Detection of bacterial contamination in food matrices by integration of quorum sensing in a paper-strip test.

There are an estimated 48 million cases of foodborne illness in the United States every year. In general, these illnesses are the result of unintentional contamination and improper food handling. Because bacterial contamination plays a major role in food spoilage and, hence, in foodborne illnesses, it is important to design easy, portable methods to detect bacteria in food.

Transcriptional regulatory proteins as biosensing tools.

We have developed sensing systems employing different classes of transcriptional regulatory proteins genetically and chemically modified to incorporate a fluorescent reporter molecule for detection of arsenic, hydroxylated polychlorinated biphenyls (OH-PCBs), and cyclic AMP (cAMP). These are the first examples of optical sensing systems based on transcriptional regulatory proteins.

Whole-Cell Biosensors as Tools for the Detection of Quorum-Sensing Molecules: Uses in Diagnostics and the Investigation of the Quorum-Sensing Mechanism.

Genetically engineered bacterial whole-cell biosensors are powerful tools that take advantage of bacterial proteins and pathways to allow for detection of a specific analyte. These biosensors have been employed for a broad range of applications, including the detection of bacterial quorum-sensing molecules (QSMs). Bacterial QSMs are the small molecules bacteria use for population density-dependent communication, a process referred to as quorum sensing (QS).

Deciphering bacterial universal language by detecting the quorum sensing signal, autoinducer-2, with a whole-cell sensing system.

Bacteria communicate with neighboring bacteria of the same species or of other species by means of chemical signaling molecules. The concentration of such signaling molecules is proportional to the bacterial population size; upon reaching a threshold concentration, corresponding to a threshold cell density, certain specialized genes are expressed. This system of communication among bacteria is known as quorum sensing (QS).

Bioluminescence inhibition assay for the detection of hydroxylated polychlorinated biphenyls.

Hydroxylated polychlorinated biphenyls (OH-PCBs) are an important class of contaminants that mainly originate from polychlorinated biphenyl metabolism. They may conceivably be as dangerous and persistent as the parent compounds; most prominently, OH-PCBs are endocrine disruptors. Due to increasing evidence of the presence of OH-PCBs in the environment and in living organisms, including humans, and of their toxicity, methods of detection for OH-PCBs are needed in the environmental and medical fields.

Engineered cells as biosensing systems in biomedical analysis.

Over the past two decades there have been great advances in biotechnology, including use of nucleic acids, proteins, and whole cells to develop a variety of molecular analytical tools for diagnostic, screening, and pharmaceutical applications. Through manipulation of bacterial plasmids and genomes, bacterial whole-cell sensing systems have been engineered that can serve as novel methods for analyte detection and characterization, and as more efficient and cost-effective alternatives to traditional analytical techniques. Bacterial cell-based sensing systems are typically sensitive, specific and selective, rapid, easy to use, low-cost, and amenable to multiplexing, high-throughput, and miniaturization for incorporation into portable devices.

Investigating the effect of antibiotics on quorum sensing with whole-cell biosensing systems.

Quorum sensing (QS) allows bacteria to communicate with one another by means of QS signaling molecules and control certain behaviors in a group-based manner, including pathogenicity and biofilm formation. Bacterial gut microflora may play a role in inflammatory bowel disease pathogenesis, and antibiotics are one of the available therapeutic options for Crohn's disease. In the present study, we employed genetically engineered bioluminescent bacterial whole-cell sensing systems as a tool to evaluate the ability of antibiotics commonly employed in the treatment of chronic inflammatory conditions to interfere with QS.

A protein switch sensing system for the quantification of sulfate.

Protein engineering has generated versatile methods and technologies that have been instrumental in advancements in the fields of sensing, therapeutics, and diagnostics. Herein, we demonstrate the employment of rational design to engineer a unique bioluminescence-based protein switch. A fusion protein switch combines two totally unrelated proteins, with distinct characteristics, in a manner such that the function of one protein is dependent on another.

Bacterial spores as platforms for bioanalytical and biomedical applications.

Genetically engineered bacteria-based sensing systems have been employed in a variety of analyses because of their selectivity, sensitivity, and ease of use. These systems, however, have found limited applications in the field because of the inability of bacteria to survive long term, especially under extreme environmental conditions. In nature, certain bacteria, such as those from Clostridium and Bacillus genera, when exposed to threatening environmental conditions are capable of cocooning themselves into a vegetative state known as spores.

Integration of spore-based genetically engineered whole-cell sensing systems into portable centrifugal microfluidic platforms.

Bacterial whole-cell biosensing systems provide important information about the bioavailable amount of target analytes. They are characterized by high sensitivity and specificity/selectivity along with rapid response times and amenability to miniaturization as well as high-throughput analysis. Accordingly, they have been employed in various environmental and clinical applications.

Packaging sensing cells in spores for long-term preservation of sensors: a tool for biomedical and environmental analysis.

