WO2018045449A1

WO2018045449A1 - Method and apparatus for detection and treatment of e. coli in water

Info

Publication number: WO2018045449A1
Application number: PCT/CA2017/000200
Authority: WO
Inventors: Sushanta Kumar MITRA; Naga Siva Kumar Gunda; Saumyadeb DASGUPTA
Original assignee: Mitra Sushanta Kumar; Naga Siva Kumar Gunda; Dasgupta Saumyadeb
Priority date: 2016-09-09
Filing date: 2017-09-08
Publication date: 2018-03-15

Abstract

A water treatment system uses a process of 'fishing, trapping and killing' to treat Escherichia coli (E. coli). D-glucose, adsorbed onto paper strips may facilitate the 'fishing' of E. coli. The porous nature of the paper strip facilitates the 'trapping' of the bacteria. A portion of the strip containing Moringa oleifera cationic protein (MOCP) can 'kill' the 'trapped' E. coli through prolonged contact. A similar strip-based device may also be used for detecting E. coli in water. One end of the strip is coated D-glucose (dextrose) 'fish' for E. coli. The E. coli in the paper strip will migrate towards the other end of the paper strip by capillary action. The E. coli bacteria will concentrate near the opposite end of the paper strip containing a hydrophobic barrier and react with the chemicals added to the strip to produce a visually observable colour change to indicate presence of E. coli.

Description

METHOD AND APPARATUS FOR DETECTION AND TREATMENT OF E. COLI IN

WATER

FIELD

[0001] The present disclosure relates generally to the field of detecting and treating pathogens in liquid samples, and, more particularly, to methods and apparatuses treating

Escherichia coli (E. coli) in water samples.

BACKGROUND

[0002] Contaminated water sources continue to pose a major challenge in the twenty-first century towards providing clean and safe drinking water to the local communities

Freshwater resources all over the world are plagued by contaminants like traditional chemical wastes from agricultural and industrial sites, heavy metals, fecal coliforms, viruses as well as new-age pollutants such as endocrine disruptors and nitrosoamines⁵⁷'⁵⁸. Notably, potable water sources contaminated by deleterious pathogens pose significant public health concerns which impact nearly 1.8 billion people globally⁵⁹. The significant public health concerns is

compounded by the limitations in implementing reliable water treatment solutions^60-64 particularly in limited-resource communities where chlorine based disinfection procedures are still in vogue despite their considerable drawbacks and long-term health effects

[0003] Emerging water treatment techniques including those that use nanoparticles^69-72 and photocatalysts^73-75 show considerable promise for point-of-use purposes. However, the long term health impacts of these techniques remain under continued scrutiny^76-79. Hence, there is a pressing need for methods and devices able to remove harmful pathogens without creating toxic by-products in already compromised water sources. Effective point-of-use devices and techniques which are cheap, easily available, portable and non-toxic with minimal energy requirements can serve this purpose towards providing clean and safe drinking water to limited- resource and marginalized communities around the world.

[0004] Routine testing of microbiological water quality is also another very important aspect for maintaining the health and safety of the general public, due to increased risk of water- borne diseases^1-6. Some presently known methods for evaluating the quality of water are generally laboratory-based, which can be complex and time-consuming¹'^7-12. Moreover,

13 laboratory testing can be expensive because trained technicians are required to perform the test ¹⁵. Therefore, testing of water samples for bacterial contamination is generally not performed on a daily basis, and the water suppliers and regulators normally perform testing in annual or semiannual basis or whenever testing may be required ''¹⁶ This kind of minimal evaluation does not ensure the quality of water sources. Hence, field test kits have been developed to simplify the testing procedures¹⁵'¹⁷. These field test kits can be used to test the water samples for assessing microbial quality of water at the point of source itself. In both traditional laboratory-based methods and present field test kits, specialized instrumentation such as an incubator are still needed. Furthermore the time required to obtain results can vary from 8 hours to 48 hours It is therefore desired to reduce the wait times to get the test results in a more immediate basis to allow for early prevention of water borne diseases.

[0005] A litmus test is an indicative test that is used in chemistry to find the general acidity or alkalinity of the substance (liquid or gas) using litmus paper. A litmus paper is made of a dye based on lichens and it turns pink or red in an acid (pH < 6.0) whereas it turns blue in a base (pH > 8.0). There will be no colour change in a solution with pH between 6.0 and 8.0. Litmus paper is inexpensive and is used to differentiate acids and bases. Similar kinds of inexpensive litmus tests are not available in biology to identify or detect the biomolecules of interest for samples being tested.

[0006] Recent developments in paper-based biosensing technology have introduced new techniques and methods of creating simple, low-cost and rapid detection devices ^29~34. For example, paper-based biosensors exploit known antigen-antibody interactions have been developed to detect the target analytes of interest in water, soil, urine, blood or saliva samples ^l" ³⁴. Devices built on paper based sensing technologies are numerous, ranging from sensors for testing of infectious diseases in blood samples, testing of grains in agriculture to testing of chemical contaminants in water and soil^29"34. Hossain et al.³⁵ developed a paper-based micro fluidic device to detect presence/absence of bacteria using chromogenic substrates. In such a device, the bacteria in water samples are pre-concentrated using antibody-coated immune- magnetic nanoparticles and the concentrated samples are then tested with the paper-based microfluidic device. The microfluidic device was able to detect bacterial at concentrations of 5- 20 CFU/mL within 30 min. using the paper-based system without culturing the bacteria. The same device was able to detect bacterial concentration of 1 CFU/lOOmL in 8 hrs with a culturing step. However, as a skilled person may appreciate, the use of nanoparticles and culturing steps increases the complexity of the disclosed detection method.

