Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems

This chapter is a brief introduction to pathogenic microorganisms and also discusses virulence factors. An understanding of virulence factors is important, as they represent potential targets for the detection of microbial pathogens. Sources and routes of infection are also briefly discussed with reference to specific examples. There are a number of ways in which infection could be acquired, including via contaminated food and water; hospital acquired infection; “naturally acquired” infection; and intentional infection, for example, through the use of biological warfare agents. The focus of the review is predominantly on human pathogens. However, there are a range of other microbial pathogens of particular importance in other areas; for example, animal and plant pathogens, which will not be discussed. Finally, a brief overview of the detection of pathogenic bacteria is presented.

Rapid microbial testing is more and more preferred worldwide. Conventional time-consuming methods with detection times taking up to several days are being replaced by rapid tests that take only a few hours. With the development of new, rapid, and accurate methods for the detection of bacterial contaminants, the requirements for sample preparation techniques are more and more challenging. In fact, sample preparation is the critical step with respect to the applicability of novel methods. Sample preparation comprises sampling/sample drawing, sample handling, and sample preparation. To fulfil the demands of modern microbiology the ideal procedure should permit rapidly providing the processed sample in a small volume which contains the analyte in the highest concentration possible. The analyte has to be free of substances interfering with the detection method to be finally applied. Additionally, sample processing procedures used should not result in any loss of the bacterial analyte, thereby enabling quantitative measurements.

Techniques for the preparation of samples subjected to microbiological examination are described, especially focusing on the methods applied to investigate the occurrence of pathogenic organisms in foods as well as in the food processing environment.

Sample drawing methods for the monitoring of air and surfaces are outlined. Moreover, different sample preparation methods intended to be carried out prior to the detection of intact bacterial cells or bacterial nucleic acids are discussed in detail. Special attention is paid to magnetic particle-based separation methods, as these tools have gained increasing importance due to their outstanding advantages.

Successful pathogen detection depends on analyst’s understanding of the nature of the matrix and the properties of the targeted microorganism. The matrix could be simple (e.g., drinking water) and easy to analyze for pathogens, or complex (e.g., fermented meat products or fecal samples) and requires an elaborate method to isolate the targeted microorganism. Some pathogens are recovered easily on common laboratory media but others may need time-consuming resuscitation on specialized media with incubation under strictly controlled conditions. Currently used methods for detecting pathogens rely on culture, immunological, genetic, and other techniques. These methods often include a preliminary step to amplify the pathogen’s population or a signal representing this microorganism. Enrichment is the most commonly used, but highly unpopular, technique to accomplish the amplification just described. In culture-based detection methods, the targeted pathogen is isolated from the enrichment using selective and differential media, then identified on the basis of multiple biochemical properties. Alternatively, the identification is accomplished by immunological or genetic techniques. Identification as commonly done does not prove the pathogenicity of the targeted organism, a deficiency that needs to be rectified in future detection methods. Rapid detection of pathogens in real time by means that are not destructive to the matrix is an idealistic goal that may materialize in near future.

In recent years there have been significant advances in microbiology, achieved through the sister sciences of chemistry, molecular biology, and computer aided imaging. These have resulted in a significant increase in the methods available for the detection, enumeration, and identification of microorganisms in the laboratory. This chapter provides a brief overview of the types of technologies available and the premise of how they work.

Modern biosensor technologies can provide rapid quantification of bacterial pathogens. Surface plasmon resonance (SPR) sensors are an optical platform capable of highly sensitive and specific measuring of biomolecular interactions in real-time. This label-free technology can quantify the kinetics, affinity and concentration of surface interactions. SPR sensors have been used to detect bacterial pathogens in clinical and food-related samples. This chapter discusses the fundamental theory behind SPR sensors and state-of-the-art SPR instrumentation, surface chemistries, molecular recognition elements and detection strategies, as well as specific challenges associated with bacterial detection using SPR sensors. SPR-based detections of bacterial cells, genetic markers and antibody biomarkers are reviewed and discussed.

Detection and identification of bacteria is an important aspect of our world today. Outbreaks of pathogenic bacteria, either occurring naturally in food or possibly being used as weapons by bioterrorists for contamination of food, air, and water, are constantly in the news. Identifying the specific bacteria responsible for these outbreaks and their potential source is of great importance. Biosensors, specifically evanescent wave-based fluorescence biosensors, are evolving to meet these challenges. In evanescent wave sensors, light is launched into an optical waveguide at such an angle that the light is internally reflected completely at the interface of the waveguide and the surrounding medium. An electromagnetic wave is generated at the surface that penetrates 100–200 nm into the surrounding medium (air, buffer). Fluorophores that are bound within this region by the target bacteria and a recognition molecule are excited. Many different configurations of this method have been developed and will be discussed in this chapter.