Whole-cell sensing systems have successfully been employed for detection of various biologically and environmentally important analytes. A limitation to their use for on-field analysis is the paucity of preservation methods for long-term storage and transport. For that, we have previously developed spore-based genetically engineered whole-cell sensing systems that are able not only to maintain the activity of the sensing cells but also to preserve it for long periods of time in normal and extreme environmental conditions.

Fluorescent and bioluminescent cell-based sensors: strategies for their preservation.

Luminescent whole-cell biosensing systems have been developed for a variety of analytes of environmental, clinical, and biological interest. These analytical tools allow for sensitive, rapid, simple, and inexpensive quantitative detection of target analytes. Furthermore, they can be designed to be nonspecific, semispecific, or highly specific/selective.

Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples.

Bacteria communicate among themselves using certain chemical signaling molecules. These signaling molecules generally are N-acyl homoserine lactones (AHLs) in Gram-negative bacteria and oligopeptides in Gram-positive bacteria. In addition, both Gram-positive and Gram-negative bacteria produce a family of signaling molecules known as autoinducer-2 that they employ for their communications.

Construction of spores for portable bacterial whole-cell biosensing systems.

Whole-cell sensing systems based on living genetically engineered bacteria are known to have high sensitivity, selectivity, and rapid response times. Although these systems have found applications in biomedical and environmental analyses, their limited shelf life and transportability still restrict their use for on-site monitoring of analytes. To that end, we have developed a new method for the long-term preservation, storage, and transport of whole-cell biosensing systems that is based on bacterial spores, a dormant form of life.

Hydroxylated polychlorinated biphenyl detection based on a genetically engineered bioluminescent whole-cell sensing system.

The metabolites of polychlorinated biphenyls (PCBs), such as hydroxylated PCBs (OH-PCBs), have been identified as environmental contaminants. Various studies have shown that some OH-PCBs can potentially contribute to health problems. Detection of these compounds in environmental and biological samples could provide useful information about their levels and lead to a better understanding of their apparent toxicity.

Biosensing systems for the detection of bacterial quorum signaling molecules.

Bacterial quorum sensing (QS) is a cell-to-cell communication phenomenon that allows bacteria to control the expression of certain specialized genes depending on their cell population size. Signaling molecules such N-acylhomoserine lactones (AHLs) mediate the communication, and their concentration reflects the bacterial population density. Quorum sensing regulates several processes including bacterial pathogenicity.

Regulation of ileal bile acid-binding protein expression in Caco-2 cells by ursodeoxycholic acid: role of the farnesoid X receptor.

Ursodeoxycholic acid (UDCA) is beneficial in cholestatic diseases but its molecular mechanisms of action remain to be clearly elucidated. Other bile acids, such as chenodeoxycholic (CDCA), are agonists for the nuclear farnesoid X receptor (FXR) and regulate the expression of genes relevant for bile acid and cholesterol homeostasis. In ileal cells CDCA, through the FXR, up-regulates the expression of the ileal bile acid-binding protein (IBABP), implicated in the enterohepatic circulation of bile acids.

Biotechnological applications of bioluminescence and chemiluminescence.

Recent progress in molecular biology has made available several biotechnological tools that take advantage of the high detectability and rapidity of bioluminescence and chemiluminescence spectroscopy. These developments provide inroads to in vitro and in vivo continuous monitoring of biological processes (e.g.

Use of a gas-sensor array for detecting volatile organic compounds (VOC) in chemically induced cells.

An application of gas sensors for rapid bioanalysis is presented. An array of temperature-modulated semiconductor sensors was used to characterize the headspace above a cell culture. Recombinant Saccharomyces cerevisiae yeast cells, able to respond to 17 beta-estradiol by producing a reporter protein, were used as a model system.

Bioluminescence and chemiluminescence in drug screening.

Drug screening, that is, the evaluation of the biological activity of candidate drug molecules, is a key step in the drug discovery and development process. In recent years, high-throughput screening assays have become indispensable for early stage drug discovery because of the developments in synthesis technologies, such as combinatorial chemistry and automated synthesis, and the discovery of an increasing number of new pharmacological targets. Bioluminescence and chemiluminescence represent suitable detection techniques for high-throughput screening because they allow rapid and sensitive detection of the analytes and can be applied to small-volume samples.

A rapid and sensitive 384-well microtitre format chemiluminescent enzyme immunoassay for 19-nortestosterone.

We developed a competitive chemiluminescent (CL) enzyme immunoassay for rapid, sensitive analysis of 19-nortestosterone (19-NT) in bovine urine. Anti-19-NT polyclonal antibodies were raised in rabbits using a 19-NT-hemisuccinate derivative conjugated with ovalbumin; the derivative was also conjugated with horseradish peroxidase (HRP) as a label. Antibodies were immobilized on 384-well black polystyrene microtitre plates and HRP-labelled 19-NT activity was measured using an efficient chemiluminescent substrate (SuperSignal ELISA Femto) after 3 min incubation.