[0007] Recently, Silver Lake Research Corporation (Azusa, CA, USA) released a rapid bacteria testing product called WaterSafe® that can be used to detect E. coli in water samples within 15 minutes. The product is based on antigen-antibody interaction on paper strips similar to lateral flow tests. Water quality is evaluated by dipping the paper strip into a water sample. The formation of two colour bands on the paper strip represents the existence of E. coli in the water sample. These paper strips are simple to use, inexpensive and provide rapid results.

However, these paper strips are not specific to E. coli, fecal, or total coliform, but detects other non-coliform bacteria as well. Moreover, the sensitivity of this kind of paper devices are debatable. These paper strips do not following the United States Environmental Protection Agency (US EPA) standards of 100 mL sample volume. Rather, these paper strips use a lower sample volume for detection so that the accuracy of these test kits are uncertain and do not provide good repeatability. Lastly, preparation of such antibody-based paper-based devices require assembly of varying types of paper strips, treatment reagents, and microparticles, making the manufacture of these papers strips more expensive and complex.

[0008] Gunda et al.^24-28 developed two versions of test kits for rapid detection of indicator bacteria, Escherichia coli (E. coli), in water samples within in one hour. However, these test kits require a housing of filter papers and chemicals within a plastic enclosure to perform the simultaneous detection and quantification (of concentration) of bacteria. While these new test kits are simple to use, portable and low cost, disposal of used plastic kits and chemicals after testing can contribute to the buildup of waste and potentially introduce a new source of environmental contamination.

[0009] Accordingly, an improved method and apparatus for detecting E. coli is desired.

SUMMARY

[0010] In one broad aspect, a water treatment apparatus for treating water contaminated with bacteria, the apparatus comprising: a first attraction zone having a first chemoattractant for attracting bacteria from the contaminated water; a treatment zone having a bactericide for neutralization of the bacteria, the treatment zone operatively connected to the first

chemoattractant zone to receive bacteria from the first attraction zone; and a second attraction zone having a second chemoattractant operatively connected to the treatment zone to receive bacteria from the treatment zone, the first and second attraction zones being separated by the treatment zone.

[0011] In another broad aspect, a method of treating water contaminated with bacteria with a water treatment apparatus, the method comprises: establishing a chemoattractant concentration gradient in the water to induce a chemotactic response of the bacteria to migrate towards a first attraction zone of the water treatment apparatus; directing progression of the bacteria to a treatment zone of the treatment apparatus, the treatment zone operatively connected to the first attraction zone and having a bactericide; entrapping the bacteria within the treatment zone; and exposing the bacteria to the bactericide in the treatment zone to neutralize the bacteria.

[0012] In another broad aspect, a test strip for detecting bacteria in a water sample, the test strip comprises: an attraction zone at one end of the test strip having a chemoattractant for receiving water and bacteria contained in the water; a reaction zone connected to the attraction zone and located between the one end and the other end of the test strip to receive bacteria from the attraction zone for detection; and a barrier zone at the other end of the test strip to maintain water and bacteria within the reaction zone by inhibiting progression of water and bacteria along the test strip, the barrier zone being separated from the attraction zone by the reaction zone.

[0013] In another broad aspect, A method of testing a water sample for bacterial contamination using a test strip, the method comprises: inducing a chemotactic response of the bacteria present in the water towards an attraction zone at one end of the test strip having a chemoattractant by establishing a chemoattractant concentration gradient in the water; directing progression of the water and migration of the bacteria from the attraction zone at the one end of the test strip to a reaction zone; and testing the water for the presence of bacteria in the reaction, the reaction zone configured to provide a visual indicator due to presence of the bacteria.

[0014] Additional aspects of the present invention will be apparent in view of the description which follows. Other features and advantages will be apparent from the specification and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The foregoing and other aspects of the invention will become more apparent from the following description of specific embodiments thereof and the accompanying drawings which illustrate, by way of example only, the principles of the invention. In the drawings:

[0016] Figures 1 A and IB are schematic drawings of a water treatment apparatus according to at least one embodiment;

[0017] Figure 1C is a schematic diagram of a modified chemotaxis assay for using with a paper strip treated with D-glucose solution;

[0018] Figure 2 is a schematic of a water treatment system showing three paper strips attached end to end with the lower edge of the paper strip dipped in sample water according to at least one embodiment;

[0019] Figure 3 is a graph showing bacteria removal efficiency of different glucose trips

(GS) as a function glucose concentration;

[0020] Figure 4 is a graph showing efficiency of a 0.1M glucose strip (GS) as a function of bacterial concentration;

[0021] Figure 5 are images taken of a Moringa paper strip (MS) and an NS after overnight incubation in a solution of yellow-green fluorescent particles;

[0022] Figure 6 are images showing positions of the liquid/air interface and the corresponding bacterial distribution at different experimental times are shown in the top and the bottom panel, respectively;

[0023] Figure 7 are scanning electron microscopy (SEM) images of the control strip (NS) showing bacterial deposition;

[0024] Figure 8 are SEM images of the TGS, MS and BGS taken under high vacuum.

