The development of sensors for detecting foodborne pathogens has been motivated by the need to produce safe foods and to provide better healthcare. However, in the more recent times, these needs have been expanded to encompass issues relating to biosecurity, detection of plant and soil pathogens, microbial communities, and the environment. The range of technologies that currently flood the sensor market encompass PCR and microarray-based methods, an assortment of optical sensors (including bioluminescence and fluorescence), in addition to biosensor-based approaches that include piezoelectric, potentiometric, amperometric, and conductometric sensors to name a few. More recently, nanosensors have come into limelight, as a more sensitive and portable alternative, with some commercial success. However, key issues affecting the sensor community is the lack of standardization of the testing protocols and portability, among other desirable elements, which include timeliness, cost-effectiveness, user-friendliness, sensitivity and specificity. [...] Full article

Antibody engineering has allowed for the rapid generation of binding agents against virtually any antigen of interest, predominantly for therapeutic applications. Considerably less attention has been given to the development of diagnostic reagents and biosensors using engineered antibodies. Recently, we produced a novel pentavalent bispecific antibody (i.e., decabody) by pentamerizing two single-domain antibodies (sdAbs) through the verotoxin B subunit (VTB) and found both fusion partners to be functional. Using a similar approach, we have engineered a bispecific pentameric fusion protein consisting of five sdAbs and five cellulose-binding modules (CBMs) linked via VTB. To find an optimal design format, we constructed six bispecific pentamers consisting of three different CBMs, fused to the Staphylococcus aureus-specific human sdAb HVHP428, in both orientations. One bispecific pentamer, containing an N-terminal CBM9 and C-terminal HVHP428, was soluble, non-aggregating, and did not degrade upon storage at 4 ºC for over six months. This molecule was dually functional as it bound to cellulose-based filters as well as S. aureus cells. When impregnated in cellulose filters, the bispecific pentamer recognized S. aureus cells in a flow-through detection assay. The ability of pentamerized CBMs to bind cellulose may form the basis of an immobilization platform for multivalent display of high-avidity binding reagents on cellulosic filters for sensing of pathogens, biomarkers and environmental pollutants. Full article

A novel miniature cell biosensor detection system for the detection of Hepatis B virus (HBV)-associated antigens and anti-HBV is described. The biosensor is based on “membrane-engineered” Vero fibroblast cells immobilized in an alginate matrix. The membrane-engineering process involved the electroinsertion of anti-HBV specific antibodies (anti-HBs, anti-HBe) or antigens (HBsAg) in the membranes of the Vero cells. The attachment of a homologous antigen to the electroinserted antibody (or, respectively, of the antibody to the electroinserted antigen) triggered specific changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the Bioelectric Recognition Assay (BERA). The sensor was used for screening 133 clinical blood serum samples according to a double-blind protocol. Considerably higher sensor responses were observed against HBV-positive samples, compared with responses against negative samples or samples positive for heterologous hepatitis viruses such as Hepatitis C (HCV) virus. Detection of anti-HBs antibodies was made possible by using a biosensor based on immobilized Vero cells bearing the respective antigen (HBsAg). The observed response was rapid (45 sec) and quite reproducible. Fluorescence microscopy observations showed that attachment of HBV particles to cells membrane-engineered with anti-HBs was associated with a decrease of [Ca²⁺]cyt. The perspectives for using the novel biosensor as a qualitative, rapid screening, high throughput assay for HBV antigens and anti-HBs in clinical samples is discussed. Full article

