Nanotechnology and Detection of Microbial Pathogens

Chapter 28

Nanotechnology and Detection of Microbial Pathogens Rishi Shanker*, Gulshan Singh*, Anurag Jyoti*, Premendra Dhar Dwivedi† and Surinder Pal Singh** *Nanotherapeutics & Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research, Lucknow, U.P., India, †Food Toxicology, CSIR-Indian Institute of Toxicology Research, Lucknow, U.P., India, **CSIR-National Physical Laboratory, New Delhi, India

Chapter Outline Summary525 What You Can Expect to Know 525 History and Methods Introduction526 Indicators of Microbial Water Quality 526 Need for Detection of Water- and Food-Borne Pathogens 527 Conventional Methods to Detect Fecal Indicator Organisms and Other Pathogenic Bacteria 528 Most Probable Number Method 529 Membrane Filtration Method 529 Defined Substrate Methods 529 Rapid Detection Using Chromogenic Substrates 529 Immunological Methods 529 Antibody-Based Detection 529 Immunomagnetic Separation and Other Rapid Culture-Based Methods 530 Molecular Methods Based on Genetic Signature of Target Pathogen 530 Polymerase Chain Reaction Technique and Quantitative PCR 530 Nanotechnology and Its Promise 531 Metallic Nanoparticles 532 History532 Detection Principle 533 Methodology533

Synthesis of Gold Nanoparticles 533 Computation of ssDNA Sequences for Functionalization of Gold Nanoparticles 534 Functionalization of Gold Nanoparticles with Thiol-Modified DNA 534 Examples534 Colorimetric Detection of DNA of Shiga Toxin Producing Escherichia coli (Using Bio-Conjugated Gold Nanoparticles) 535 Colorimetric Detection of Enterotoxigenic Escherichia Coli (ETEC) Gene Using Gold Nanoparticle Probes 535 Clinical Significance of Nanoparticle-Based Detection 536 Ethical Issues 536 Translational Significance 537 Future Approaches 537 World Wide Web Resources 537 References538 Further Reading 539 Glossary539 Abbreviations539 Long Answer Questions 539 Short Answer Questions 539 Answers to Short Answer Questions 539

SUMMARY

WHAT YOU CAN EXPECT TO KNOW

The optical characteristics and functionalization of gold nanoparticles with DNA can be exploited to generate conjugated DNA probes for the detection of pathogens. Gold nanoparticle probes with low-cost instrumentation (or a “spot and read” system) can be a viable alternative for on-site detection and monitoring of pathogens.

The consumption of unsafe water and food in developing countries is one of the major causes of infectious disease outbreaks. The existing methods for the detection of pathogens prevalent in water and food samples are expensive, time consuming, and highly diverse. A majority of these pathogens often escape detection by conventional

Animal Biotechnology. http://dx.doi.org/10.1016/B978-0-12-416002-6.00028-6 Copyright © 2014 Elsevier Inc. All rights reserved.

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methods. The detection of target pathogens requires the development of innovative, simple, rapid, sensitive, and highly specific methods to overcome existing drawbacks for the management of infectious disease outbreaks. The recent advancements in nanotechnology have led to the development of nanoparticle-based facile assays for specific detection of the bioanalytes of clinical interest. Gold nanoparticles (GNPs) with unique optical properties and high surface area are being extensively used for facile detection of bioanalytes of interest in the samples. The outstanding physicochemical properties of GNPs have proved advantageous over conventional detection methods for diagnostic purposes. The colloidal solution of GNPs exhibits intense red and blue/purple colors depending on the size, shape, and degree of aggregation of nanoparticles. The present chapter encompasses the application of nanotechnology in the field of pathogen detection, and provides insight on how nanotechnology can be exploited to overcome the problems related to existing methods.

HISTORY AND METHODS INTRODUCTION Rapid population growth and industrialization have led to the deterioration of the microbiological quality of water, adversely affecting human health and sustainable development. Water plays a significant role in the transmission of human diseases. Typhoid fever, infectious hepatitis, cholera, traveler’s diarrhea, amebic and bacillary dysenteries, and other gastrointestinal diseases are waterborne. The occasional outbreaks of waterborne diseases point towards the need for strict monitoring and management of the “water quality” from public and private water supplies. “Water quality” is a technical term that is based upon the characteristics of water in relation to guideline values of what is best for human consumption and for all usual domestic needs, including personal hygiene. Microbial, biological, chemical, and physical aspects are important components of water quality. When referring to microbial aspects, microorganisms that are known to be pathogenic should be absent in drinking (potable) water. Potable water is water that has been either treated, cleaned, or filtered, and meets established drinking water standards as set by regulatory authorities like the World Health Organization (WHO), the Bureau of Indian Standards (BIS), the American Public Health Association (APHA), and the United States Environmental Protection Agency (USEPA). This water is expected to be realistically free from harmful bacteria and contaminants, and considered “safe” for drinking, or cooking and baking purposes. Municipal water that has been UV-irradiated, filtered, distilled, or purified, falls into the category of potable water. Hence, water treatment regimens that employ disinfection methods and the execution of bacteriological

surveillance programs have resulted in decreased occurrences of water-related illness. Management of the frequency of waterborne disease outbreaks has become a challenging task. Globally, the source of almost two-thirds of the drinking water consumed is surface water, which may be easily contaminated by sewage discharges, animal defecation, and municipal and industrial wastes. Fecal wastes from domestic animals, wildlife, and humans (to varying extents) are incorporated into the soil. These fecal wastes can also enter the water stream directly, or through poorly processed sewage effluents, by percolation of water pipelines, malfunctioning septic tanks, and seepage from sanitary landfills. Different pathogenic viruses, bacteria, and parasites may be found in the feces of domestic animals, wild animals, and humans, along with the non-pathogenic bacteria and parasites that exist in large numbers in the feces of animals as well as in soil and water. Hence, it is important to identify the etiological agent for appropriate treatment, interventions, and control. However, the identification and monitoring of specific pathogens in low concentrations or doses in the presence of a large number of background microflora is a daunting task. The presence of “indicator organisms,” generally non-pathogenic microorganisms, points towards the presence of enteric pathogens in a sample. Indicator organisms play an important role in predicting the probability of the occurrence of pathogens that are quite low in number. There are a few criteria for a microorganism to be declared an “indicator.” It must be present when pathogens are present in water, absent in uncontaminated water, present in higher numbers than pathogens in contaminated water, must survive better in water than pathogens, and must be easy to analyze. Therefore, the need to identify, classify, and delineate the permissible limits of “indicators” of microbial water quality in different sectors of water have been described in this chapter.