Rapid and specific identification of bacteria is critical for clinical and biosafety applications. Fiber optic biosensors (FOBs) are increasingly being applied to the detection of bacteria in food and water supplies, food processing facilities, and homeland security operations. These biosensors can be used for multiplexed pathogen detection or to confirm the results of other techniques, often in less than one hour. FOBs offer several advantages over conventional culture-based techniques, or polymerase chain reaction (PCR)-based assays, in terms of speed, specificity, and depth of information content. In addition, some sensor platforms have been developed into portable systems capable of emergency field deployment. In this chapter, we will discuss the detection of bacteria using fiber optic immunosensors, nucleic acid-based FOBs in various assay formats, and several applications of these technologies.

Rapid, specific, and sensitive detection of pathogenic bacteria is very important in areas like food safety, medical diagnostics, hospital infection, and biological warfare. Optical evanescent wave sensors are evolving to meet these challenges. Evanescent wave biosensors generate an electromagnetic wave at the sensor surface that penetrates 100–200 nm into the surrounding medium, and have proven to be a highly sensitive tool to monitor interactions in the close vicinity of the sensor surface. However, the use of such waveguides for bacterial detection is problematic for several reasons. These include the short penetration depth of the evanescent field of these waveguides (100–200 nm) compared to the typical size of a bacterium (1–5 μm), which places the majority of the bound cell outside the evanescent field. In addition, the low refractive index contrast between the bacterium cytoplasm and the aqueous environments in which detection is usually performed, as well as the availability and accessibility of antigens on the bacterium surface binding to the biorecognition elements. Finally, the sensor performance can be limited due to (1) the mass transport of large analytes like bacteria, which limits the binding to the immobilized recognition receptors; (2) non-specific binding; and (3) long analysis time.

This chapter will focus on the development of different configurations of deep-probe optical evanescent wavesensors such as metal-clad leaky waveguides (MCLW) and waveguide sensors with low-index substrates for bacterialdetection. In addition, two complete detection systemsintegrated with physical force fields to overcome these problems will be presented. These sensor systems are basedon MCLW sensors and integrated with, respectively, an electric field and ultrasound standing waves as a physical force to concentrate and enhance the capture of bacteria spores into immobilized antibodies on the sensor surface.The integration improves the detection limit by a few orders of magnitude and shortens the analysis time significantly.

Every chemical reaction or interaction causes a change in refractive index, including such bioconjugate interactions as antibody/antigen, DNA hybridization and enzyme/substrate interactions. Interferometry is an optical method for measuring refractive index changes. With the proper choice of sensing film, an interferometer can identify and quantify the presence of a biological moiety. An interferometer compares optically two almost equivalent light paths – one that interrogates the refractive index change caused by a bioconjugate interaction, and the other that serves as a reference that cancels out any nonspecific interactions. Interferometers have the capability of detecting refractive index changes of 10

-7

, which corresponds to ppb concentrations of small molecules, pg/mL concentrations of toxins and proteins, and 100s–1000s of whole cells, viruses and spores. Several optical interferometric designs are described. Most configurations combine a bioconjugate reaction isolated on a rigid support with a long interaction length of mm to cm to achieve high sensitivity. The most common interferometric configuration utilizes a planar optical waveguide. The evanescent field associated with a wave-guided beam extends above the waveguide surface where the bioreceptor is immobilized. The bioconjugate interaction perturbs the propagating beam and the extent of this perturbation is measured by comparing the phase of the light traveling along the sensing channel with that traveling along a reference channel that is not functionalized with the bioreceptor. The phase change is measured by optically combining the two beams at the output of the interferometer to create an interference pattern, a series of dark and light fringes that is caused by constructive and destructive interference. By proper choice of receptor molecule and calibration, both the identity and the quantity of a specific bioentity can be measured with the interferometric biosensor.