The thixotropic-like properties of saline/glycerol drops, containing biotinylated capture antibodies, on streptavidin-coated glass slides have been investigated, along with their implications for bacterial detection in a fluorescent microarray immunoassay. The thixotropic-like nature of 60:40 saline-glycerol semisolid droplets (with differing amounts of antibodies) was observed when bacteria were captured, and their presence detected using a fluorescently-labeled antibody. Semisolid, gel-like drops of biotinylated capture antibody became liquefied and moved, and then returned to semisolid state, during the normal immunoassay procedures for bacterial capture and detection. Streaking patterns were observed that indicated thixotropic-like characteristics, and this appeared to have allowed excess biotinylated capture antibody to participate in bacterial capture and detection. When developing a microarray for bacterial detection, this must be considered for optimization. For example, with the appropriate concentration of antibody (in this study, 0.125 ng/nL), spots with increased diameter at the point of contact printing (and almost no streaking) were produced, resulting in a maximal signal. With capture antibody concentrations greater than 0.125 ng/nL, the excess biotinylated capture antibody (i.e., that which was residing in the three-dimensional, semisolid droplet space above the surface) was utilized to capture more bacteria. Similarly, when the immunoassay was performed within a hydrophobic barrier (i.e., without a coverslip), brighter spots with increased signal were observed. In addition, when higher concentrations of cells (~10⁸ cells/mL) were available for capture, the importance of unbound capture antibody in the semisolid droplets became apparent because washing off the excess, unbound biotinylated capture antibody before the immunoassay was performed reduced the signal intensity by nearly 50%. This reduction in signal was not observed with lower concentrations of cells (~10⁶ cells/mL). With increased volumes of capture antibody, abnormal spots were visualized, along with decreased signal intensity, after bacterial detection, indicating that the increased droplet volume detrimentally affected the immunoassay. Full article

Immunomagnetic beads (IMB) were synthesized using anti-Escherichia coli O157 antibodies and magnetic beads of two different sizes (1 mm and 2.6 to 2.8 mm) that contained a streptavidin coating, activated carboxyl groups or tosylated surfaces. The synthesized IMB, together with a commercially available IMB, were used to capture different strains of E. coli O157:H7 and E. coli O157:NM. The E. coli capture was measured by the time resolved fluorescence (TRF) intensity using a sandwich assay which we have previously demonstrated of having a sensitivity of 1 CFU/g after 4.5 hour enrichment [1]. The analyses of measured TRF intensity and determined antibody surface concentration indicated that larger beads provided higher response signals than smaller beads and were more effective in capturing the target of interest in pure culture and ground beef. In addition, while each type of IMB showed different favorable capture of E. coli O157:H7, streptavidin-coated IMB elicited the highest response, on average. Streptavidin-coated IMB also provided an economic benefit, costing less than $0.50 per assay. The results could be used to guide the proper choice of IMB for applications in developing detection processes for E. coli O157:H7. Full article

In animal diets optimal amino acid quantities and balance among amino acids is of great nutritional importance. Essential amino acid deficiencies have negative impacts on animal physiology, most often expressed in sub-optimal body weight gains. Over supplementation of diets with amino acids is costly and can increase the nitrogen emissions from animals. Although in vivo animal assays for quantification of amino acid bioavailability are well established, Escherichia coli-based bioassays are viable potential alternatives in terms of accuracy, cost, and time input. E. coli inhabits the gastrointestinal tract and although more abundant in colon, a relatively high titer of E. coli can also be isolated from the small intestine, where primary absorption of amino acids and peptides occur. After feed proteins are digested, liberated amino acids and small peptides are assimilated by both the small intestine and E. coli. The similar pattern of uptake is a necessary prerequisite to establish E. coli cells as accurate amino acid biosensors. In fact, amino acid transporters in both intestinal and E. coli cells are stereospecific, delivering only the respective biological L-forms. The presence of free amino- and carboxyl groups is critical for amino acid and dipeptide transport in both biological subjects. Di-, tri- and tetrapeptides can enter enterocytes; likewise only di-, tri- and tetrapeptides support E. coli growth. These similarities in addition to the well known bacterial genetics make E. coli an optimal bioassay microorganism for the assessment of nutritionally available amino acids in feeds. Full article