INDICATORS OF MICROBIAL WATER QUALITY In water quality assessment, the fecal indicator bacteria (FIB) are used to measure the sanitary quality of water for recreational, industrial, agricultural, and water supply purposes. FIB’s are natural inhabitants of the gastrointestinal tract of humans and other warm-blooded animals. Generally harmless, these are released into the environment with feces, and on exposure to a variety of the environmental factors (Ashbolt et al., 2001). In general, it is believed that the fecal indicator adapted to live in the gastrointestinal tract cannot grow in natural environments. However, survival of fecal indicator bacteria in water is influenced by environmental factors like sunlight, temperature, nutrient competition with bacteria naturally inhabiting the water, predation by protozoa and other small organisms, and toxic

Chapter | 28  Nanotechnology and Detection of Microbial Pathogens

industrial wastes. Studies have shown that FIB survive from a few hours up to several days in water, but may survive for days or months in sediments where they may be protected from sunlight and predators. The survival time of fecal indicator bacteria in water is a function of many environmental influences, and there is no common factor that applies collectively to all water bodies, or even at different seasons in a year for a single body of water. It is assumed that the mortality of pathogens and FIB are equal. Therefore, the presence of relatively high numbers of FIB in the environment indicates the likelihood of the presence of other pathogens as well. The environmental indicators of water quality are described in Figure 28.1. Coliforms and related pathogens are broadly categorized into total coliforms (TC), fecal coliforms (FC), or thermo-tolerant coliforms and other Indicators of Water Quality

Fecal Indicators Pathogen +/– Indicates the presence of faecal contamination Thermo tolerant coliforms or Escherichia coli

Process Indicators Process efficacy +/– Demonstrates the efficacy of a process Total heterotrophic bacteria or total coli forms for chlorine disinfection

Index or Model Organism Specific Pathogen +/– Indicator of a particular pathogen presence & its behaviour. F-RNA coliphages as models of human enteric viruses

FIGURE 28.1  Types of water indicators.

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indicator organisms (Figure 28.2). Studies show that 60 to 90% of total coliforms are fecal coliforms, and 90% of fecal coliforms, are Escherichia coli. The permissible limits of “indicators” of water quality are summarized in Table 28.1.

NEED FOR DETECTION OF WATER- AND FOOD-BORNE PATHOGENS “Indicators” are useful to access pathogenic microorganisms, but why is detection of pathogens so urgently needed? The most frequently encountered water- and food-borne diseases (i.e. traveler’s diarrhea, typhoid, and cholera) are caused by consumption of foodstuffs, including meat products and contaminated water. In the developing world, particularly southeast Asia, communities use untreated water for drinking, food preparation, and other domestic purposes, particularly in urban, suburban, and rural environments. It has been reported that the presence of water- and foodborne bacteria are a major cause of the economic burden on the food industry in developing countries (Wang et al., 2010). It is therefore necessary to detect these pathogens at an early stage to circumvent the spread of disease and epidemics. At this juncture, it is essential to know about the major players in the world of pathogenic microbes. The pathogenic group of bacteria includes pathotypes of Escherichia coli, such as ETEC (Enterotoxigenic E. coli) and EHEC (Enterohemorrhagic E. coli), Salmonella spp., Vibrio cholerae, Campylobacter spp. (including antibiotic-resistant

FIGURE 28.2  Different types of indicator organisms.

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TABLE 28.1  Summary of Water Quality Criteria for Microbiological Indicators Water Use

Escherichia Coli *CFU

Enterococci *CFU

Fecal Coliforms *CFU

Raw Drinking Water – no treatment

0/100 mL

0/100 mL

0/100 mL

Raw Drinking Water – disinfection only

≤ 10/100 mL 90th percentile

≤ 3/100 mL 90th percentile

≤ 10/100 mL 90th percentile

Raw Drinking Water – partial treatment

≤ 100/100 mL 90th percentile

≤ 25/100 mL 90th percentile

≤ 100/100 mL 90th percentile

Raw Drinking Water – complete treatment None applicable

None applicable

None applicable

Livestock – free range animals

None applicable

None applicable

None applicable

Livestock – general livestock use

200/100 mL maximum

50/100 mL maximum

200/100 mL maximum

Livestock – closely confined (no treatment)

0/100 mL maximum

0/100 mL Maximum

0/100 mL Maximum

Livestock – closely confined (disinfection only)

≤ 10/100 mL 90th percentile

≤ 10/100 mL 90th percentile

≤ 10/100 mL 90th percentile

Livestock – closely confined (partial treatment)

≤ 100/100 mL 90th percentile

≤ 100/100 mL 90th percentile ≤ 100/100 mL 90th percentile

Livestock – closely confined (complete treatment)

None applicable

None applicable

None applicable

*CFU: Colony Forming Unit. (Modified from Water Quality Criteria for Microbiological Indicators (Warrington et al., 2001))

Campylobacter jejuni). Among the E.coli pathotype family, EHEC (foremost representative of E. coli 0157:H7) are the major culprits of water- and food-borne diseases in humans. These bacterial strains are characterized by the production of one or more types of cytotoxins that cause tissue damage in humans and animals. Enterotoxigenic Escherichia coli (ETEC), the causative agent of travelers’ diarrhea, is one of the important pathogens in the farming industry, and is found in cattle and weaning piglets. ETEC strains from humans cause mild or severe watery diarrhea by producing a heat-labile enterotoxin (LTI), similar in structure to cholera toxin and heat-stable enterotoxins (ST IA and/or ST Ib). The heat-labile enterotoxins of E. coli are oligomeric toxins with two major serogroups, LTI and LTII. LTI is expressed by E. coli strains that are pathogenic for both humans and animals. Salmonella, another typical pathogen, causes gastroenteritis and typhoid in humans. Typhoid caused by the Salmonella enterica serotype Typhi remains an important public health problem in developing countries. It has been reported that South Asian countries exhibit a high burden of typhoid fever. Furthermore, India, Indonesia, Bangladesh, and Pakistan have been identified as high infection zones (caused by Salmonella spp.). The severity of infections of Salmonella is due to their infective dose, which can be as low as 15–100 CFU. This high vulnerability to waterborne Salmonella infections in Asia and other developing countries is due to the lack of potable water and the dependence of a large population on natural resources for daily water requirements.

Vibrio cholerae and Vibrio parahaemolyticus are pathogens that cause diarrhea in humans. V. parahaemolyticus is an invasive organism affecting primarily the colon, whereas V. cholerae is non-invasive, affecting the small intestine through secretion of an enterotoxin. Vibrio cholerae causes a globally prevalent gastrointestinal disease, cholera, which remains a persistent problem in many countries. These pathogens occur in both marine and freshwater habitats, and are associated with aquatic animals. Campylobacter jejuni has been associated with dysentery-like gastroenteritis, as well as with other types of infection, including bacteremic and central nervous system infections in humans. Based on the potential risk posed by the above-mentioned pathogens, it is clear that the detection of these organisms is the key to the prevention of water- and food-borne epidemics or diseases in humans and animals. It is therefore useful to understand the advantages and limitations of the existing state-of-the-art detection methods used to detect indicator organisms and identify pathogenic variants of such microbes.

CONVENTIONAL METHODS TO DETECT FECAL INDICATOR ORGANISMS AND OTHER PATHOGENIC BACTERIA Conventional techniques like culture-based methods have been recommended and used routinely for decades for identification and detection of pathogens. In culture-based

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methods, microbes grow on specific culture media at a specific temperature for a particular time period. Their characterization is based on the morphology of bacterial colonies and confirmation by biochemical tests. The most prevalent conventional methods, including culture-based methods, for pathogen detection are described in the following sections.

Most Probable Number Method The most probable number (MPN) technique is an important technique for estimating microbial populations in soils, waters, food matrices, and agricultural products. Many soils are heterogeneous, therefore exact cell numbers of an individual organism are impossible to determine. The MPN technique is used to estimate microbial population where heterotrophic counts are difficult. This technique does not rely on quantitative assessment of individual cells; instead it relies on specific qualitative attributes of the microorganism being counted. The MPN technique estimates microbial population sizes in a liquid substrate; the method is tedious, and takes 24 to 48 hours.