Luminescence-based techniques for the detection of microbial pathogens are extensively employed in industrial setting where the continuous monitoring of bacterial contamination is of great importance. The primary advantage of all luminescence-based assays is their rapidity and sensitivity. Here we describe two different types of luminescence systems that have been adapted for commercial use, bioluminescence (BL) and chemiluminescence (CL). BL is a naturally occurring process by which living organisms convert chemical energy into light. Light-emitting pathways have been identified in bacteria, insects, and other eukaryotic organisms. Bacterial (

lux

) systems have been extensively studied and have been engineered for a variety of purposes. In the most common adaptation of the

lux

genes for the microbial detection, luciferase reporter phages are constructed for the direct and specific identification of many bacterial species including

Salmonella

spp.,

Listeria

, and

E. coli

O157:H7. Central to the

lux

reaction is that bioluminescence is dependent on higher-level energy intermediates, allowing levels of light to be correlated to changes in bacterial metabolism. The firefly (

LUC

) luciferase is also widely used in biotechnology. Since all living things possess intracellular pools of ATP, many applications of the

LUC

system capitalize on the ATP-dependency of this luminescence reaction for the detection of microbial populations in situ. The

LUC

system is also useful in determining the efficacy of sanitizing agents, as decreases in BL are proportional to the number of active bacteria within a defined matrix. Other eukaryotic luciferases, such as those from marine copepod

Gaussia princeps

and Jamican click beetle, are currently been explored as alternative means for bacterial detection in extreme environmental conditions, and in situations where the simultaneous detection of multiple bacterial species is desired. CL is generally defined as the production of light by chemicals during an exothermic reaction, and CL differs from BL in that light production is not catalyzed by biological reactions. Although not as widely used in industrial applications, CL is sometimes preferred to BL-based detection systems due to the relative simplicity of the reaction and the elimination of certain steps sometimes required for the optimization of BL. CL has been used mainly for the detection of foodborne pathogens in combination with immunoassays. Using CL-linked antibodies specific for certain bacterial antigens, allows the simultaneous detection of

E. coli

O157:H7,

Yersinia enterocolitica, Salmonella typhimurium

, and

Listeria monocytogenes

. Luminescence-based techniques are proven effective agents in the detection of contaminating microbial populations, and with increases in the sensitivity and simplicity of such techniques, their application in numerous industrial and commercial settings will only grow.

The development of novel sensors able to produce a response directly upon binding of a target biomolecular analyte remains a major area of research in materials science and analytical chemistry. As the primary “raw material” for the microelectronics industry, and because of its biocompatibility and optical properties, silicon has drawn considerable attention in this field. This chapter discusses the current state of efforts in our group and others to develop porous and planar sensors based on silicon. Porous silicon, so-called because of its complex three-dimensional network structure, provides a high internal binding surface and allows for observation of binding by changes in the reflectivity or luminescence spectra of single- or multi-layer devices. Planar silicon sensors, exemplified here by Arrayed Imaging Reflectometry (AIR), do not have the high surface area of porous silicon yet still respond with a high degree of sensitivity to the binding of analytes to the sensing surface. Examples are presented for the use of both types of sensors for the detection of DNA, proteins, and pathogenic bacteria.

This chapter is focused on the development and use of acoustic wave biosensor platforms for the detection of bacteria, specifically those based on the thickness shear mode (TSM) resonator. We demonstrated the mechanical and electrical implications of bacterial positioning at the solid-liquid interface of a TSM biosensor and presented a model of the TSM with bacteria attached operating as coupled oscillators. The experiments and model provide an understanding of the nature of the signals produced by acoustic wave devices when they are used for testing bacteria. The paradox of “negative mass” could be a real threat to the interpretation of experimental results related to the detection of bacteria. The knowledge of the true nature of “negative mass” linked to the strength of bacteria attachment will contribute significantly to our understanding of the results of “weighing bacteria.” The results of this work can be used for bacterial detection and control of processes of bacterial settlement, bacterial colonization, biofilm formation, and bacterial infection in which bacterial attachment plays a role.

Biosensor technology has the potential to speed the detection of food pathogen and to increase specificity and sensitivity of the analysis. 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 electrochemical biosensor can be based on potentiometric, amperometric or impedimetric/conductimetric transducers. In this chapter, amperometric transducers will be described in detail. In particular amperometric biosensors for food pathogen will be reviewed as microbial metabolism-based, antibody-based (immunosensor), and DNA-based biosensor.

In this chapter, potentiometric detection methods for microbial DNA involved recognition events by use of genetic field effect devices will be described. Fundamental principles of field effect devices and the technical background with their ongoing applications in the field of bio-sensor technologies, termed bio-FET, will be first introduced. Then concept of genetic field effect transistor will be described with emphasis on their fabrication, characteristics, and recent applications to microbial Single Nucleotide Polymorphysms (SNPs) Analysis as well as DNA sequencing. By comparing to other conventional methods, technical significance and future perspective of the genetic field effect transistor will also be discussed in detail.

Electrochemical impedance spectroscopy (EIS) is an important detection technique for biosensors. In the field of immunosensors, and particularly pathogen detection, it is one of the preferred electrochemical techniques because it does away with the use of enzyme labels or redox mediators. This chapter provides an introduction to the fundamentals of EIS and basic data analysis, with an emphasis on the most common features found in immunosensors and possible experimental limitations.

