assay to 39 fresh meat samples, 16 samples were found to be contaminated by either 1 or 2 of these pathogens. The co-occurrences of Salmonella and E. coli O157:H7, Salmonella and L. monocytogenes in the same meat samples were also observed. Overall, the microarray combined with multiplex PCR method was able to effectively screen single or multiple pathogens in food samples and to provide important genotypic information related to pathogen virulence.
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A microfabricated flow cytometer was used to demonstrate multiplexed detection of bacteria and toxins using fluorescent coded microspheres. Antibody-coated microspheres bound biothreat targets in a sandwich immunoassay format. The microfluidic cytometer focused the microspheres in three dimensions within the laser interrogation region using passive groove structures to surround the sample stream with sheath fluid. Optical analysis at four different wavelengths identified the coded microspheres and quantified target bound by the presence of phycoerythrin tracer. The multiplexed assays in the microflow cytometer had performance approaching that of a commercial benchtop flow cytometer. The respective limits of detection for bacteria (Escherichia coli, Listeria, and Salmonella) were found to be 10(3), 10(5), and 10(4) cfu/mL for the microflow cytometer and 10(3), 10(6), and 10(5) cfu/mL for the commercial system. Limits of detection for the toxins (cholera toxin, staphylococcal enterotoxin B, and ricin) were 1.6, 0.064, and 1.6 ng/mL for the microflow cytometer and 1.6, 0.064, and 8.0 ng/mL for the commercial system.
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An improved qualitative cell cytotoxicity assay for the detection of Bacillus cereus emetic and enterotoxin is described. The presence of toxin in culture supernatant fluids was detected by measurement with the tetrazolium salt MTT, as it adversely affects the metabolic status of cultured CHO cells. Psychrotrophic B. cereus isolates (65) were assessed for toxin production using the cytotoxicity assay, and 91% of culture supernatant fluids were cytotoxic. Toxin assessment using BCET-RPLA and ELISA immunoassays indicated that 51% and 85% of the cultures, respectively, were toxigenic. There were pronounced strain differences in the amount of toxin produced by the B. cereus isolates. Some isolates of B. circulans, B. laterosporus/cereus, B. lentus, B. licheniformis, B. mycoides, B. subtilis and B. thuringiensis were also toxigenic.
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An improved McCoy cell cytotoxicity assay for Bacillus cereus diarrhoeal toxin, which includes a staining procedure in addition to visual examination, was developed and the method was compared with two commercially available kits (Oxoid BCET-RPLA and Tecra BDE-VIA). A total of 71 strains of 15 different Bacillus, Brevibacillus and Paenibacillus species, including 16 strains of B. cereus from outbreaks of food-borne illness, were evaluated for toxin production. Eleven of the outbreak strains exhibited cytotoxicity, including all six B. cereus strains associated with diarrhoeal illness. Several other isolates of B. cereus, and its relatives B. anthracis, B. mycoides and B. thuringiensis, exhibited similar cytotoxicity. The other species showed no cytotoxicity, with the exception of certain B. subtilis strains. The cytotoxicity assay was more sensitive than the Oxoid kit and unlike the Tecra kit, did not give false positive results with supernatant fluids heat-treated to destroy the toxin.
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The hybridoma Ped-2E9 based cytotoxicity assay was developed to distinguish virulent from avirulent Listeria species in 6 hr. The cytotoxicity effect on Ped-2E9 was reported to be primarily due the cytolytic action of listeriolysin O (LLO), produced by L. monocytogenes. In this study, the effect of a reducing agent, dithiothreitol (DTT, 0-2 mM) that is known to activate LLO was investigated to make the Ped-2E9 based cytotoxicity assay an even more sensitive and rapid. Also, we examined the effect of fetal bovine serum (FBS, 0-50%), a common ingredient of tissue culture media on cytotoxicity. A DTT concentration of 0.5 mM gave an optimum cytotoxicity effect, which could be measured by both alkaline phosphatase (AP) and lactate dehydrogenase (LDH) assays in just 1.5-2 hr. FBS, at levels between 10 to 50%, significantly inhibited Listeria-mediated cytotoxicity. Concentrated culture filtrates from L. monocytogenes or LLO producing recombinant L. innocua (prfA+ hlyA+) strain also caused cytotoxicity effects, which were observed by scanning electron microscopy or a cytotoxicity assay in 2-3 hr. Interestingly, addition of DTT to culture filtrates produced 100% cell cytotoxicity in just 15 min. This indicated that LLO activity, which is responsible for Ped-2E9 cytotoxicity, was augmented several folds with the addition of a reducing agent. Examination of Listeria isolates belonging to different serogroups from clinical sources or naturally contaminated meat products with DTT gave cytotoxicity results in 2 hr, which were comparable to the 5-hr assay analyzed concurrently without DTT. These results indicated that DTT, which activated the LLO, could be used in the cytotoxicity assay to enhance Listeria-mediated Ped-2E9 cell cytotoxicity. This knowledge will greatly assist us to develop a user-friendly rapid assay to screen cytopathogenic properties of Listeria species.
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Different classes of cell-based biosensors are introduced. These include devices to measure cell-cell contact and set-ups to determine metabolic products. Main emphasis is put on sensors based on nerve-cell networks which are able to detect neuro-active compounds. The different experimental set-ups are explained and examples for typical applications are given. A main point concerns new achievements and prospects for future developments.
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Some Listeria monocytogenes strains not related to clinical cases have been found to exhibit a low virulence level in mice as well as in an in vitro test using Caco-2 cells. The purpose of this study was to validate a new in vitro test of virulence based on a plaque-forming assay (PFA) using a HT-29 cell monolayer with 118 Listeria strains. The use of HT-29 cells in 96-well tissue culture plates allowed the testing of 30 strains per day and providing results in 24 h. In addition. statistical analyses demonstrated the reproducibility and repeatability of the PFA. No quantitative relationship was observed between the virulence of the strains and the hemolytic titer or the cytotoxic effects on HT-29 cells. In contrast, good agreement was observed between virulence assessed after subcutaneous (SC) infection and virulence obtained by PFA. Three groups of L. monocytogenes strains (avirulent, hypovirulent and fully virulent) were established by comparison of the clinical origin of the strains, the number of immunocompetent contaminated mice and the numbers of Listeria strains recovered in the spleen after SC infection. With one exception, i.e. a clinical case of L. seeligeri (sensitivity 0.98), the PFA successfully detected the virulent strains only (specificity 1). Decision-tree algorithms performed by SAS and S-Plus demonstrated that this tissue culture assay discriminated between the avirulent and hypovirulent strains and the virulent strains. This test could therefore be an alternative to in vivo tests, allowing grading of virulence.