Exploitation of Nanotechnology for the Monitoring of Waterborne

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Exploitation of Nanotechnology for the Monitoring of Waterborne Pathogens: State-of-the-Art and Future Research Priorities Helen Bridle,*,† Dominique Balharry,‡,§ Birgit Gaiser,‡ and Helinor Johnston‡ †

Downloaded by SUNY UPSTATE MEDICAL UNIV on September 3, 2015 | http://pubs.acs.org Publication Date (Web): September 2, 2015 | doi: 10.1021/acs.est.5b01673

Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS, United Kingdom ‡ School of Life Sciences, Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS, United Kingdom § Centre for Genomics and Experimental Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, United Kingdom ABSTRACT: Contaminated drinking water is one of the most important environmental contributors to the human disease burden. Monitoring of water for the presence of pathogens is an essential part of ensuring drinking water safety. In order to assess water quality it is essential to have methods available to sample and detect the type, level and viability of pathogens in water which are effective, cheap, quick, sensitive, and where possible high throughput. Nanotechnology has the potential to drastically improve the monitoring of waterborne pathogens when compared to conventional approaches. To date, there have been no reviews that outline the applications of nanotechnology in this area despite increasing exploitation of nanotechnology for this purpose. This review is therefore the first overview of the state-of-the-art in the application of nanotechnology to waterborne pathogen sampling and detection schemes. Research in this field has been centered on the use of engineered nanomaterials. The effectiveness and limitations of nanomaterial-based approaches is outlined. A future outlook of the advances that are likely to emerge in this area, as well as recommendations for areas of further research are provided. labor-intensive.7,8 The exploitation of nanotechnology offers the potential to improve pathogen sampling and detection to provide quicker, and more sensitive approaches. This paper reviews the state-of-the-art regarding the application of nanotechnology to either enhance existing monitoring methods or to enable novel sampling or detection approaches. First, introductions to waterborne pathogens, conventional monitoring methods, and nanotechnology are provided. Second, the use of nanotechnology in both sample processing and detection are reviewed. Finally, the paper concludes with recommendations for future research priorities to ensure that nanotechnology can deliver the benefits anticipated for waterborne pathogen detection. 1.1. Waterborne Pathogens. Waterborne pathogens can be divided into three main categories; viruses, bacteria, and parasites. These pathogens reach water sources when infected people or animals shed microbes in faeces. This can occur when untreated or undertreated sewage is released as this allows pathogens to enter water sources. Alternatively, either runoff to source water, or permeation of animal faeces or sewage utilized as fertilizer into groundwater can occur. Many waterborne pathogens are zoonotic, capable of infecting both humans and animals. Table 1 provides an overview of some key pathogens of concern for water quality management and disease

1. INTRODUCTION Contaminated drinking water is one of the most important environmental contributors to the human disease burden worldwide; it has been estimated that in 2008, diarrheal disease (transmitted by contaminated water) claimed the lives of 2.5 million people, and for children under five the number of deaths exceeded those caused by both HIV/AIDS and malaria combined.1 The World Health Organisation (WHO) consider that microbial hazards (i.e., pathogens) represent the main challenge in the delivery of safe drinking water.2 In addition to the significant disease burden, the worldwide negative economic impact of waterborne pathogens is large. For example, lost productivity in the U.S. alone due to diseases caused by these pathogens has been estimated at US$20 billion per annum.3 Furthermore, a recent study estimated the cost of Escherichia coli O157 human infections just in The Netherlands to be 9.1 million Euros per year.4 Furthermore, there are also costs relating to treatment and emergency measures that add to the economic burden of adverse health effects associated with waterborne pathogens.5,6 Monitoring of water for the presence of pathogens is an essential part of ensuring drinking water safety. Determining risk also requires assessment of the potential infectivity of pathogens, which considers speciation, and viability determination. However, there are many shortcomings with existing conventional methods of waterborne pathogen detection including, for example, their inability to determine pathogen species and viability, their lack of sensitivity, the poor recovery rate of pathogens, and the fact they are time-consuming and © XXXX American Chemical Society

Received: September 12, 2014 Revised: August 14, 2015 Accepted: August 24, 2015

A

DOI: 10.1021/acs.est.5b01673 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

Table 1. Overview of Selected Problematic Waterborne Pathogens From the WHO List of Relevant Waterborne Pathogens2a microorganism

morphology

infectivity

persistence in water

resistance to disinfection

incidence/outbreaks (worldwide unless otherwise stated)

norovirus

Viruses 35−40 nm; single-stranded RNA in a nonenveloped icosahedral capsid

high

long

high

high

long

high

9000 cases in 21 outbreaks (.S., 1990s); 2400 cases (Sweden, 2008) 50−60% of all hospitalisations of children with acute gasteroenteritis