Ample evidence exists showing that eukaryotic signal molecules synthesized and released by the host can activate the virulence of opportunistic pathogens. The sensitivity of prokaryotes to host signal molecules requires the presence of bacterial sensors. These prokaryotic sensors, or receptors, have a double function: stereospecific recognition in a complex environment and transduction of the message in order to initiate bacterial physiological modifications. As messengers are generally unable to freely cross the bacterial membrane, they require either the presence of sensors anchored in the membrane or transporters allowing direct recognition inside the bacterial cytoplasm. Since the discovery of quorum sensing, it was established that the production of virulence factors by bacteria is tightly growth-phase regulated. It is now obvious that expression of bacterial virulence is also controlled by detection of the eukaryotic messengers released in the micro-environment as endocrine or neuro-endocrine modulators. In the presence of host physiological stress many eukaryotic factors are released and detected by Gram-negative bacteria which in return rapidly adapt their physiology. For instance, Pseudomonas aeruginosa can bind elements of the host immune system such as interferon-γ and dynorphin and then through quorum sensing circuitry enhance its virulence. Escherichia coli sensitivity to the neurohormones of the catecholamines family appears relayed by a recently identified bacterial adrenergic receptor. In the present review, we will describe the mechanisms by which various eukaryotic signal molecules produced by host may activate Gram-negative bacteria virulence. Particular attention will be paid to Pseudomonas, a genus whose representative species, P. aeruginosa, is a common opportunistic pathogen. The discussion will be particularly focused on the pivotal role played by these new types of pathogen sensors from the sensing to the transduction mechanism involved in virulence factors regulation. Finally, we will discuss the consequence of the impact of host signal molecules on commensally or opportunistic pathogens associated with different human tissue. Full article

Optical phenomena such as fluorescence, phosphorescence, polarization, interference and non-linearity have been extensively used for biosensing applications. Optical waveguides (both planar and fiber-optic) are comprised of a material with high permittivity/high refractive index surrounded on all sides by materials with lower refractive indices, such as a substrate and the media to be sensed. This arrangement allows coupled light to propagate through the high refractive index waveguide by total internal reflection and generates an electromagnetic wave—the evanescent field—whose amplitude decreases exponentially as the distance from the surface increases. Excitation of fluorophores within the evanescent wave allows for sensitive detection while minimizing background fluorescence from complex, “dirty” biological samples. In this review, we will describe the basic principles, advantages and disadvantages of planar optical waveguide-based biodetection technologies. This discussion will include already commercialized technologies (e.g., Corning’s EPIC^® Ô, SRU Biosystems’ BIND^™, Zeptosense^®, etc.) and new technologies that are under research and development. We will also review differing assay approaches for the detection of various biomolecules, as well as the thin-film coatings that are often required for waveguide functionalization and effective detection. Finally, we will discuss reverse-symmetry waveguides, resonant waveguide grating sensors and metal-clad leaky waveguides as alternative signal transducers in optical biosensing. Full article

This review presents a deep insight into the Surface Generated Acoustic Wave (SGAW) technology for biosensing applications, based on more than 40 years of technological and scientific developments. In the last 20 years, SGAWs have been attracting the attention of the biochemical scientific community, due to the fact that some of these devices - Shear Horizontal Surface Acoustic Wave (SH-SAW), Surface Transverse Wave (STW), Love Wave (LW), Flexural Plate Wave (FPW), Shear Horizontal Acoustic Plate Mode (SH-APM) and Layered Guided Acoustic Plate Mode (LG-APM) - have demonstrated a high sensitivity in the detection of biorelevant molecules in liquid media. In addition, complementary efforts to improve the sensing films have been done during these years. All these developments have been made with the aim of achieving, in a future, a highly sensitive, low cost, small size, multi-channel, portable, reliable and commercially established SGAW biosensor. A setup with these features could significantly contribute to future developments in the health, food and environmental industries. The second purpose of this work is to describe the state-of-the-art of SGAW biosensors for the detection of pathogens, being this topic an issue of extremely importance for the human health. Finally, the review discuses the commercial availability, trends and future challenges of the SGAW biosensors for such applications. Full article

The detection and identification of pathogen microorganisms still rely on conventional culturing techniques, which are not suitable for on-site monitoring. Therefore, a great research challenge in this field is focused on the need to develop rapid, reliable, specific, and sensitive methods to detect these bacteria at low cost. Moreover, the growing interest in biochip development for large scale screening analysis implies improved miniaturization, reduction of analysis time and cost, and multi-analyte detection, which has nowadays become a crucial challenge. This paper reviews multiplexed foodborne pathogen microorganisms detection methods based on electrochemical sensors incorporating microarrays and other platforms. These devices usually involve antibody-antigen and DNA hybridization specific interactions, although other approaches such as the monitoring of oxygen consumption are also considered. Full article