Membrane Filtration Method The membrane filter (MF) technique is used to test relatively large volumes of sample, and yields numerical results more rapidly than the MPN method. The membrane filter technique is extremely useful in monitoring drinking water and a variety of natural waters. On the basis of the MF technique, the coliform group may be comprised of all aerobic and many facultative anaerobic, Gram-negative, nonspore-forming, rod-shaped bacteria; these develop a red colony with a metallic sheen within 24 hours at 35°C on an endow-type medium containing lactose. Some members of the total coliform group may produce a dark red or nucleated colony without a metallic sheen, and are classified as typical coliform colonies after verification. Pure cultures of coliform bacteria produce a negative cytochrome oxidase (CO) and positive β-galactosidase (ONPG) reaction. Generally, all red, pink, blue, white, or colorless colonies that lack sheen are considered non-coliforms by this technique. However, the MF technique has limitations, particularly when testing waters with high turbidity or a presence of non-coliform bacteria. Hence, for such waters, or when the membrane filter technique has not been used previously, it is desirable to conduct parallel tests with the multiple-tube fermentation technique to demonstrate applicability.

Defined Substrate Methods Media without harsh selective agents but specific enzyme substrates provide significant improvements in recoveries and identification of target bacteria such as coliforms and E. coli. Furthermore, the enzyme-based methods appear to

Conventional Methods

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Most Probable Number Method Membrane filteration Method Defined Substrate Methods

Modified Methods

Chromogenic Culture media Antibody Based Methods Immunomagnetic separation

Molecular Methods

RFLP, RAPD FISH PCR, qPCR

Laborious Non-specific Time consuming

Laborious Non-specific Time reduced Complex Low Sensitivity

Highly Sensitive Expensive instrumentation More Complex

FIGURE 28.3  Different methods of pathogen detection.

pick up traditionally non-culturable Coliforms. Total coliforms are members of genera or species within the family Enterobacteriaceae, capable of growth at 37°C, which and that possess β-galactosidase. This defined substrate approach has been advocated by the International Standards Organization for miniaturized MPN-based methods for Coliforms/E. coli and enterococci (ISO/FDIS, 1999). Certain methods in vogue are presented in Figure 28.3.

Rapid Detection Using Chromogenic Substrates Chromogenic compounds added to conventional or newly devised media are used for the isolation of indicator bacteria. These chromogenic substances are modified either by enzymes (which are typical for the respective bacteria) or by specific bacterial metabolites. After modification, the chromogenic substance changes its color or fluorescence, thus enabling easy detection of colonies displaying the metabolic activity. In this way, these substances are used to avoid the need for isolation of pure cultures and confirmatory tests. The time required for the determination of different indicator bacteria can be reduced to between 14 to 18 hours. When necessary, the surface antigens can be selected and used as the pathogen recognition element for confirmation.

Immunological Methods Antibody-Based Detection Antibodies are immunoglobulins secreted by B-cells, and are recruited by the immune system to identify and neutralize foreign objects such as bacteria and viruses. Antibodies possess highly specific binding and recognition domains that can be targeted to specific surface structures of a pathogen. Immunological methods using antibodies are widely used to detect pathogens in clinical, agricultural,

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and environmental samples. As always with immunological techniques, the specificity of the reagents and optimization of their use is a critical issue. Although total Coliforms are a broad group and likely to be unsuitable immunological targets in environmental waters, E. coli could be identified from other Coliforms. It is evident that the aforementioned methods and approaches are either laborious and time-consuming, or non-specific with a probability of false positives. Another major disadvantage of the above methods is the failure to detect non-cultivable microbial communities that are viable but non-culturable (VBNC). The VBNC state of a bacterium is its ability to cause the disease and yet fail to respond to enumeration through classical culture procedures. The VBNC state of bacterial enteropathogens poses a potential threat to human and animal health because the failure to culture such organisms in current resuscitation protocols leads to incorrect estimation.

Immunomagnetic Separation and Other Rapid Culture-Based Methods Immunomagnetic separation (IMS) offers an alternative approach to the rapid identification of culturable and nonculturable microorganisms. The principles and application of the method are simple, but rely on suitable antibody specificity under the experimental conditions. Purified antigens are typically biotinylated and bound to streptoavidin-coated paramagnetic particles (e.g. DynalTM beads). The raw sample is gently mixed with the immunomagnetic beads, then a magnet is used to hold the target organisms against the wall of the recovery vial, and non-bound material is poured off. If required, the process can be repeated, and the beads can be removed by simple vortexing. Target organisms can then be cultured or identified by direct means. The IMS approach may be applied to recovery of indicator bacteria from water, but is possibly more suited to replace labor-intensive methods for specific pathogens. E. coli O157 recovered from water samples were detected using this technique in some studies. Furthermore, E. coli O157 detection following IMS can be improved by electrochemiluminescence detection. However, the IMS/culture methods are also accompanied by disadvantages, such as the ability of non-specific binding, the need for physicochemical conditions such as pH and temperature, sensitivity to chemicals in the samples, the high cost of monoclonal antibody production, and limited shelf life.

MOLECULAR METHODS BASED ON GENETIC SIGNATURE OF TARGET PATHOGEN The nucleic acid sequences are unique to all living organisms. These genetic signature sequences are the potential

targets to differentiate one organism from another and to diagnose various disease-causing agents. In the postgenomic era, large numbers of microorganisms have been sequenced. In early 2013, ∼18,000 prokaryotic genomes have been sequenced (NCBI Genome database, http://www. ncbi.nlm.nih.gov/genome/browse/). This sequence database has made it possible to analyze microbial pathogens at the molecular level. The application of molecular methods has to be considered within the framework of quality management for potable water. The new methods will influence epidemiology and outbreak investigations more than the routine testing of processed drinking water. Certain molecular approaches like Restriction Fragment Length Polymorphism (RFLP), Random Amplification of Polymorphic DNA (RAPD), and Florescence In Situ Hybridization (FISH) are extensively used for pathogen detection, but each has limitations. In FISH-based detection, gene probes with a fluorescent marker are used, typically targeting the 16S ribosomal RNA (16S rRNA). Concentrated and fixed cells are permeabilized and mixed with the probe. The stringency of the homology between the gene probe and the target sequence are influenced by incubation temperature and the addition of chemicals. A single fluorescent molecule within a cell does not allow detection, and target sequences with multiple copies in a cell have to be selected (e.g. there are 102–104 copies of 16S rRNA in active cells). Low-nutrient environments may result in cells entering a non-replicative, viable, but non-culturable (VBNC) state for many pathogens. Such a state may give a false result that makes culture-based methods unreliable and can be overcome using molecular approaches.