[0025] Figure 9 is a schematic illustration of the use of a DipTreat device to clean water in accordance with at least one embodiment; [0026] Figure 10 is an illustration of an experimental setup used to demonstrate the

'fishing, trapping and killing' of E. coli;

[0027] Figure 1 1 is an illustration of a fluorescence and imaging arrangement used to capture the liquid/air interface and bacteria cells within the paper strips;

[0028] Figure 12 are images depicting fluorescence observed on paper under different set of excitation wavelengths for different time instants during an experiment;

[0029] Figures 13A and 13B are schematic diagrams of an the E. coli detection device in accordance with at least one embodiment;

[0030] Figures 14A and 14B are illustrations showing a comparison the E. coli detection device of Fig. 13A tested with deionized water (a) and with E. coli contaminated water (b);

[0031] Figure 15 shows the development of colour in samples with decreasing

concentrations of E. coli (CFU/lOOmL) tested with the E. coli detection device of Fig. 13 A;

[0032] Figure 16 is a graph of response times for an embodiment of the E. coli detection device as a function of various known concentrations of E. coli;

[0033] Figures 17A and 17B show a comparison of the colouration on MWK strips according to at least one embodiment after 90 minutes when E. coli concentration is at (a) 10⁶ CFU/mL and (b) 10⁵ CFU/mL;

[0034] Figure 18 shows a comparison of MWK strips according to at least one embodiment;

[0035] Figure 19 shows the uneven nature of the wax coating on paper according to an embodiment;

[0036] Figure 20 shows the colour observable on a test strip of at least one embodiment after having been dipped in water with an E. coli concentration of approximately 10⁵ CFU/mL;

[0037] Figure 21 shows the colour observable on a test strip of at least one embodiment after having been dipped in water with an E. coli concentration of approximately 10⁶ CFU/mL;

[0038] Figure 22 shows the colour observable on a test strip of at least one embodiment after having been dipped in water with lower and higher concentrations of E. coli after 1 hour; [0039] Figure 23 A is a scanning microscope image of a porous paper matrix of the a detection device of at least one embodiment;

[0040] Figure 23B is an image the detection device of Fig. 23 A after dipping in deionized water at room temperature;

[0041] Figure 23C is an image of the detection device of Fig. 23A tested with E. coli contaminated water (2 x 10⁴ CFU/mL) at room temperature, with the appearance of colour on the used device representing the presence of E. coli;

[0042] Figure 24 are images showing the development of pinkish red colour on the E. coli detection device according to at least one embodiment after 2 hours based on the indicated concentrations of E. coli (CFU/mL) in which the control shows no colour as it is dipped in deionized water;

[0043] Figure 25 is a graph illustrating wait (response) times for the appearance of the pinkish red colour with respect to various dip times for various known concentrations of E. coli contaminated water samples;

[0044] Figure 26 illustrates an embodiment of the E. coli detection device to test the water sample for the presence of E. coli bacteria; and

[0045] Figure 27 is a chart summarizing the results for 40 different water samples tested using an embodiment of the E. coli detection device according to at least one embodiment.

DETAILED DESCRIPTION

[0046] The description which follows, and the embodiments described therein, are provided by way of illustration of examples of particular embodiments of the principles of the present invention. These examples are provided for the purposes of explanation, and not limitation, of those principles and of the invention.

[0047] In respect of treatment of water samples, a device and process of water treatment which involves effective 'fishing, trapping and killing' of Escherichia coli (E. coli) in contaminated water samples is described. As will be described in greater detail subsequently, a chemoattractant, such as naturally occurring sugar D-glucose, may be been adsorbed onto a substrate material such as a blotting paper or other porous substrate material to facilitate the 'fishing' of E. coli cells by inducing their chemotactic response towards the chemoattractant (glucose) concentration gradient. Various types of material other than blotting paper can be used, including but not limited to, polymers, ceramics, fabrics, and the like. In one example

embodiment, as described in greater detail subsequently, the 'fishing, trapping and killing' mechanisms can be established by using porous paper substrates in the form of a strip. By dipping such paper strips laced with 0.1M D-glucose as the chemoattractant into a sample of contaminated water, bacterial removal efficiency of up to 99.5% is obtainable after a 90 minute duration. The porous nature of the paper strip of the described example embodiment can facilitate the 'trapping' of the bacterial cells within its inherently porous structure. Antimicrobial capabilities may be added to the substrate through the adsorption of a suitable bactericide or antibiotic thereon. For example, to treat water contaminated with E. coli using one example embodiment, Moringa oleifera cationic protein (MOCP) can be adsorbed onto the paper substrate to 'kill' the 'trapped' E. coli through prolonged contact, as described in more detail below. Finally, these individual 'fishing', 'trapping' and 'killing' processes may be combined into an integrated 'fishing, trapping, and killing' water treatment apparatus, or a "DipTreat" apparatus, whereby the designated portions of the water treatment apparatus may be

functionalized to carry out the 'fishing, trapping and killing' mechanisms . Aspects of this disclosure may allow a new generation of inexpensive and portable water treatment devices which can passively remove and neutralize deleterious pathogens from water.