This chapter then discusses a series of functionalisation approaches that can be used in the development of an immunosensor for the detection of bacteria. This is followed by a selection of impedance-based immunosensor examples from the literature.

There is an increasing public awareness and concern regarding the safety of our food supply. The complexity of the US food supply chain provides numerous entry points and routes in which pathogens and other disease-causing organisms can be introduced into the nation’s food system. Foodborne illness and product recalls have been increasing in incidence. It is not easy to address the hundreds of microbial contaminants associated with microbial foodborne diseases. Conventional methods of identifying these pathogens require 2–7 days. Although these methods are highly sensitive and specific, they are elaborate and laborious. The use of biosensors as emerging technologies could revolutionize the study and detection of these foodborne microorganisms. The development of biosensors will further serve the food industry, agricultural sector, regulatory community, and public health. Biosensor techniques will play an extensive role in understanding the occurrence of contamination at the source during the next decade and help forecast the potential for risk and mitigation before foodborne outbreaks occur. This chapter describes emerging and novel biosensor technologies for rapid and sensitive detection of pathogens of concern to the food supply. Particularly, molecular nanowires as transducers in biosensor devices are covered. Antibody and DNA based biosensors are reviewed and two illustrations on immunosensors are presented.

In situations of widespread infectious disease an action that might result, the rapid diagnosis of pathogenic states will assist first responders in implementing prompt treatments, in a huge reduction in the number of illnesses and deaths. Currently available detection/diagnostic procedures are either time-consuming (8–48 h) and require enrichment and culturing of bacteria before testing, or provide only qualitative results. Magnetic immunoassay technology appears to have particularly superior performance over other immunodetection methods. A typical magnetic immunoassay entails a capture part and a detection part, between which the target is immobilized. The capture part of the immunoassay consists of magnetic particles functionalized to capture the target from the sample. The immobilized target is then sandwiched between the capture and detection complexes and subjected to a detection process that will provide accurate and rapid results, most of the time in a matter of minutes. Another important advantage that a sensitive magnetic immunoassay confers is the reduced volume of samples and reagents needed. This chapter discusses the elements associated with a magnetic immunoassay specifically designed for the rapid detection of pathogens. The chapter presents a review of the different techniques used in the synthesis and encapsulation of magnetic particles, as well as strategies for the immobilization and detection of the targeted pathogen. Several magnetic separation strategies are also discussed.

In this chapter we summarize briefly the use of cantilever sensors for pathogen detection. Both micro- and macro-cantilever sensors have been investigated for detecting pathogens in liquid samples. In this review we examine previous work and summarize progress on using piezoelectric-excited millimeter-sized cantilever (PEMC) sensors developed in the author’s laboratory. PEMC sensors immobilized with an antibody specific to the target pathogen has been shown to be very highly sensitive for detecting one cell per mL in one liter samples and 10 cells per mL in 10,mL samples, both in buffers and at similar concentrations in food matrices. After a brief introduction, the physics of sensing is reviewed, followed by a characterization of PEMC sensors, and finally the results from detection experiments are described.

In this chapter, we explore technology developments for the rapid detection, identification, and viability assessment of endospore-forming pathogens with a focus on

Bacillus anthracis

. First, we introduce various toxin-producing species and their role as bioinsecticides, probiotics, and bioweapons. We also review the role of endospores as biological indicators (i.e., dosimeters) for evaluating sterilization regimens, such as autoclaving and wastewater remediation. Monitoring the effectiveness of cleaning and sterilization regimens to maintain good hygiene is required in several major industries, including health care, food, and pharmaceutical industries. In the next section, we review recent developments in DNA-, immuno-, and dipicolinic acid assays, and their applications for detection and monitoring of

Bacillus anthracis

and other endospore-forming pathogens. Finally, we review viability assays capable of rapid validation of endospore inactivation after sterilization, including assays based on ATP synthesis during stage II germination, and DPA release during stage I germination.