Polymerase Chain Reaction Technique and Quantitative PCR The powerful molecular technique, Polymerase Chain Reaction (PCR), allows amplification of target DNA to generate multiple copies that can be detected. PCR has been validated by the International Organization for Standardization (ISO), and is now used for testing of food-borne pathogens (Malorny et al., 2008). One problem with PCR is that the assay volume is on the order of microliters and requires that the sample be concentrated to the microliter range. The water sample has to be concentrated and purified using adequate methods, as natural water samples often contain inhibitory substances such as humic acids and iron that concentrate with the nucleic acids. Hence, it is critical to have positive and negative controls with each environmental sample PCR to check for inhibition and specificity. It may also be critical to find out whether the signal obtained from the PCR is due to naked nucleic acids, or living and dead microorganisms. The sensitivity of the PCR is often not sufficient, and post-PCR processing and

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analysis is needed. Additionally, PCR requires technical equipment and laboratory setup, which is not suitable for on-site diagnostics. Quantitative PCR (qPCR), also called Real-Time PCR, a fluorescence-based detection format, is more sensitive than conventional PCR. Samples can be analyzed in real time with higher specificity and sensitivity. No post-PCR processing is required. The technique has been applied to the location of non-point sources of pathogen contamination and environmental risk assessment (Singh et al., 2010). Fluorescence labeling is a commonly used and wellestablished method, but has disadvantages like a lower stability of fluorescent dyes and the requirement of expensive readout systems. The fluorescence-based detection systems have many significant drawbacks, including susceptibility to photobleaching, complexity, sensitivity to contamination, cost, and reliance on relatively expensive equipment to probe their presence in an assay. Although diverse, the existing methods have certain disadvantages that limit their use in point-of-care settings and field situations due to the requirements of sophisticated instrumentation and trained personnel. Faster, simpler, and more reliable detection methods would largely provide support to help protect consumers. Direct detection methods that provide quick, accurate, simple, and cost effective devices to be used on-site are highly desired. Advances in human and animal science are placing increasingly stringent demands on diagnostic and clinical tests to enhance sensitivity, specificity, and thresholds. There is an extensive need for the selection of a unique system that will allow the pathogen recognition element to be accepted for an ideal detection format. It is becoming evident that new pathogen detection methodologies enabled by nanotechnology-based approaches have the potential to provide better options.

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The surface functionalization of nanomaterials by biomolecules has led to the development of new interdisciplinary research areas like biomedical nanotechnology, nanomedicine, diagnostic devices, theranostics, contrast agents, nanobiosensors, and targeted drug delivery vehicles. Functionalities can be added to nanomaterials by interfacing them with biological molecules or structures (Figure 28.4a); that makes nanomaterials useful in various technological areas as well as in both in vivo and in vitro applications. Nanotechnology has played a significant role in the development of affinity sensors (e.g. antibody–antigen interaction-based biosensors and ultrasensitive DNA hybridization detection). At the nanoscale some materials have been shown to exhibit extraordinary optical properties relative to their bulk counterparts. The light-scattering power of nanoparticles is orders of magnitude greater than fluorescent labels. Moreover, the optical signals generated from these nanoparticles are not prone to photobleaching, compared to their organic dye counterparts. Semiconductor quantum dots are one of the pioneering examples that distinctly show size-dependent emission of different colors with high quantum yield when excited with a single wavelength. The ability to simultaneously tag multiple biomolecules with these quantum dots has provided the opportunity to develop new optical diagnostic tools and the ability to observe complex cellular changes and associated events. On the other hand, noble metal nanoparticles have attracted much interest because of their unique physico-chemical properties, including large optical field enhancements that result in the strong scattering and absorption of light in the visible region due to the presence of surface plasmon resonances (SPRs). Biochemical assays based on light-scattering signals from metal nanoparticles have been widely used in the determination of the affinity interaction between DNA, proteins, and drugs, and have led to the development of easy-to-use optical sensing devices.

NANOTECHNOLOGY AND ITS PROMISE The term nano, derived from the Greek word for dwarf, is usually combined with a noun to form words such as nanometer, nanorobot, and nanotechnology. In the last two decades, nanoscience and nanotechnology have seen a plethora of new developments in almost every field of science and technology, especially in biology and medicine. Nanotechnology has set high expectations in biological and medical sciences to solve key questions concerning bio-systems that operate at the nanoscale. It deals with the creation of functional materials, devices, and systems on the nanometer scale length of 1–100 nm. The ability to manipulate and engineer materials at the nanoscale (atomic, molecular, and macromolecular) enables one to tune the physical and chemical properties of desired materials according to specific applications.

FIGURE 28.4A  Functionalization of nanoparticles.

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FIGURE 28.4B  Nanomaterial applications in pathogen detection.

(C)

Nanotubes

Quantum Dots Noble metals

Magnetic nanoparticles

Nanomaterials

Silver nanoparticles

Silica nanoparticles

Biosensor

Aptamers

Carbohydrate

Recognition Elements Peptides

Antibodies

Magnetic

Electrochemical

Signal Transduction Optical

Flourescence

FIGURE 28.4C  Nanomaterial components involved in pathogen detection. (Modified from Vikesland et al., 2010.)

Metallic Nanoparticles Colloidal metal nanoparticles (e.g. gold and silver nanoparticles) have attracted tremendous research interest. Metallic nanoparticles in colloidal solution appear colored; for example, colloidal gold nanoparticles generally appear ruby red, purple, blue, and orange, depending upon their shape, size, and synthesis conditions. Similarly, titanium (Ti) and platinum (Pt) nanoparticles give blue and dark brown colors. The different colors of metal nanoparticles arise from SPRs and their confinement. Among metals, gold nanoparticles exhibit excellent biocompatibility, electronic, optical, and catalytic properties. Gold nanoparticles (GNPs) have found a distinguished place in bio-diagnostics due to their size-dependent optical properties, variety of surface coatings, and biocompatibility (Mirkin et al., 1996; Storhoff et al., 2004; Sato et al., 2007, Pandey et al., 2008). Spherical gold nanoparticles exhibit SPR-related optical absorption at 520 nm, which strongly depends on particle size and morphology. The rich surface chemistry of gold nanoparticles allows surface modifications with various biofunctional

groups, such as nucleic acids, sugars, and proteins, via the strong affinity of the gold surface with thiol ligands (Schofield et al., 2007); this creates multi-functionality to tailor the needs of biomedical applications including imaging, diagnostics, and therapy. The conjugation of nanoparticles with biomolecules (e.g. proteins and DNA) can be done either by direct covalent linkage or by noncovalent interactions. Biomolecules are often covalently linked to ligands on the nanoparticles’ surface via traditional coupling strategies such as carbodiimide-mediated amidation and esterification. The high surface-to-volume ratio of GNPs makes surface electrons sensitive to minor changes in the dielectric (refractive index) constant of the medium. Therefore, changes to the surface chemistry and environment of these particles (surface modification, aggregation, medium refractive index, etc.) lead to colorimetric changes of the dispersions that become the basis for detection of any analyte of interest. This has further facilitated the application in bio-detection via numerous methods (Boyer et al., 2002). The various synthetic methods for gold nanoparticles are discussed in the methodology section.

HISTORY The detection of microbial pathogens remains a challenging task despite great strides made in the past three decades. The most frequently used culture-based methods have undergone diverse modifications, including specific substrates for enzymes present in target organisms. However, the issues of specificity and sensitivity directed towards low doses of organism, viable but non-culturable states, and long incubation periods, still elude the culture technique. The immunological methods using antibodies to detect pathogens in different domains of an environment, as well as in clinical settings, followed culture techniques. Immunomagnetic separation evolved to improve detection, but

Chapter | 28  Nanotechnology and Detection of Microbial Pathogens

faced specificity and limited shelf-life problems. The understanding of nucleic acid structure and function led to the era of gene sequencing, DNA hybridization, and consequent genetic signature-based detection of organisms. A milestone in molecular biology, the polymerase chain reaction (PCR) technique developed by Kary Mullis in 1983 led to a burst in PCR use in pathogen detection between 1992 and 1999. This was followed by advancements in PCR, including real-time PCR or quantitative PCR (qPCR) based on fluorescence chemistries. This technique has been widely used for quantitative enumeration of pathogens in different domains from 1999 until today. Fluorescence-based detection is complex, and has issues of photo-bleaching, contamination, cost, and dependence on expensive instrumentation that limit applications for “on-site detection.” These methodologies delineate the need for simple assays and less expensive detection system. Nanoscience provides new horizons in bio-nanotechnology for the detection of pathogens based on genetic signatures. In the last decade, nano-based approaches that exploit the unique properties of nanoparticles have shown potential for the development of novel pathogen-detection systems.