[0048] In respect of detection of E. coli in water samples, the present disclosure describes a relatively simple and relatively low-cost testing device that may be used to detect E. coli bacteria within 100 mL volume water samples. The testing device may be put in contact with a water sample such that bacteria in that water may cause the device to generate an observable indication of the presence of the bacteria. Specifically, illustrative examples of E. coli detection

77 78

capabilities similar to the Mobile Water Kit (MWK) ' can be implemented on relatively simple test strips, as will be described in more detail subsequently in the section entitled "Testing of Water for Bacterial Contamination", below.

[0049] In some embodiments, the strip- based testing device may be made of a porous material, such as Grade GB003, Whatman absorbing gel blotting paper and the like, with one edge being coated with a hydrophobic material, such as wax and the like, to create a hydrophobic barrier and another edge (hereinafter the "attraction zone") can be coated with a bacterial chemoattractant, such as with D-glucose (dextrose) solution and the like, for insertion into a water sample.

[0050] The strip-based testing device also comprises a reaction zone between the two ends of the strip, immediately below the hydrophobic barrier on the strip (if oriented vertically for insertion into water), and can be coated with enzymatic substrates and other ingredients (e.g. compounds similar to the chemicals used in the MWK and plunger tube assembly) for bacteria detection. During use, the attraction zone can be dipped into the possibly contaminated water for detection of E. coli bacteria to perform a "DipTest".

[0051] Similar to the DipTreat apparatus, a chemoattractant provided in the attraction zone can be used to attract the bacteria in the water being tested to migrate towards the strip- based testing device³⁶. For example, D-glucose may be used to target E. coli bacteria. Water, along with attracted bacteria, can flow along the strip (e.g. based on capillary action) from the attraction zone to the reaction zone until it is inhibited from travelling further by the hydrophobic barrier.

DipTreat Treatment of Water Contaminated with Bacteria

[0052] The approach towards removing bacteria from water in the present disclosure may be implemented using the phenomenon of directed movement of bacterial cells (chemotaxis) towards a favorable chemical agent. The chemotactic behavior of bacterial cell dispersions has been widely studied^80-86. In the presence of favorable chemical agents, called chemoattractants, the bacterial cells have been found to migrate from regions of lower chemoattractant

concentration towards regions of higher chemoattractant concentration along a chemoattracting gradient. Certain amino acids⁸⁷ (like aspartate and serine) and sugars⁸⁸ (like glucose, fructose and lactose) may function as chemoattractants with varying degree of effectiveness depending upon its concentration and the type of bacterial species⁸¹'⁸⁹. Such controlled behavior has been used in a number of applications ranging from increasing the efficiency of contact-killing surfaces⁹⁰ to regulated mixing in microfluidic systems⁹¹ and payload delivery in micro-robotics⁹².

Incorporation of a suitable chemoattractant on a porous substrate material of the water treatment apparatus to lure bacterial contaminant such as Escherichia coli (E. coli)out of water may be useful for the purposes of water treatment. [0053] Based on previous research ' , D-glucose or dextrose, a naturally occurring sugar, may be used as a chemoattractant to lure bacteria out of water. For example, bacteria such as E. coli is known to be attracted to D-glucose. This approach may be viewed as implementing a mechanism of 'fishing' that uses a chemoattractant to 'bait' and to capture bacteria from water. The use of a porous substrate like paper and other suitable materials known to a person skilled in the art, instead of an impervious one, can facilitate the 'trapping' of the 'fished' bacterial cells within its internal porous network. In the examples described below, the inventors found a maximum bacterial removal of 99.5% from a sample of water using this 'fishing' and 'trapping' procedure.

[0054] In some embodiments, the water treatment apparatus can 'kill' the trapped bacteria to provide a complete water treatment solution. There are a number of natural materials that may be used, such as plant essential oils⁹⁵'⁹⁶, allicin⁴⁹, curcumin⁹⁸ and the like, which are known to have significant bactericidal properties. In the inventors' previous work focusing on nature-inspired solutions for water treatment,³⁸ the inventors have established the efficiency of Moringa oleifera seed extract for reducing bacterial loads in non-turbid water. Moringa oleifera is a multipurpose medicinal plant found in regions with tropical climate, especially in southeast Asia and Africa⁹⁹. Besides having a high nutritional value, crude Moringa seed extract has been shown to contain a water soluble flocculating cationic protein which has been characterized^100-103 and used in the treatment of highly turbid water sources^104"-106. Moreover, this Moringa oleifera cationic protein (MOCP), present in the seed extract, has been shown to exhibit antimicrobial activity by causing bacterial cell death through the mechanism of membrane fusion · . Owing to its effectiveness as a bactericide and nontoxic nature, MOCP may be a candidate for incorporation into the water treatment apparatus for killing of the trapped bacteria. In some embodiments, other types of antibiotics or bactericides can be used with the water treatment apparatus to provide killing of the trapped bacteria. In yet other embodiments, combinations of bactericides and/or antibiotics may incorporated to kill one or more types of trapped bacteria.