2 × 0.5 μm; gram negative, rod-shaped bacterium

high

moderate

low

4 × 1 μm; gram negative, nonmotile, rod-shaped bacterium

high

short

low

Vibrio cholerae

2 × 0.5 μm; gram negative, comma shaped bacterium

low

short−long

low

Cryptosporidium

Protozoa hard-shelled oocyst encasing four sporozoites; size varies with species; human pathogenic species parvum and hominis approx 5 μm

high

long

high

cyst enclosing 2 trophozoites; around 8 × 12 μm in size

high

moderate

high

rotavirus

80 nm; segmented double stranded RNA in a nonenveloped icosahedral capsid with outer shell that has a wheel-like appearance Bacteria


Escherichia coli (Enterohemorrhagic) Shigella

Giardia

2300 cases and 7 deaths (Walkerton, Canada, 2000) 2 million cases occur annually, resulting in an estimated 600 000 deaths 3−5 millions cases and 100 000 deaths annually 250−500 million cases annually; 400 000 cases (Milawaukee, WI, 1994) 2 million cases annually; > 1000 cases in 2004 (Norway)

a Relative infectivity is categorized as low if the infective dose is greater than 104 pathogens, moderate for doses between 102 and 104 and high for doses between 1 and 102. There is no indication in the WHO list of what probability of infection this infective dose represents. Persistence in water is defined as short for pathogen survival periods of less than 1 week, moderate for times between a week and a month, and high for pathogens capable of survival in the environment for longer than one month. Resistance to disinfection is considered low if 99% of organisms are inactivated in 1 min, moderate if 99% inactivation takes 1−30 min and high for times longer than 30 min (for freely suspended organisms, at conventional doses and pH 7−8).

Figure 1. Schematic demonstrating the process of direct detection using filtration, centrifugation, immunomagnetic separation and fluorescent microscopy to identify Cryptosporidium oocysts. The figure indicates the numerous steps required to monitor for pathogen presence from large samples (though these will vary depending upon the pathogen type and the end detection method chosen; this procedure was selected as an example as it is the only direct detection routinely performed by water companies) and the challenges in terms of time required and process losses/low recovery rates. Figure is reproduced with permission from "Detection of Cryptosporidium in miniaturised fluidic devices", Bridle et al., Water Research, 46 (6), 2012.

prevention. These were selected from the WHO list of Relevant Waterborne Pathogens2 due to high and/or recent incidence/ outbreaks and should not be taken as an exhaustive list of priority pathogens. For more detailed information on these pathogens, and others, see references2 and9 1.2. Current Approaches to Monitoring of Waterborne Pathogens. During the 20th century the measurement of faecal indicator bacteria in water has contributed to a significant improvement in drinking water safety.10,11 This approach detects bacterial species as indicators of possible

sewage contamination of water, due to the fact that these organisms are commonly found in faeces. The bacteria detected are not necessarily pathogens. Water samples are taken and then analyzed in the laboratory (often requiring expensive equipment and trained specialists to perform the analysis), and thus results are not obtained immediately and the analysis is costly. Accordingly, the development of online monitoring systems has expanded over recent years (e.g., for early warning of an outbreak) with many companies entering this market (e.g., http://speedybreedy.com/; http://www.vienna-waterB


none described, but positively charged surface

none described

nanoaluminia fibres, fibres are 2 nm in diameter, approximately 0.3 μm long, surface area 500 to 600 m2/g

cellulose nanocrystals, 90 ± 10 nm length, 8 ± 1 nm diameter

virus; disc filters

bacteria; depletion flocculation

Fe3O4, 17−50 nm diameter

Fe3O4, 20 nm diameter

CdTe quantum dots and Fe3O4 Sizes not reported

bacteria; magnetic nanomaterials

E. coli; magnetic nanomaterials

E. coli and S. typhimurium; magnetic nanomaterials

C

coating with E. coli and S. typhimurium antibodies

amine-functionalized, then conjugation to antibodies or avidin

anti-E. coli or Anti-S. typhimurium antibodies at different concentrations

acetate-stabilized, positive charge at pH 3.5

zirconia nanopowder Crystalline/amorphous Zr(OH)x, 5−10 nm diameter extensive characterization of filters containing nanomaterials

bacteriophage; surface modified filters

E. coli; magnetic nanomaterials

functionalization

type of NM

pathogen/method

not described in detail

not described in detail

19−45 mg/mL

nanocrystal:bacteria ratio of 100:1 and 100,000:1

2−5 mL

1−2 mL

20 μL

103−108 cfu/mL

103−107 cfu/mL

not described

1L

1 L or more

sample volume

103−108 cfu/mL

108 cells/ml

102−103 pfu/L

107 plaque forming units (pfu) per liter

90% of filter pore volume filled with zirconia not described

pathogen concentration

NM concentration

Table 2. Literature Reported Examples of the Use of NMs in Sample Processing of Waterborne Pathogens sample Type