DETECTION PRINCIPLE The efficient use of nanomaterials in biological systems relies on knowledge of the nano–bio interface. Gold nanoparticles have found widespread applications in the life sciences, and serve as excellent standards to understand more general features of the nano–bio interface because of their many advantages over other inorganic materials. The bulk material is chemically inert. Gold’s background concentration in biological systems is low, which makes it relatively easy measure at the parts-per-billion level or lower in water. The unique optical and electronic properties of GNPs enable them to conjugate biological molecules like RNA and DNA and serve as scaffolds for nanostructures. GNP interactions with light are governed by size, environment, and physical dimensions. The fluctuating electric fields of a light ray promulgating near a colloidal nanoparticle interact with free electrons to cause the intense oscillation of electron charge that is in resonance with the frequency of visible light. These resonant oscillations are known as surface plasmons. Surface plasmon resonance for monodispersed gold nanoparticles (∼30 nm) causes light absorption in the blue– green portion of the spectrum (∼450 nm), while red light (∼700 nm) is reflected, yielding a rich red color. The wavelength of surface plasmon resonance-related absorption shifts to longer wavelengths, with an increase in particle size. Red light is then absorbed, and blue light is reflected, resulting in solutions with a pale blue or purple color. The change in optical properties as a result of shape, size, and aggregation pave the way for the development of colorimetric and sensitive detection systems for specific bioanalytes.

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As an example, the DNA hybridization event using GNP is recognized by a change in color that appears as a result of DNA hybridization, which brings GNPs in close proximity. Several proof-of-concept studies demonstrate the use of GNPs in biomedical applications like chemical sensing, biological imaging, drug delivery, and cancer treatment. A number of factors, namely size and shape of the nanoparticle, refractive index of the surrounding media, and inter-particle distance, are taken into account for use in colorimetric detection of DNA (Baptista et al., 2008). Mirkin and co-workers (1996) reported the colorimetric detection of DNA targets based on the cross-linking mechanism use of GNP probes. The two different batches of probes are designed to target the DNA. Thus, upon the addition of target DNA, a polymeric network of GNP probes is generated due to aggregation, turning the solution from red to blue (Mirkin et al., 1996). This aggregation mechanism is mainly applied to detect small-sized targets. The GNP aggregation induced by interparticle cross-linking is a relatively slower process. The relatively slow aggregation is due to the nature of the interparticle cross-linking aggregation mechanism. In general, the aggregation is driven by random collisions between nanoparticles with relatively slow Brownian motion (Sato et al., 2003). Similarly, DNA detection based on a non-cross-linking mechanism has been well documented. Sato and co-workers described a non-cross-linking mechanism of GNP aggregation for DNA detection. The single-stranded DNA can be immobilized on GNPs above the physiological temperature. The GNP probes aggregate together at a considerably high salt concentration when the target DNA is perfectly complementary to the probe (Sato et al., 2007). In non-cross-linking aggregation systems, the van der Waals force of attraction dominate. Many parameters, including surface charge properties (e.g. charge density, the amount of associated counter ions) and entropy factors are also involved in aggregation. Compared to interparticle cross-linking aggregation systems, the non-cross-linking aggregation mechanism has some attractive features. Aggregation induced by the noncross-linking process is very rapid, leading to the development of faster assays. The interparticle attractive forces (van der Waals forces) dominate over the interparticle repulsive forces, which results in rapid aggregation (Sato et al., 2003). The use of GNP probes for the colorimetric detection of DNA targets represents an inexpensive and simple workable alternative to fluorescence- or radioactivity-based assays (Storhoff et al., 2004).

METHODOLOGY Synthesis of Gold Nanoparticles Gold nanoparticles can be synthesized using various synthetic routes. In a typical synthesis, tetrachloroauric acid

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alignment of the retrieved conserved region (http://www. ebi.ac.uk/Tools/msa/clustalw2/) is followed by computation of oligonucleotide probes using web-based or dedicated software such as Primer 3, Lasergene, or Beacon Designer. An analysis for cross homology and secondary structure is carried out by BLAST (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) and Mfold (www.bioinfo.rpi.edu/applications/ mfold) servers. The computed ssDNA sequences are then synthesized in a DNA synthesizer for functionalization of GNPs.

Functionalization of Gold Nanoparticles with Thiol-Modified DNA FLOW CHART 28.1  1. This is the first step of synthesis of GNPs by the Turkevitch and Brust method. Citric acid reduces gold chloride (i.e. AuCl3) and triggers the nucleation of Au ions to form nanoparticles, followed by its adsorption to the surface, which provides colloidal stability to nanoparticles due to its negative charge. 2.  In this second step, GNPs are functionalized with ssDNA. Gold nanoparticles are incubated with thiol-modified ssDNAs for 16–20 hrs, and unbound ssDNA is removed by centrifuging the solution. The pellet of DNA-conjugated GNPs is washed and stored in 0.3 M trisacetate NaCl (pH 8.2) at room temperature. 3. Next is hybridization of the formed GNP probes with targets and subsequent detection. After hybridization with the target, the GNP probes come in close proximity, and as a result, the particles aggregate and the solution appears purple/blue. 4. This is the last step, in which visible color change appears as a red to blue/purple change in the SPR band, and is recorded by UV-visible spectroscopy.

(HAuCl4) is mixed with a reducing agent, which leads to the reduction of Au ions to form nanoparticles. In the most reliable and popular method by Turkevich and Brust, citric acid first reduces gold ions from HAuCl4 and triggers the nucleation to form nanoparticles, followed by its adsorption to the surface, which provides colloidal stability to nanoparticles due to its negative charges (Brust et al., 1998). This method produces monodisperse spherical gold nanoparticles with a diameter in the 10–20 nm range. (Step 1 in Flow Chart 28.1)

Computation of ssDNA Sequences for Functionalization of Gold Nanoparticles The GNPs are bio-functionalized with the thiol-modified single-stranded DNA (ssDNA) or oligonucleotides to generate a GNP probe for target DNA detection. The bioinformatics tools are used to compute ssDNA sequences or probes complementary to the target gene sequence of the pathogen of interest. The first step towards this is the retrieval of nucleotide sequences of targeted genes of selected organisms from GenBank (www.ncbi.nlm.nih.gov). Multiple sequence

The computed oligonucleotides are synthesized with modification of the thiol group (–SH) at their 5′ or 3′end. These modified oligonucleotides are then attached to the GNPs through chemisorption of the thiol group onto the surface of the GNPs; GNP probes are then generated. In order to functionalize with DNA, gold nanoparticles (selected size ∼20±0.2 nm diameter) are incubated with different batches (number varies with the target size) of thiol-modified ssDNAs separately for 16–20 hours with an oligonucleotide concentration of 2 μM. The unbound ssDNA is then removed by centrifugation at 16,000 × g for 15 min, and the pellet of DNA-conjugated GNPs is washed in 0.1 M Tris-acetate and NaCl (pH 8.2) buffer. The ssDNA-grafted GNP is then stored in 0.3 M Tris-acetate NaCl (pH 8.2) at room temperature. The absorption spectra of the bio-functionalized GNPs are recorded by UV-visible spectroscopy for confirmation of DNA immobilization (Steps 2–4 in Flow Chart 28.1). The basic steps of DNA detection using gold nanoparticle probes are depicted in Figure 28.5a

EXAMPLES Enterohemorrhagic E. coli (EHEC) serotype O157:H7 is one of the most deadly pathogens. EHEC produces stx1- and stx2type enterotoxins and symptoms, such as abdominal pain and watery diarrhea. Many patients develop life-threatening diseases, such as hemorrhagic colitis (HC) and hemolyticuremic syndrome (HUS), the deadly consequences of these cytotoxins. The natural reservoirs of EHEC are domestic and wild ruminant animals, which shed the bacteria along with their feces into the environment. The products of animal origin, such as meat and milk, are at risk of contamination with EHEC originating from animals. Consumption of food containing EHEC was identified in different countries as a major route of human infection with these pathogens. Hamburger, vegetables, and fruit juices have frequently been contaminated with pathogenic E. coli, and have also been sources of infection. Apparently, cattle, the natural reservoir for pathogenic strains, have often been implicated in E. coli infections. Following are examples for detection of EHEC using GNPs.