[0055] By using a combination of 'fishing, trapping and killing' mechanisms, the inventors may develop a water treatment apparatus. In some embodiments, the materials used may be made of naturally available, biodegradable substances. For example a substrate made of highly porous, bio-degradable blotting paper (Grade GB003, Whatman) that may be laced with a chemoattractant such as D-glucose. A similarly porous substrate maybe laced with a bactericide or antibiotic such as non-toxic Moringa oleifera seed extract containing MOCP. These elements, when used together, may provide the intended 'fishing, trapping and killing' mechanisms to treat water contaminated with bacteria. In the described example, the D-glucose may work as a chemoattractant to lure the bacteria into the treatment apparatus so that the bacteria's contact with the MOCP present in the Moringa oleifera seed extract may lead to the killing of the bacteria. The use of a suitable substrate material such as blotting papers, or similar materials in some embodiments, allows a uniform capillary action facilitating the passive transport of fluid within the treatment apparatus without the need of any additional elements such as pumps, thereby reducing the overall cost of such a system. While the bacteria tested in the examples is E. coli, a common Gram-negative bacteria of the fecal coliform group, present in abundance in compromised potable water sources, other types of bacteria may be targeted by using a suitable chemoattractant in combination with a suitable bactericidal compound within the treatment apparatus.

[0056] Fig. 1 A is a schematic drawing illustrating an embodiment of water treatment apparatus 100 A usable for treating contaminated water. The water treatment apparatus may be rectangular in shape in the form of a strip having different functional regions provide over a substrate.

[0057] In the illustrated embodiment, the water treatment apparatus 100A has a first attraction zone 102 and a second attraction zone 106. The first and second attraction zones 102 and 106, as noted above, may be laced with a chemoattractant, such as D-glucose and the like, so that when the first attraction zone 102 is dipped into the water being treated, the chemoattractant may be released into the water to establish a chemoattractant concentration gradient to induce a chemotactic response of the bacteria in the water to migrate towards the first attraction zone 102, as shown in Fig. 2.

[0058] In the illustrated embodiment of water treatment apparatus 100A, the treatment zone 104 may be positioned in between the first and second attraction zones 102 and 106. Either attraction zone 102 or 106 may be dipped into the water to be treated. In the illustrated embodiment, the attraction zone that is dipped into the water being treated may be regarded as the first attraction zone, while the attraction zone not dipped in water may be regarded as the second attraction zone. In some other embodiments of the water treatment apparatus, the water treatment apparatus may comprise of one attraction zone and one and one treatment zone, each zone arranged to be adjacent to each other.

[0059] In some embodiments, the chemoattractant in the attraction zones 102 and 106 may be different compounds. Alternatively, the attraction zones 102 and 106 may be laced with the same chemoattractant compound, but each zone having a different concentration of the compound. In such embodiments, one of the attraction zones 102 and 106 may be designated specifically for dipping in water.

[0060] Referring still to Fig. 1 A, the treatment zone 104 may be laced with a bactericide or antibiotic, such as MOCP and the like, to neutralize or "kill" the bacteria that has migrated from the first attraction zone 102 into the treatment zone 104. Once the bacteria enter the treatment zone 104, contact of the bacteria with MOCP may cause killing of the bacteria via mechanisms such as membrane fusion. For the purposes of the present disclosure, "bactericide" may be understood to include all different types of antibiotics and bactericidal compounds, whether naturally occurring or otherwise, that are capable of killing bacteria or otherwise inhibiting the growth of bacteria.

[0061] Selection of a suitable substrate material for use with the apparatus may facilitate movement of water and bacteria along the water treatment apparatus 100 A. A porous substrate such as blotting paper and the like may facilitate capillary action to permit movement of water and bacteria along the apparatus. While blotting paper is used in the present embodiment as the substrate, other suitable porous substrates capable of providing capillary action can similarly be used.

[0062] As noted previously, the water treatment apparatus may have a single attraction zone and a single treatment zone. However, the addition of a second attraction zone as shown in Figs. 1A and IB may reduce the number of viable bacteria (i.e. those that were not 'killed' in the treatment zone) migrating back to the water. The addition of a second attraction zone can induce a second chemotactic response from the viable bacteria so that they may migrate towards the second attraction zone, away from the water being treated.

[0063] It may be noted that the water treatment apparatus 100A of Fig. 1 A may be fabricated on a common or "shared" substrate so as to allow movement of water and migration of bacterial from one zone to the next. However, in another embodiment, such as the embodiment shown in Fig. IB, the water treatment apparatus 100B may comprise several substrate portions 114, 116, 1 18 attached together.

[0064] In some embodiments, each substrate portion can be connected to another substrate portion to facilitate movement of water and bacteria. A connection can be established, for example, by overlapping an edge region of one substrate portion with a corresponding edge region of another substrate portion. The overlapping areas may be regarded as a contact zone, as shown by contact zones 1 10 and 1 12. A distance between the edges of the overlapping substrate portions (i.e. along a direction that is transverse to the edge) can be set to a desired value. In the embodiment depicted in Fig. IB, an edge region of the substrate portion 1 14 of the first attraction zone 102 may overlap with a corresponding edge region of the substrate portion 1 16 of the treatment zone 104 to form a first contact zone 1 10. In some embodiments, a second edge region of the substrate portion 1 16 of the treatment zone 104 may overlap with an edge region of the substrate portion 118 of the second attraction zone 106 to form a second contact zone 112. In some other embodiments, where two attraction zones and one treatment zone are present, one of the attraction zones and the treatment zone may share a common substrate in one substrate portion, while the other attraction zone may be provided on a separate substrate portion. In such an embodiment the individual substrate portions may be connected, using an overlapping connection, for example, to form the water treatment apparatus.