Raman spectroscopy is a label-free technique for generating unique spectral fingerprints from intact microorganisms. Studies conducted for more than a decade have shown that these “whole-organism fingerprints” can be used to identify pathogens, including bacteria, yeasts, and spores, at the strain level, even when the microorganisms are so closely related that they are difficult to distinguish by conventional techniques. Emerging techniques such as Raman microscopy and surface-enhanced Raman scattering (SERS) can enhance the magnitude of the signal to the point that Raman fingerprinting can achieve single-cell sensitivity. More recently, Raman microscopy and SERS have been integrated with biomolecule capture to produce a new microarray technology, dubbed “microSERS,” for rapid identification of pathogens and their toxins in complex samples, without any labels, pre-processing of the sample, or culturing. This chapter reviews the studies that have been done on Raman microscopy and SERS for pathogen identification, and innovative methods for sample collection, concentration, and manipulation that can be combined with fingerprinting techniques. It also presents recent progress on microSERS analysis for the identification of bacteria, spores, and toxins in complex samples; differentiation between viable and nonviable microorganisms; and evaluation of growth conditions on microbial phenotype and specificity/affinity for capture biomolecules.

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.

The ultimate goal in microbial testing is the ability to accurately and sensitively detect pathogens in real-time or as quickly as possible. Nucleic acid diagnostics (NAD) offer many advantages over traditional microbiological and immunological methods for the detection of infections micro-organisms. These include faster processing time as well as greater potential for intra-species identification and identification of antibiotic susceptibility and strain typing based upon unique sequences. The original techniques of PCR and gel electrophoresis are being superseded by real-time PCR while the development of integrated sample preparation and amplification devices with a simplified user interface will allow for true point-of-care disease detection and suitably tailored treatments. This chapter describes the principles of nucleic acid diagnostics including an overview of the technology’s history as well as the general properties of an ideal nucleic acid diagnostics target. Special emphasis is placed upon the detection of pathogens relevant to the food industry. While traditional culture-based methods will retain the lead position as bioanalytical test methods for food safety for the foreseeable future, rapid NAD methods will increasingly compliment or provide alternatives to these methods to meet the ever-evolving challenges in food safety. Ongoing developments in molecular detection platforms including microarrays and biosensors provide potential for new test methods that will enable multi-parameter testing and at-line monitoring for microbial contaminants.

The rapid and unambiguous detection and identification of microorganisms, historically a major challenge of clinical microbiology, gained additional importance in the fields of public health and biodefence. These requirements cannot be well addressed by classical culture-based approaches. Therefore, a wide range of molecular approaches has been suggested. Microarrays are molecular tools that can be used for simultaneous identification of microorganisms in clinical and environmental samples. Main advantages of microarrays are high throughput, parallelism and miniaturization of the detection system. Furthermore, they allow for both high specificity and high sensitivity of the detection.

Microarrays consist of set of probes immobilized on a solid surface. Even though the first application of the microarrays can be seen as relatively recent (Schena et al. 1995), the technology developed rapidly reaching the milestone of 5,000 published papers in 2004 (Holzman and Kolker 2004). This development encompasses both the successful transfer of various technological aspects as well as the expansion of the application scope. The most important technological elements of custom-made platforms as well as the characteristics of the commercially available formats are reviewed in this chapter. Furthermore, application potential is presented together with considerations about quality control.

Protein–carbohydrate interactions are involved in a wide variety of cellular recognition processes including cell growth regulation, differentiation, adhesion, cancer cell metastasis, cellular trafficking, the immune response, and viral or bacterial infections. These specific interactions occur through glycoproteins, glycolipids, polysaccharides found on cell surfaces, and proteins with carbohydrate-binding domains called lectins through cooperative multiple interactions since it is known that individual carbohydrate–protein interactions are generally weak. A common way for bacteria to accomplish adhesion is through their cellular lectins, also called fimbriae or pili, which bind to complementary carbohydrates on the surface of the host tissues. Lectin-deficient mutant bacteria often fail to initiate infection. Carbohydrate-based detection of bacterial pathogens presents an exciting alternative to standard methods for screening and detecting bacterial targets in food industry, water and environment quality control, and clinical diagnosis. Conjugated fluorescent glycopolymers such as conjugated glycopoly(p-phenylene-ethynylene)s, glycopolythiophenes, glycopoly(p-phenylene)s, and carbohydrate-bearing polydiacetylenes have been prepared for quick detection of

Escherichia coli

(

E. coli

) through cooperative multivalent interactions between the polymeric carbohydrates and the bacterial pili because they combine fluorescent scaffolding and carbohydrate reporting functions into one package and possess intrinsic fluorescence and high sensitivity to minor external stimuli. Glyconanoparticles and galactose-functionalized carbon nanotubes (Gal-SWNTs) have been used as three-dimensional systems to study their specific multivalent interactions with

E. coli

. In addition, Gal-SWNTs have also been employed to detect

Bacillus anthracis

spores through divalent cation-mediated multivalent carbohydrate–carbohydrate interactions. Carbohydrate microarrays combine the benefits of immobilized format assays with the capability of detecting thousands of analytes simultaneously and can offer a general and powerful platform for whole-cell applications because their multivalent display of carbohydrates can mimic multivalent interactions at cell–cell interfaces. A simple but very effective diagnostic carbohydrate microarray has been reported for the quick detection of

E. coli

in complex biological mixtures with detection limit of 10

5

–10

6

cells. Wang et al. reported another direct and unique approach to detect pathogenic bacteria by using a carbohydrate microarray of 48 carbohydrate-containing antigenic macromolecules for recognition of carbohydrate-binding antibodies from 20 human serum specimens.