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FIGURE 28.5A Methodology.

Colorimetric Detection of DNA of Shiga Toxin Producing Escherichia coli (Using Bio-Conjugated Gold Nanoparticles) EHEC causes bloody diarrhea in humans through the production of shiga-like toxin. The toxin is encoded by stx2 gene in E. coli. The existing methods for detection of EHEC are culturing of the bacteria on fluorogenic-substrate media, which is time-consuming. Molecular methods such as PCR and real-time PCR assays are also used, which require expensive instrumentation. In recent studies, the optical properties of gold nanoparticles (GNPs) have been exploited for detection of the nucleic acid of ­Escherichia coli. The PCR product of stx2 gene representing EHEC signature has been targeted using gold nanoparticle probes. Gold nanoparticles of 20±0.2 nm were synthesized by citrate reduction and characterized by UV-visible spectroscopy and transmission electron microscopy. Two different batches of thiolated, single-stranded DNA (19 and 22 bp) complementary to the target are grafted onto the GNPs (Table 28.2). The hybridization of GNP probes with target DNA led to a change in color from red to purple that is visible with the naked eye (Jyoti et al., 2010). The hybridization-induced aggregation was also observed by transmission electron microscopy.

Colorimetric Detection of Enterotoxigenic Escherichia coli (ETEC) Gene Using Gold Nanoparticle Probes In the present example, the colorimetric detection of heatlabile toxin gene LT1 (1257 bp) of ETEC was shown.

TABLE 28.2  Single-Stranded Thiol-Modified Oligonucleotide Probes and Their Complementary Sequences Probe/ Synthetic Nucleotide Sequence Target (5’-3’)

Length (bp)

sx2F

HS-(C)6-GGAGTTCAGTGGTAATACAATG-

22

sx2R

HS-(C)6-GCGTCATCGTATACACAGG-

19

(Modified From Jyoti et al., 2010.)

Multiple probes were used to target the gene to increase specificity towards the target as well as the aggregation of gold nanoparticles. A total of eight GNP probes were used to target the different locations on the target DNA sequence. The oligonucleotides were computed based on the conserved signature gene of ETEC. The GNPs were functionalized with thiol-modified single-stranded DNAs to facilitate hybridization with the target. After hybridization, change in the surface plasmon resonance-related band was observed by UV-visible spectroscopy. This led to a visible colorimetric change of reaction assay mixture from red (λmax = 524 nm) to purple (λmax = 552 nm) that is clearly visible to the naked eye. TEM was used to evaluate the aggregation and reduction in the interparticle distances of gold nanoparticles. TEM confirms the hybridization, aggregation, and reduction in the interparticle distances of the GNP probes in the presence of target DNA (Figure 28.3). In addition, the assay shows its specificity by differentiating the DNA of

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FIGURE 28.5B  (A) UV–Vis spectra of unmodified GNPs (black) and hybridized DNA–GNPs (red); (B) Transmission electron micrograph of the (1) monodispersed GNPs (20±0.2 nm) and (2) hybridized polymer network corresponding to the change in solution color from red/pink to purple.

Enterohemorrhagic E. coli (EHEC) from ETEC, a closely related pathotype of E. coli. The aggregation and the spectral shift in the plasmon band leading to a change in color observed with target DNA indicates the possibility of a simple and rapid colorimetric “spot and read” test in contrast to amplification-based fluorescence detection methods (Figure 28.5b).

Clinical Significance of Nanoparticle-Based Detection In recent years, nano-based approaches have been exploited for detection of other pathogens of clinical significance, like mycobacterium and other pathogenic species that affect humans and animals as causative agents of tuberculosis, leprosy, and paratuberculosis. Direct detection of unamplified DNA from pathogenic mycobacteria using DNAderivatized gold nanoparticles was reported by (Liandris et al., 2009). Different nanodiagnostics systems have been developed for the molecular diagnosis of tuberculosis-like nanoparticle-based systems, such as gold, silver, silica, and quantum dots (QDs); they have been the most widely used for TB diagnostics due to their unique physicochemical properties. For detection of multiple bacterial genomic DNA, gold nano-rod probes were used; they have high sensitivity and excellent specificity for reading the decrease in the sensitive longitudinal absorption band (Wang et al., 2012). The development of nano-enabled assays and on-site detection systems for EHEC and ETEC can perhaps help in the reduction of morbidity and mortality in children in rural ecozones. In summary, detection systems with high

sensitivity and selectivity, and that are easy-to-use and rapid, are of immediate need; nanoparticle-based detection shows great promise for the development of such nanodevices.

ETHICAL ISSUES Containment of hazards in laboratory experiments is one key to safety. Microbiologists have gained valuable experience over decades of handling extremely dangerous natural organisms, such as smallpox virus and cholera bacteria. According to Biosafety in Microbiological and Biomedical Laboratories (CDC, 2009), safe handling and containment of infectious microorganisms and hazardous biological materials are the mandatory principles of working with microbes. The basics of containment include the use of safety equipment, proper microbiological practices, and safeguarded facilities that protect laboratory workers, the sterile environment, and the public from exposure to infectious microorganisms that are handled and stored in the laboratory. This inclusion of risk assessment in our routine practice can prevent laboratory-associated infections. Individual workers handling the pathogenic microorganisms must understand the containment conditions under which infectious agents can be safely maneuvered and secured. Correct information and the use of appropriate techniques and equipment will enable the microbiological and biomedical communities to prevent personal, laboratory, and environmental exposures to potentially infectious agents or biohazards. Furthermore, limited understanding of the adverse impact of engineered nanomaterials or

Chapter | 28  Nanotechnology and Detection of Microbial Pathogens

Animal Breeding & Genetics

Identity preservation and Tracking of feed and food

537

Veterinary medicine

Nanotechnology in animal Biotechnology

Feed improvement and waste safety

Pathogen detection & removal from poultry FIGURE 28.6  Applications of nanotechnology in animal biotechnology.

nanoparticles in biological systems demands safe handling via basic laboratory safety norms, and disposal as per international guidelines (Dhawan et al., 2011).

detects animal cytochrome b genes in food or feed products. Nanosensors can detect very small amounts of chemical contaminants, viruses, or bacteria in food systems.