[0065] While the embodiment of Fig. 1 B makes use of substrate portions having substantially the same widths, the size of each substrate do not have to be the same. Other shapes and sizes of the substrate portions may also be used. For example, in some embodiments, one of the attraction zones may be wider, longer or both wider and longer relative to the dimensions of the treatment zone 104 and/or the other attraction zone. Furthermore, each substrate portion may be fabricated using different substrate materials. For instance, the attraction zones may be made of paper while the treatment zone may be made of woven fabric. The dimensions of the substrate portions may be dictated by the characteristics of the substrate material being used, for example, in respect of its porosity and how well the material absorbs water. Example Preparation and Empirical Characterization of the DipTreat Water Treatment Apparatus

[0066] The following sections provided herein describe example embodiments illustrating the example implementations and example methods in respect of a water treatment apparatus that makes use of the "fishing, trapping, killing" mechanisms to treat contaminated water samples. The bacteria of interest in the examples described below are E. coli, although other types of bacteria may be considered. The presented examples and their characterization are intended to be provided for the purposes of explanation, and not limitation, of the present invention.

Materials and Equipment For Characterization of DipTreat Water Treatment Apparatus

[0067] Sample water was obtained from Barnstead Nanopure water purifier (Model

No. Dl 1971, Thermo Scientific, Waltham, Massachusetts). The model microorganism,

Escherichia coli (K-12 strain), used in this study was obtained from New England Biolabs, Ipswich Massachusetts, USA. The blotting paper (Catalogue no. WHA 10427804) used to prepare the paper strips was procured from Sigma Aldrich, Canada. D-glucose or dextrose (Catalogue no. 3260-1 -70) was obtained from Caledon Laboratories Ltd, Ontario, Canada.

Lauryl Tryptose Broth (LTB) (Catalogue no. CA90001-768) was used as the growth medium for E. coli. Luria Bertani (LB) agar (Catalogue no. CA90002-674) was used to plate the bacteria for the purposes of enumeration. All bacterial growth media were purchased from VWR, Canada. Sodium chloride (NaCl) obtained from Sigma Aldrich, Canada, was used to prepare a 0.85% concentrated solution which was used as a medium to suspend bacteria prior to fluorescent staining. Dry, unshelled Moringa oleifera seeds were procured from Purelife Herbs, USA.

Standard issue cardboard and adhesives were used to prepare the device. 100 mL glass beakers (Catalogue no. 89000-200) from VWR, Canada were used to conduct the experiments. A two- pronged clamp (Catalogue no. 80063-610) from VWR, Canada was used to hold the paper strip combinations in place during all experiments, as shown in Figure 10. E. coli were stained for fluorescence using Live/DeadR BacLight™ bacterial viability kit (Catalogue no. L7012) from ThermoFisher Scientific, USA. Yellow-green fluorescent nanoparticles, 200 nm in diameter, (Catalogue no. F8848, Thermo Fisher Scientific, USA) were used to determine the protein adsorption on paper fibers. All bacterial growth media were autoclaved (Primus Sterilizer) according to experimental requirements.

Imaging and Characterization System

[0068] An imaging station, as depicted in Fig. 1 1 was built to perform fluorescence imaging of the stained bacteria. A Digital Single-Lense Reflex (DSLR) camera (Nikon D5200) was used to capture all the images used in this study. The images of the paper strips after the experiments were obtained inside a dark enclosure (details provided further below). The mounted LED light source (Catalogue no. M490L3) used to observe the fluorescence from stained bacterial cells was attached to an adjustable collimator lens (Catalogue no. SM2P50-A) and connected to a LED driver (Catalogue no. LEDD1B) which controlled the current input. A 575 nm bandpass filter was used in conjunction with the camera to capture the fluorescence from stained E. coli cells. All lighting and filter accessories were obtained from Thorlabs, USA.

[0069] A scanning electron microscope (Quanta 3D FEG, FEI) was used to characterize bacterial distributions within the paper strips. For the purpose of scanning electron microscopy (SEM) imaging, small portions of the paper strips were cut from the regions of interest and attached to SEM stubs using double sided carbon tape. The samples were then sputter coated with gold and imaged under high vacuum (10^"5 Torr) and high voltage (20 kV) with the electron microscope. A confocal laser scanning microscope (Zeiss LSM 700) was used for the purposes described in this study. An optical upright microscope (Omax Epi -fluorescent Trinocular Compound Microscope) was used to capture bacterial distribution in the chemotaxis assay.

Bacterial Growth Characterization and Fluorescent Staining

[0070] E. coli was grown overnight (24 hours) in 100 raL of autoclaved lauryl tryptose broth (LTB) at 37°C inside an incubator under a constant shaking speed of 150 rpm. The resulting culture was enumerated using the plate counting technique following a serial dilution protocol. For the plate counting procedure, luria broth (LB) agar plates were used and the plated bacteria were incubated overnight (24 hours) at 37°C before enumeration. The concentration of bacterial culture prepared before each set of experiments was maintained at 109 CFU/mL. For staining purposes, the BacLight™ bacterial viability kit was used. Prior to staining, E. coli cells were doubly washed in deionized (DI) water according to manufacturer guidelines and re- suspended in 0.85% sodium chloride solution (NaCl). The fluorescent dyes Syto 9 and propidium iodide (PI) were mixed in a 1 : 1 ratio and 3 mL of the mixture was added per mL of bacterial culture and mixed thoroughly using a vortex shaker. Before using it in the experiments, the stained culture was kept in dark for 15 minutes at room temperature. Only freshly stained cultures were used in the experiments to ensure uniformity in results.