PBS

apple juice and milk

spinach wash and isolated from milk into PBS

ground beef wash

10 mM NaCl solution

seawater; RO membrane treated seawater permeate; secondary treated sewage effluent

DI water with NaCl added to reach 400 μS/cm conductivity


recovery rate

not reported

76% to 126% depending on sample type

not investigated; study focused on specificity and LOD

Agrawal42

Cheng39

Ravindranath38

Varshney37

Sun36

80−100% flocculation

94%

Li35

Wegmann34

40−90%, depending on water type and starting numbers

99.99%

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nanotechnology-based applications, however, an overview of NM toxicity will not be provided here, as a number of reviews on this topic are already available (e.g., Johnston et al. 2013,20 Oberdorster et al. 2007,21 Donaldson et al. 2013,22 Nel et al. 2006, Scwon et al, 201023). Accordingly, this review will solely focus on the properties of NMs which may be exploited for sampling, and detecting waterborne pathogens. Recent reviews of nanotechnology for the detection of pathogenic infectious agents24 have mainly focused on medical, bioterrorism or food-borne pathogens, rather than the particular challenge of detecting pathogens in water.25,26 Monitoring of pathogens is an important contribution to ensuring the safety of drinking water, and this review will therefore outline how nanotechnology may be exploited to improve water quality by (i) improving sample processing and/ or (ii) enabling pathogen detection. More recently, there has also been increasing interest in the use of NMs for the removal of waterborne pathogens, however this topic will not be covered as it out-with the scope of this review and instead readers are referred to existing reviews.27−29

monitoring.com/index.php/en/products/coliminder; http:// www.colifast.no/). However, there are concerns about the degree of correlation between the detection of indicators (which are typically not pathogenic) and presence of microbial pathogens, as well as the fact that this approach does not allow a valid identification of the pathogen.12−14 Microbial source tracking (MST) has emerged as an alternative to using faecal detection as an indicator, to associate faecal sources with human health risk and provide a better correlation between MST marker and pathogen presence.15 In contrast, direct detection approaches look for the presence of specific pathogens (Figure 1) and will be the main focus of this review, although the techniques enhancing molecular detection of pathogens discussed could also be applied to MST markers. Direct microbiological testing presents a unique challenge due to low numbers of pathogens in water.16 Methods of detection include specific culturing of organisms (when possible), molecular methods for both speciation and viability determination, optical and electrical detection technologies, and biosensors.9 Many emerging monitoring techniques do not offer sufficient sensitivity and therefore signal amplification is essential. Nanotechnology represents one potential solution to this challenge. For example, assessment of pathogen viability is a critical parameter when assessing water quality, and nanotechnology-based approaches can enhance the sensitivity of existing methods. In addition, as current approaches used to detect water pathogens are time-consuming (e.g., due to the many steps required for sample processing and detection) more efficient schemes could be enabled by more sensitive, and novel nanotechnology based methods. 1.3. Nanotechnology & Nanotoxicology. Nanotechnology encompasses the exploitation of engineered nanomaterials (NMs) in a variety of applications and products,17 as well as processes performed at the nanoscale (1−100 nm) and the use of “nanostructured surfaces”, which are designed to have active elements at the nanoscale. Generally, a nanomaterial is defined as a material with at least one dimension in the “nanoscale” of 1−100 nm (BSI/British Standards, European Commission, National Nanotechnology Initiative18). Unique properties derive from the large surface area per mass unit of materials produced at the nanoscale and higher surface reactivity at the increased surface to volume ratio present in NMs.19 In parallel to the increased production and use of NMs within a variety of applications it is necessary to evaluate their toxicity to ensure responsible innovation and ensure the safety of products and applications containing NMs. This is also an important consideration when developing novel NM based approaches to water quality monitoring. The production and use of NMs for water quality monitoring is likely to lead to the direct exposure of humans and wildlife, including organisms vital to food chains. Additionally, if monitoring is conducted in situ, NMs will be in direct contact with water samples and care must be taken to quantify and minimize the risk associated with their use and potential leakage into the environment and water supplies. NM toxicity cannot be extrapolated from existing information on the behavior of their larger “bulk” counterparts, which leads to uncertainty surrounding the hazards posed by NMs to human health and the environment. Therefore, the discipline of nanotoxicology (the study of the toxicity of NMs) has grown considerably over recent years to better understand the potential adverse effects associated with NMs. Nanotoxicology studies provide vital information for the safe development of

2. NANOTECHNOLOGY IN SAMPLE PROCESSING Sample processing is a key part of any monitoring strategy, and is required to concentrate samples from liters down to microlitre or milliliter sized samples, which can be processed by detection technologies. Large initial sample volumes are required to gain a more representative sample as well as to increase the likelihood of detecting pathogens which are often present in water at very low concentrations. For example, human exposure to a few oocysts (

Exploitation of Nanotechnology for the Monitoring of Waterborne

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