TRANSLATIONAL SIGNIFICANCE

FUTURE APPROACHES

Nanomaterial-based DNA detection and biosensors for pathogens are attracting much attention due to their comparatively high sensitivity and non-complexity (Figure 28.4b). The replacement of fluorescently labeled DNA probes with a class of metallic nanoparticle-conjugated probes appears promising because it can minimize or eliminate the necessity of using expensive and complex instrumentation. Nano-sized, multipurpose sensors are being developed to detect almost everything from physiological parameters to toxic compounds. Nanosensors can detect very small amounts of a chemical contaminant, virus, or bacteria in food systems. Carbon nanotubes are being investigated as biosensors to detect glucose, ethanol, hydrogen peroxide, immunoglobulins, and as electrochemical DNA hybridization biosensors (Figure 28.4c). Nanotechnology has the tremendous potential to revolutionize the agricultural and livestock sectors. It can provide new tools for molecular and cellular biology, biotechnology, veterinary physiology, animal genetics, and reproduction; this would greatly increase the sensitivity of detection of biological materials with quantities typically in the nano-to-picoliter range. (Figure 28.6) Nanoparticles are being used to remove Campylobacter and E. coli from poultry products (Manuja et al., 2012). Listeria monocytogenes, another food-borne pathogen, was detected in spiked milk samples by magnetic nanoparticlebased immune-magnetic separation combined with realtime PCR. Gold nanoparticles have been used to detect contamination of melamine in raw milk samples by the naked eye, with no sophisticated instruments required. The method is also promising for detection of melamine contamination in other foods, such as eggs and animal feed. A fluorescent, bio-barcoded DNA assay has been developed for the rapid detection of Salmonella enteritidis based on two nanoparticles (Zhang et al., 2009). A hand-held chip

Nanotechnology has the potential to revolutionize biomedical science of the 21st century through a paradigm shift in diagnostics, drug discovery and delivery, vaccine development, and tissue engineering. In the domain of veterinary science, nanotechnology has improved animal reproduction, hygiene and health, and nutrition. Food science and technology has seen new nanoscience-based developments in food storage, especially in animal food like red meat and fish, by increasing shelf life and fortified nutritional value. In microbiology, nanomaterials are paving the way as new bactericides promising to fight even multidrug resistance. Nanotechnology tools could enable the understanding of how bacteria work, while providing new opportunities to probe the dynamic and physical aspects of molecules, molecular assemblies, and intact microbial cells, whether in isolation or under in vivo conditions. Furthermore, developments in nanoscience are leading to new, sensitive, and faster methods for pathogen detection in the form of nanosensors. The detection of relatively large-sized double-stranded DNA using stable DNA-functionalized gold nanoparticles is a quite simple colorimetric approach, without the use of complex instrumentation. However, the developed colorimetric approaches still need research advancements in terms of sensitivity and on-site instrumentation before they will be ready for commercialization.

WORLD WIDE WEB RESOURCES 1. NCBI Genome Database: http://www.ncbi.nlm.nih.gov/ genomes/lproks.cgi. 2. Multiple sequence alignment: http://www.ebi.ac.uk/ Tools/msa/clustalw2.

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3. BLAST (Basic Local Alignment Search Tool): http:// blast.ncbi.nlm.nih.gov/Blast.cgi. 4. mfold: www.bioinfo.rpi.edu/applications/mfold. 5. Wikipedia: http://en.wikipedia.org/wiki/Surface_plasmon_resonance. The NCBI houses genome sequencing data in GenBank, and an index of biomedical research articles in PubMed Central and PubMed, as well as other information relevant to biotechnology. ClustalW is a program for global multiple sequence alignment. It constructs pairwise sequence alignments. This heuristic method does a pairwise progressive sequence alignment for all the sequence pairs that can be constructed from the sequence set. A dendrogram (guide tree) of the sequences is then generated according to the pairwise similarity of the sequence. Finally, a multiple sequence is constructed by aligning sequences in the order defined by the guide tree. BLAST is an algorithm for comparing primary sequence information, such as nucleotides of DNA sequences or the amino acid sequences of different proteins. A BLAST search compares a query sequence with a library or database of sequences, and identifies library sequences that resemble the query sequence above a certain threshold. Different types of BLASTs are available according to the query sequences. In silico is an expression used to mean “performed on computer or via computer simulation.” The phrase is coined from the Latin phrases in vivo and in vitro, and is commonly used in biology; it refers to experiments done in living organisms and outside of living organisms, respectively. The in silico approach reduces the time and expense involved in the bench work used for experimentally testing several PCR primers to validate a protocol. mfold is one of the earliest systems designed for molecular biology computations. The program uses mainly thermodynamic methods to predict the secondary structures of RNA and DNA.

REFERENCES Ashbolt, N. J., Grabow, W. O. K., & Snozzi, M. (2001). Indicators of microbial water quality. Water Quality: Guidelines, Standards and Health. World Health Organization (WHO). Water Quality: Guidelines, Standards and Health. In Lorna Fewtrell & Jamie Bartram (Eds.), London UK: IWA Publishing. ISBN: 1900222 28 0. Baptista, P., Pereira, E., Eaton, P., Doria, G., Miranda, A., Gomes, I., Quaresma, P., & Franco, R. (2008). Gold nanoparticles for the development of clinical diagnosis methods. Analytical and Bioanalytical Chemistry, 391, 943–950. Boyer, D., Tamarat, P., Maali, A., Lounis, B., & Orrit, M. (2002). Photothermal imaging of nanometer-sized metal particles among scatterers. Science, 297, 1160–1163. Brust, M., Bethell, D., Kiely, C. J., & Schiffrin, D. J. (1998). Self-assembled gold nanoparticles thin films with non-metallic optical and electronic properties. Langmuir, 14(19), 5425–5429.

Centers for Disease Control and Prevention, National Institutes of Health. (2009). Biosafety in Microbiological and Biomedical Laboratories. (BMBL 5th Ed.U.S. Department of Health and Human Services, pp. 21–1112). Public Health Service., HHS Publication No. (CDC). Dhawan, A., Shanker, R., Das, M., & Gupta, K. C. (2011). Guidance for Safe Handling of Nanomaterials. Journal of Biomedical Nanotechnology, 7, 218–224. ISO/FDIS. (1999). Water Quality – Detection and enumeration of Escherichia coli and coliform bacteria in surface and waste water – Part 3. Miniaturised method (Most Probable Number) by inoculation in liquid medium. Geneva: International Standards Organization. 9308–3. Jyoti, A., Pandey, P., Singh, S. P., Jain, S. K., & Shanker, R. (2010). Colorimetric detection of nucleic acid signature of shiga toxin producing Escherichia coli using gold nanoparticles. Journal of Nanoscience and Nanotechnology, 10, 4154–4158. Liandris, E., Gazouli, M., Andreadou, M., Comor, M., Abazovic, N., Sechi, L. A., & Ikonomopoulos, J. (2009). Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles. Journal of Microbiological Methods, 78, 260–264. Malorny, B., Löfström, C., Wagner, M., Krämer, N., & Hoorfar, J. (2008). Enumeration of Salmonella bacteria in food and feed samples by real-time PCR for quantitative microbial risk assessment. Applied Environmental Microbiology, 74, 1299–1304. Manuja, A., Kumar, B., & Singh, R. K. (2012). Nanotechnology developments:opportunities for animal health and production. Nanotechnology Development. DOI: 10.4081/nd.2012.e4. Mirkin, C. A., Letsinger, R. L., Mucic, R. C., & Storhoff, J. J. (1996). A DNA-based method for rationally assembling nanoparticles into ­macroscopic materials. Nature, 382, 607–609. Pandey, P., Singh, S. P., Arya, S. K., Sharma, A., Datta, M., & Malhotra, B. D. (2008). Gold nanoparticle–polyaniline composite films for glucose sensing. Journal of Nanoscience and Nanotechnology, 8, 3158–3163. Sato, K., Hosokawa, K., & Maeda, M. (2003). Rapid aggregation of gold nanoparticles induced by non-cross-linking DNA hybridization. Journal of the American Chemical Society, 125, 8102–8103. Sato, K., Hosokawa, K., & Maeda, M. (2007). Colorimetric biosensors based on DNA–nanoparticle conjugates. Analytical Science, 23, 17–20. Schofield, C. L., Field, R. A., & Russell, D. A. (2007). Glyconanoparticles for the colorimetric detection of cholera toxin. Analytical Chemistry, 79, 1356–1361. Singh, G., Vajpayee, P., Ram, S., & Shanker, R. (2010). Environmental reservoirs for enterotoxigenic Escherichia coli in south Asian gangetic riverine system. Environmental Science and Technology, 44, 6475–6480. Storhoff, J. J., Lucas, A. D., Garimella, V., Bao, Y. P., & Muller, U. R. (2004). Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes. Nature Biotechnology, 22, 883–887. Vikesland, P. J., & Wigginton, K. R. (2010). Nanomaterial enabled biosensors for pathogen monitoring – a review. Environment and Science and Technology, 15, 3656–3669. Wang, S., Singh, A. K., Senapati, D., Neely, A., Yu, H., & Ray, P. C. (2010). Rapid colorimetric identification and targeted photothermal lysis of Salmonella bacteria by using bioconjugated oval-shaped gold nanoparticles. Chemistry – A European Journal, 16, 5600–5606.