Pathogenic bacteria possess a cell surface capsular polysaccharide (CPS) or lipopolysaccharide (LPS) shell, or both, which helps the pathogen initiate an infection. Lectin microarrays have been utilized as a very important and powerful tool to detect

E. coli

, profile diverse glycan structures of

E. coli

and discover dynamic changes in surface glycosylation of bacteria in response to environmental stimuli through specific multivalent interactions of lectins and the bacterial LPSs.

DNA and RNA are well-known polymers that are central to the existence of every known form of life. Once thought to be strictly passive templates containing genetic information, it has since become clear that these nucleic acids are capable of much more. Synthetic biologists are working to exploit this potential, creating a wide spectrum of “functional nucleic acids”. These molecules can be divided into two broad categories: catalysts (deoxyribozymes and ribozymes) and receptors (aptamers). This chapter begins by providing a background on the field of functional nucleic acids with an emphasis on aptamer technology. One major application of aptamers is their use as recognition elements in sensors of interesting molecules and cell types. Some common designs of these sensors are profiled, explaining how the aptamer-target binding is converted into a detectable signal. The chapter concludes with a discussion of aptamer-based sensors of bacteria, including some of the relevant targets, the progress to date and the future prospects.

Protein-based microarrays is a novel, rapidly evolving proteomic technology with great potential for analysis of complex biological samples. The technology will provide miniaturized set-ups enabling us to perform multiplexed profiling of minute amounts of biological samples in a highly specific, selective, and sensitive manner. In this review, we describe the potential and specific use of protein microarray technology, including both functional protein microarrays and affinity protein microarrays, for the detection and identification of bacteria, bacterial proteins as well as bacterial diseases. To date, the first generations of a variety of set-ups, ranging from small-scale focused biosensors to large-scale semi-dense array layouts for multiplex profiling have been designed. This work has clearly outlined the potential of the technology for a broad range of applications, such as serotyping of bacteria, detection of bacteria and/or toxins, and detection of tentative diagnostic biomarkers. The use of the protein microarray technology for detection and identification of bacterial and protein analytes is likely to increase significantly in the coming years.

Methods for detection of bacterial pathogens have to be rapid, sensitive, specific, inexpensive, easy to perform, and robust. Traditional culture-based plating techniques are hampered by time-consuming enrichment steps. This and other problems are tackled by culture-independent detection methods. The use of bacteriophage or parts thereof for bacterial detection attracts increasing attention, as reflected by a multitude of different phage-based techniques recently reported. Bacterial viruses have been optimized by evolution to specifically target their host organisms and are therefore ideal tools for detection of these microbes. For this purpose, every stage in the replication cycle of phages, from adsorption to host cell lysis, has been exploited.

Phage amplification assays are among the easiest methods to harness the host specificity of phages; the use unmodified phage particles to infect target organisms, followed by amplification of the infection as a signal by addition of helper cells. The capacity of phages to infect and lyse their host cells is utilized in assays which detect the release of cytoplasmic molecules. Cell wall recognition, phage adsorption, and injection of phage DNA into the host bacterium have also been exploited by detection methods, including capture of cells by immobilized phages and labeling of target organisms by fluorescently tagged phage particles. Phage encoded high affinity molecules such as tail fiber proteins or cell wall binding domains of phage endolysins have proven to be suitable for this purpose, especially when coupled with magnetic separation of captured bacteria. For bacterial detection, genetically modified reporter phages introduce reporter genes into their hosts. Upon infection, these gene products are produced in the target cells and can be detected with high sensitivity. Other detection methods employ phages without making use of their own host specificity. Phage display is a popular technique that can be used for production of non-phage derived high affinity molecules for recognition of pathogenic bacteria. Filamentous phages present entire libraries of randomized peptides on their surfaces, and the most suitable ones can be selected by repeated rounds of screening. In conclusion, the use of phages for detection of pathogenic bacteria offers interesting alternatives and advantages compared to traditional analytical methods.