EXAMPLE 1- CHEMOATTRACTANT AND BACTERICIDE PREPARATION

[0071] An example embodiment of a water treatment apparatus comprising D-glucose and Moringa oleifera seed extract is described for the purpose of illustrating the preparation of the chemoattractant and bactericide, respectively. In the present example embodiment, blotting paper is used as the substrate material for the empirical characterization, (and obtained from Sigma Aldrich, Canada). As noted previously, other suitable porous substrates may be used instead of blotting paper in other embodiments of the treatment apparatus device. The blotting paper substrate can be cut to a desired size. For example, in the described embodiment, the substrate is cut into 55 mm x 14 mm strips. Chemoattractant of varying concentrations may be prepared for inclusion into the substrate strips. In the present example embodiment, solutions of D-glucose of five different concentrations (0.0001 M, 0.001 M, 0.01 M, 0.1 M and 1 M) were prepared by adding requisite amounts of D-glucose in DI water. The paper strips were completely immersed in these solutions and kept on a shaker for 2 hours at room temperature. In some cases, two hours of immersion may be sufficient. In other cases the immersion time may be less than two hours or greater than two hours, depending on the type and nature of the chemoattractant. Thereafter, the wet strips were removed from the solutions and dried overnight at room temperature and used as glucose paper strips (GS) in subsequent characterization.

[0072] Bactericide may also be prepared in a similar manner for adsorption into the substrate material. In the presently described embodiment, Moringa oleifera seed extract (5% concentration, w/v) was selected as the bactericide and prepared using previously reported procedures . As noted previously, other bactericide or antibiotic compounds such as plant essential oils⁹⁵'⁹⁶, allicin⁴⁹, curcumin⁹⁸ and the like may be used, depending on the bacteria of interest. As with D-glucose, blotting paper strips were immersed in the seed extract for a sufficient amount of time under constant shaking. In some cases, two hours of immersion may be sufficient. In other cases the immersion time may be less than two hours or greater than two hours, depending on the type and nature of the bactericide. The dimensions of the paper strips containing Moringa may be the same as those containing the D-glucose. However in other embodiments, the dimensions may be different. The strips were dried overnight prior to use in the characterization procedure and may be referred to as Moringa paper strips (MS). The 2 hour immersion time was found to be sufficient, in the present example embodiment, for the adsorption of glucose and Moringa oleifera cationic protein (MOCP) onto the paper surfaces.

EXAMPLE 2 - 'FISHING ' FOR BACTERIA WITH D-GLUCOSE AS CHEMOATTRACTANT

[0073] The effect of the use of a chemoattfactant in the water treatment apparatus to attract or 'fish' bacteria is characterized. In the present example D-Glucose, for example, in the form of D-glucose Strips (GS) prepared in the previous example, was qualitatively analyzed relative to the strips without a chemoattractant. A conventional capillary chemotaxis assay⁹³ was modified as shown in modified chemotaxis assay lOOC of Fig. 1C. Specifically, the conventional chemotaxis assay was modified by replacing the capillary containing chemoattractants normally used with the assay with a GS on the glass slide 122. The rest of the chemotaxis setup, as reported by Adler , was kept unchanged. The setup comprises of a U-shaped spacer wire 124 covered by a coverslip 126 to create the bacterial chamber 128. To examine the effect of the chemoattractant, 20 mL of bacterial culture of concentration 10⁹ CFU/mL was placed carefully inside the chamber 128 to establish a bacterial suspension. Thereafter, a small piece of GS 120 was inserted carefully inside the bacterial chamber 128. After 5 minutes, photographic images of bacterial dispersion near the edge of the paper strip 120 were obtained using a camera attached to an optical microscope (details provided further below). As a control, a similar piece of paper without glucose (NS) was also inserted in the modified chemotaxis assay lOOC and images of a corresponding region around the edge of the NS were taken after 5 minutes. The images were then compared to determine the density of bacteria around the edges of the GS and NS.

[0074] Different samples of GS, as discussed previously, were prepared using five different D-glucose concentrations (1 M, 10^"1 M, 10^"2 M, 10^"3 M, 10^"4 M) and the effectiveness of the GS to fish for bacteria were characterized in water samples containing a known initial bacterial concentration of 1.5 X 10⁶ CFU/mL. In the present example, an end of a vertical GS in 60 mL of model contaminated water sample with a known initial bacterial concentration as shown in Fig. 10. A clamp 1002 was used to hold the GS in a stable position (similar to the arrangement shown in Fig. 10 and described further below). The GS was removed from water after 90 minutes followed by the enumeration of bacterial count in the residual water sample by serial dilution and plate counting techniques. LB agar plates were incubated at 37°C for 24 hours before counting the number of colonies. Control experiments were conducted using a NS in lieu of the GS were similarly conducted to assess the effectiveness of the chemoattractant in respect of 'fishing' the bacteria out of the contaminated water sample.