Chapter | 28  Nanotechnology and Detection of Microbial Pathogens

Wang, X., Yuan, L., Wang, J., Wang, Q., Xu, L., Juan, D., Yan, S., Zhou, Y., Fu, Q., Wanga, Y., & Zhan, L. (2012). A broad-range method to detect genomic DNA of multiple pathogenic bacteria based on the aggregation strategy of gold nanorods. Analyst, 137, 4267. Warrington, P. D. (2001). Water Quality Criteria for Microbiological Indicators Prepared pursuant to Section 2(e) of the Environment Management Act, 1981. Zhang, D., Carr, D. J., & Alocilja, E. C. (2009). Fluorescent bio-barcode DNA assay for the detection of Salmonella enterica serovar Enteritidis. Biosensors and Bioelectronics.2009, 24, 1377–1381.

FURTHER READING Fournier-Wirth, C., & Coste, J. (2010). Nanotechnologies for pathogen detection: Future alternatives? Biologicals, 38(1), 9–13. Kuzma, J. (2010). Nanotechnology in animal production – Upstream assessment, of applications. Livestock Science, 130, 14–24. Lu, Y., & Liu, J. (2006). Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes. Nature Protocols, 1, 246–252. Mousumi, Debnath, Bisen, Prakash S., & Prasad, Godavarthi B. K. S. (2010). Molecular Diagnostics: Promises and Possibilities. USA: Springer. Sekhon, B. S. (2012). Nanoprobes and their Applications in veterinary Medicine and Animal Health. Research Journal of Nanoscience and Nanotechnology, 2, 1–16.

GLOSSARY DNA Detection  Identification and detection of DNA molecules using molecular techniques. Gold Nanoparticles  Suspension of nanometer-sized particles of gold in a fluid having intense red color (less than 100 nm). Nanotechnolgy  The engineering of functional systems at the molecular scale. Thiol-Modified DNA  Single-stranded DNA with thiol modification (–SH group) at either the 5′ or 3′ terminal. Surface Plasmon Resonance (SPR)  The resonant, collective oscillation of valence electrons in a solid stimulated by incident light.

ABBREVIATIONS APHA  American Public Health Association BIS  Bureau of Indian Standards BLAST  Basic Local Alignment Search Tool CO  Cytochrome Oxidase FC  Fecal Coliforms FIB  Fecal Indicator Bacteria FISH Fluorescence In Situ Hybridization GNPs  Gold Nanoparticles MF  Membrane Filter MPN  Most Probable Number NSET  Nanoparticle Surface-Energy-Transfer Ruler PCR  Polymerase Chain Reaction QDs  Quantum Dots qPCR  Quantitative Polymerase Chain Reaction RAPD  Random Amplification of Polymorphic DNA

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RFLP  Restriction Fragment Length Polymorphism SERS  Surface-Enhanced Raman Spectroscopy SPR  Surface Plasmon Resonances TC  Total Coliforms TEM  Transmission Electron Microscopy TPS  Two-Photon Scattering Method USEPA  United States Environmental Protection Agency VBNC  Viable But Non-Culturable WHO  World Health Organization

LONG ANSWER QUESTIONS 1. Define nanotechnology. 2. What is nanobiotechnology? 3. Discuss the following applications of nanotechnology. a.  Medical. b.  Nanotoxicology. c.  Nanotechnology and environment. 4. Discuss in detail the principle of using gold nanoparticles for DNA detection. 5. Describe the applications of nanomaterials for pathogen detection.

SHORT ANSWER QUESTIONS 1. What is the significance of particle size in the nanodomain? 2. Briefly talk about metal nano-particle synthesis by the colloidal route. 3. What is the principle behind the color change in a hybridization solution? 4. Differentiate the use of cross-linking and non-crosslinking patterns of GNP probes in DNA detection. 5. How does electron microscopy confirm the detection of target DNA?

ANSWERS TO SHORT ANSWER QUESTIONS 1. Nanotechnology deals with the creation of functional materials, devices, and systems on a nanometer scale length (1–100 nm). Small particles exhibit a high surface-to-volume ratio compared to their bulk counterparts, and become more reactive due to the presence of large numbers of atoms on their surface. Quantum confinement effects at a nanoscale level impart exceptional physico-chemical properties to nanoparticles, which make them promising for various technological applications. In biology, these small-size particles find immense potential for improving our understanding of cell functioning, as most biological activity happens at the nanoscale. 2. Metal nanoparticles are synthesized by reduction of metal cations using appropriate reducing agents in

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aqueous and/or non-aqueous solvents. A stabilizer is also used during the synthesis to prevent aggregation of nanoparticles. The synthesis can be generalized with the following equation: M++ ne− + stabilizer → M° 3. After DNA hybridization, nanoparticles come closer to each other and act like an aggregate. This aggregate of nanoparticles causes a shift in light scattering that leads to a change in color. 4. The method based on GNP cross-linking involves attachment of non-complementary DNA oligonucleotides capped with thiol groups to the surfaces of two batches of GNPs. A polymer network is formed when DNA, complementary to the two grafted oligonucleotides, is added to the solution. This condensed network self-assembles the conjugated GNPs into aggregates with a concomitant change of color from red to purple. This technique is most suitable for tracking small synthetic target sequences of up to 50 bp. Another GNP

aggregation system induced by non-cross-linking DNA hybridization involves immobilization of singlestranded DNA on gold nanoparticles above the physiological temperature. The nanoparticles conjugated with oligonucleotide probes aggregate together at considerably higher salt concentrations when the target DNA is perfectly complementary to the probe. This can help in tracking only the short synthetic oligonucleotides (up to 20–30 bp) targets, which, again, do not represent the sequences of a pathogenic DNA or PCR product. 5. In transmission electron microscopy (TEM), a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through. TEMs are capable of imaging at a significantly higher resolution, so if there is hybridization of GNP probes with target DNA, the GNP probes come in close proximity, and as a result, the particles aggregate. This aggregation can be visualized by TEM imaging.