There exists a great need for detecting bacterial pathogens in food, water, environmental, and patient samples. Although significant developments are being made in nucleic acid-based detection, e.g., real time-PCR, there are advantages to detecting microbial antigens using a variety of assays such as enzyme-linked immunosorbent assay (ELISA), antibody arrays, fiber optics, and surface plasmon resonance. Immunological reagents used to detect microbial antigens have mostly consisted of antisera or monoclonal antibodies. However, over the past two decades new methods have been developed that use bacteriophages (phages), viruses of bacteria, as tools to express antibody fragments or random peptides that can detect microbes. The most widely used antibody fragment is the single chain F variable (scFv) portion that includes the antigen-binding regions of the heavy and light chains. The genes encoding antibodies can be obtained either from unimmunized animals or from animals immunized with the target antigen or microbe. scFv libraries are usually constructed in specialized plasmids called phagemids that create fusions of the antibody fragment to a phage coat protein. Phagemids must be packaged into phage particles by the use of helper phages during infection of

E. coli

hosts. Random peptides are usually constructed in phage genomes directly; hence, they do not require helper phages. DNA sequences encoding random peptides and antibodies are most often fused to the

gIII

gene of a filamentous phage such as M13 so that a hybrid pIII protein is expressed on the phage particle. Phage particles that display a fusion peptide that recognizes the desired antigen are selected from the library by a process called panning. Panning involves binding phages to the antigen, washing away unbound phages, eluting the specific phages, and amplification by infection of an

E. coli

host. A major advantage of phage display is that panning and screening of clones can be accomplished in only a few days, as opposed to months for antisera and monoclonal antibodies. Once a phage displaying an antibody fragment or random peptide of desired specificity is isolated, the gene encoding the binding peptide can be genetically manipulated. With scFvs, it is possible to have

E. coli

cells secrete the protein, rather than relying on the use of the phage particle displaying the scFv protein. This chapter reviews the literature on the use of phage display to detect bacteria in a variety of assays. There has been considerable success in the use of commercially available random peptide phage display libraries. Most reported success with scFv phage display has been from using libraries constructed from immunized animals. In addition to antibodies, other target-binding tools are under development, such as affibodies, anticalins, ankyrins, and trinectins. The ease, economy, rapidity, and genetic manipulability of phage display make it an effective tool for developing reagents to detect bacterial pathogens.

There is a trend in biohazard diagnostics to develop integrated systems to extract, concentration and detection from sample matrices. Although biological recognition agents, such as antibodies, can be applied for concentration and detection, there are several limitations. Specifically, biological recognition agents are hard to produce in large quantities, expensive and inherently unstable. Due to such limitations there has been a sustained interest in developing artificial or plastic antibodies that can be readily mass produced, highly stable and cheap. One of the most promising approaches to date has been in the area of Molecular Imprinted Polymers (MIP’s). In basic theory behind MIP’s is to form a polymer matrix around a template (analyte or structural surrogate) which is subsequently removed to leave voids with high affinity for the target analyte. To date, the majority of MIP research has focused on concentrating or detecting low molecular weight analytes in analytical chemistry. However, there has been interest in applying MIP’s to separate, concentrate or detect bioagents such as microbial metabolites, toxins, enzymes and even microbial cells. In the following chapter an overview on the principles of MIP will be outlined. The application of MIP’s as solid phase extraction matrices for separating and concentrating biological agents will be reviewed and recent advances described. The utility of MIP’s as biorecognition elements in biosensor devices will be covered. Finally, future directions in MIP research will be discussed and the main technological barriers to overcome identified.

The disruption of the membrane/coat, or lysis, of bacteria and spores is often a critical step for analyzing the intracellular molecules such as proteins and nucleic acids. In this chapter, we review recent advances in the application of microfluidic devices for lysis of bacteria and spores. We divide existent devices and methods into five categories: mechanical, chemical, thermal, laser, and electrical. We also point out future directions in this field.

The purpose of this chapter is threefold: introducing microfluidics to the general audience, describing in detail the polymerase chain reaction (a technique used for DNA amplification), and reviewing the state-of-the-art methods regarding the detection of pathogens by on-chip PCR. The first section gives a brief introduction to the field of microfluidics. Although the microfluidic technologies have been developed substantially since 1990, their existence and applications are still unknown from the general public. The history and the applications of miniaturized total analysis systems (μTAS) are therefore summarized in the first section (Microfluidics). Secondly, the polymerase chain reaction (PCR) is described in detail. The second section (DNA amplification) therefore covers a brief history of DNA and the applications, requirements, and processes of PCR. As a conclusion of this section, the different techniques available to perform PCR (namely conventional PCR, real-time PCR and on-chip PCR) are compared. Lastly a mini-review presents the state-of-the-art in terms of detection of pathogens by on-chip PCR. The polymerase chain reaction is becoming recognized by official administrations as an acceptable method for the detection of pathogens. It is therefore no surprise that the microfluidic community is also developing devices to support this transition. The last section (Minireview) provides a snapshot of the most exquisite techniques available for the on-chip detection and analysis of pathogens.