Salmonella is a leading cause of foodborne illness in the United States, with poultry and poultry products being a primary source of infection to humans. Poultry may carry some Salmonella serovars without any signs or symptoms of disease and without causing any adverse effects to the health of the bird. Salmonella may be introduced to a flock by multiple environmental sources, but poultry feed is suspected to be a leading source. Detecting Salmonella in feed can be challenging because low levels of the bacteria may not be recovered using traditional culturing techniques. Numerous detection methodologies have been examined over the years for quantifying Salmonella in feeds and many have proven to be effective for Salmonella isolation and detection in a variety of feeds. However, given the potential need for increased detection sensitivity, molecular detection technologies may the best candidate for developing rapid sensitive methods for identifying small numbers of Salmonella in the background of large volumes of feed. Several studies have been done using polymerase chain reaction (PCR) assays and commercial kits to detect Salmonella spp. in a wide variety of feed sources. In addition, DNA array technology has recently been utilized to track the dissemination of a specific Salmonella serotype in feed mills. This review will discuss the processing of feeds and potential points in the process that may introduce Salmonella contamination to the feed. Detection methods currently used and the need for advances in these methods also will be discussed. Finally, implementation of rapid detection for optimizing control methods to prevent and remove any Salmonella contamination of feeds will be considered. Full article

Electronic-nose devices have received considerable attention in the field of sensor technology during the past twenty years, largely due to the discovery of numerous applications derived from research in diverse fields of applied sciences. Recent applications of electronic nose technologies have come through advances in sensor design, material improvements, software innovations and progress in microcircuitry design and systems integration. The invention of many new e-nose sensor types and arrays, based on different detection principles and mechanisms, is closely correlated with the expansion of new applications. Electronic noses have provided a plethora of benefits to a variety of commercial industries, including the agricultural, biomedical, cosmetics, environmental, food, manufacturing, military, pharmaceutical, regulatory, and various scientific research fields. Advances have improved product attributes, uniformity, and consistency as a result of increases in quality control capabilities afforded by electronic-nose monitoring of all phases of industrial manufacturing processes. This paper is a review of the major electronic-nose technologies, developed since this specialized field was born and became prominent in the mid 1980s, and a summarization of some of the more important and useful applications that have been of greatest benefit to man. Full article

Rapid pathogen sensing remains a pressing issue today since conventional identification methodsare tedious, cost intensive and time consuming, typically requiring from 48 to 72 h. In turn, chip based technologies, such as microarrays and microfluidic biochips, offer real alternatives capable of filling this technological gap. In particular microfluidic biochips make the development of fast, sensitive and portable diagnostic tools possible, thus promising rapid and accurate detection of a variety of pathogens. This paper will provide a broad overview of the novel achievements in the field of pathogen sensing by focusing on methods and devices that compliment microfluidics. Full article

Pathogenic bacteria are one of the major concerns in food industries and water treatment facilities because of their rapid growth and deleterious effects on human health. The development of fast and accurate detection and identification systems for bacterial strains has long been an important issue to researchers. Although confirmative for the identification of bacteria, conventional methods require time-consuming process involving either the test of characteristic metabolites or cellular reproductive cycles. In this paper, we review recent sensing strategies based on micro- and nano-fabrication technology. These technologies allow for a great improvement of detection limit, therefore, reduce the time required for sample preparation. The paper will be focused on newly developed nano- and micro-scaled biosensors, novel sensing modalities utilizing microfluidic lab-on-a-chip, and array technology for the detection of pathogenic bacteria. Full article

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. Full article

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform. Full article

Microbial biofilms are a major impediment to the use of indwelling medical devices, generating device-related infections with high morbidity and mortality. Major efforts directed towards preventing and eradicating the biofilm problem face difficulties because biofilms protect themselves very effectively by producing a polysaccharide coating, reducing biofilm sensitivity to antimicrobial agents. Techniques applied to combating biofilms have been primarily chemical. These have met with partial and limited success rates, leading to current trends of eradicating biofilms through physico-mechanical strategies. Here we review the different approaches that have been developed to control biofilm formation and removal, focusing on the utilization of acoustic energy to achieve these objectives. Full article