Current technologies capable of rapidly and accurately detecting the presence of infectious diseases and toxic compounds in the human body and the environment are inadequate and new, novel techniques are required to ensure the safety of the general population. To develop these technologies, researchers must broaden their scope of interest and investigate scientific areas that have yet to be fully explored. Lithography is a common name given to technologies designed to print materials onto smooth surfaces. More specifically, micropatterning encompasses the selective binding of materials to surfaces in organized microscale arrays. The selective micropatterning of bacteria and viruses is currently an exciting area of research in the field of biomedical engineering and can potentially offer attractive qualities to biosensing applications in terms of increased sensing accuracy and reliability. This chapter focuses on briefly introducing the reader to the fundamentals of bacterial and viral surface interactions and describing several different micropatterning techniques and their advantages and disadvantages in the field of biosensing. The application of these techniques in healthcare and environmental settings is also discussed.

Microfabricated flow cytometry has been extensively investigated recently. Miniaturization of flow cytometers by adopting microfabrication techniques to fabricate microchannels and micro-nozzles in silicon, glass/quartz, and even plastic substrates has been demonstrated. When compared with their large-scale counterparts, these micro flow cytometers are more compact in size, portable, cost-effective, user-friendly, and most importantly, could have almost comparable performance. In this chapter, microfabrication techniques for these micro flow cytometers were first reviewed. The operating principles for cell transportation, focusing, detection, and sorting inside these micro flow cytometers were briefly discussed. Finally, several promising applications including environmental monitoring, rapid assessment of bacterial viability, rapid analysis of bacteria levels in food, antibiotic susceptibility testing, and diagnosis of bacterial in blood and urine were reviewed. It can be envisioned that a portable flow cytometer system can be available for point-of-care applications if these issues can be addressed properly in the near future.

It has been known for millennia that electrostatic forces can be used to manipulate particles; recently developed techniques give sufficient sensitivity, selectivity and precision for the selective trapping and manipulation of bacteria. These forces are all defined from the attraction of charge in an electric field, but exploit it in different ways to achieve different ends. Such phenomena include electrophoresis, the electrostatic attraction of particles; dielectrophoresis, the force generated by the interaction of a particle with a non-uniform, time-variant electric field; and electro-osmosis, where micro-flows are induced in fluid by non-uniform field effects. These phenomena have demonstrated a number of benefits for bacterial study, including particle filtration, preconcentration and identification. These phenomena have much to offer for the field of bacterial detection and analysis. By treating the bacterial cell as an electronic object and hence considering its electrical properties, it is possible to gain important insights into the electrophysiology of the cell. Furthermore, differences in electrophysiology can be exploited to allow the separation or concentration of bacteria prior to analysis. As these technologies all rely on similar devices, they can be integrated into a single lab on a chip device, with many potential benefits including portability and disposability in addition to the ability to detect particles at lower concentrations than ever before.

This chapter introduces the concept of using ultrasound for the manipulation of small particles in fluids for in vitro systems, and in particular how this can be applied to bacterial cells in suspension. The physical phenomena that lead to this effect are discussed, including radiation forces, cavitation, and streaming, thus allowing an appreciation of the limitations and applicability of the technique. Methods for generating ultrasound are described, together with practical examples of how to construct manipulation systems, and detailed examples are given of the current practical techniques of particle manipulation. These include filtration of particles for both batch and continuous systems, concentration of particles, cell washing from one fluid into another, fractionation of cellular populations, and trapping of material against flow. Concluding remarks discuss potential future applications of ultrasonic technology in microfluidic bacterial analysis and predict that it will be a significant tool in cell sample processing, with significant integration potential for Lab-On-Chip technologies.

The analysis of bio-aerosols poses a technology challenge, particularly when sampling and analysis are done in situ. Mass spectrometry laboratory technology has been modified to achieve quick bacteria typing of aerosols in the field. Initially, aerosol material was collected and subjected off-line to minimum sample treatment and mass spectrometry analysis. More recently, sampling and analysis were combined in a single process for the real-time analysis of bio-aerosols in the field. This chapter discusses the development of technology for the mass spectrometry of bio-aerosols, with a focus on bacteria aerosols. Merits and drawbacks of the various technologies and their typing signatures are discussed. The chapter concludes with a brief view of future developments in bio-aerosol mass spectrometry.