'Fishing ' efficiency of Glucose Strips

[0075] The inventors hypothesized that a chemoattractant concentration gradient in the bulk media surrounding the GS would cause the directed migration of bacterial cells towards and into the GS. A two-fold approach was undertaken to verify this chemoattracting efficiency of the GS. The inventors' findings with the modified chemotaxis assay of Fig. 1C confirmed that use of a GS was more efficient in luring bacteria towards to the treatment apparatus than a NS.

Specifically, on being introduced into the bacterial suspension, the GS, unlike the NS, induces a chemoattractant concentration gradient, as shown in Fig. 1C, panel B, through diffusion to promote the active movement of bacteria along the glucose concentration gradient. This was confirmed by the presence of a higher density of E. coli cells in the area near the edge of the GS after 5 minutes, as shown in Fig. 1C, Panel B, as compared to the NS, shown in Fig. 1C, Panel A. Specifically, Fig. 1C Panel A is a microscope image showing bacterial distribution near the control paper (NS, without any glucose additive) edge after 5 minutes. Figure 1C panel B shows E. coli a higher density of cells near the edge of the glucose paper strip (GS) after 5 minutes. The glucose concentration gradient is shown in Panel B. Images were taken using a 40X brightfield objective, and the scale bar is 50 mm.

EXAMPLE 3 - CHARACTERIZATION OF CORING A ON POROUS SUBSTRATE

[0076] The presence of MOCP in the dried MS was verified using yellow-green fluorescent particles (200 nm in diameter, sulphate modified) carrying a negative surface charge. A 10 mL solution of the fluorescing particles with a concentration of 2.65 X 10¹⁰ particles per mL was prepared using DI water. The MS were completely immersed in solutions containing the above-mentioned fluorescent particles and kept overnight under constant shaking. This ensured sufficient time for the electrostatic adsorption of the particles to the surface of the MS due to the underlying positive charge of the MOCP coating. Thereafter, the samples were removed from the solution, dried and imaged using a confocal laser scanning microscope. Untreated paper strips (NS) were subjected to the same experimental procedure and used as control.

[0077] In previous studies, the antimicrobial properties of MOCP were achieved on the surfaces of silica microparticles¹⁰⁹ by suspending the microparticles in a solution of Moringa oleifera seed extract. The inventors hypothesized that porous substrates, such as the blotting paper strip as described in Example 1 , would similarly be able to adsorb the water soluble MOCP from the Moringa oleifera seed extract and the protein would retain its antimicrobial properties in a dried form.

[0078] Figure 5 shows the confocal microscope images taken of a MS (Panel A) and NS

(Panel B) after overnight incubation in a solution of yellow-green fluorescent particles. Figure 5 Panel A, shows intense fluorescence observed in the MS. Negatively charged fluorescent nanoparticles (200 nm) are adsorbed onto the paper fiber surface owing to the presence of underlying MOCP coating. Figure 5 Panel B shows very low fluorescence observed in case of the control paper strip (NS) relative to the use of MOCP in Panel A, indicating significantly lower adsorption of nanoparticles. The confocal (the images at the left) and the differential interference contrast (DIC) images (at the middle) were taken using a 5X objective and merged (the images on the right) to create a perspective of the position of the nanoparticles with respect to the paper fibers.

[0079] The confocal microscopy images show relatively higher particle density, and hence, a relatively higher fluorescence intensity in the MS image compared to the NS image. These images can be used to provide support of the presence of the MOCP on the paper substrate because the negatively charged fluorescent particles are greatly adsorbed due to the cationic charge of the underlying MOCP in the MS. It may be observed from the images of the distribution of fluorescing particles along the paper fibers in the MS image. In the NS sample, devoid of any cationic surface charge, the corresponding NS image shows minimal nanoparticle retention owing to the interaction of surface forces.

EXAMPLE 4 - FABRICATION AND USE OF THE WATER TREATMENT APPARATUS

[0080] With reference to Fig. 2, an embodiment of the water treatment device similar to device 100B can be constructed using the components described in the preceding examples. Specifically, in the present embodiment, substrates laced with chemoattractant and bactericide targeting E. coli, are assembled into the water treatment apparatus as described in this section. Accordingly, similar reference numerals shall be used to describe the various components. It is noted that the water treatment apparatus described in the present section is an example to highlight one possible implementation of the 'fishing, trapping and killing' mechanisms. It may be appreciated that variations and combinations of the described elements may be used to carry out the 'fishing, trapping and killing' of bacteria of interest in a water sample.

[0081] The paper strips (GS and MS) can be supported on a cardboard scaffold and appropriate adhesives. Other types of scaffolding and adhesives known to a person skilled in the art may also be used for constructing the paper strips. Two GS and a MS of similar dimensions (55 mm by 14 mm), obtained through the processes described earlier, were fixed end to end. In some embodiments, there can be approximately 5 mm edge-to-edge overlap, along a direction that intersects both edges, between them forming the contact zones 110 and 112 so that the entire water treatment apparatus is composed of a bottom glucose strip (BGS) 102, a middle Moringa strip (MS) 104 and a top glucose strip (TGS) 106. In other embodiments, different dimensions and overlaps may be considered. The magnified images (Panels A-C) of Fig. 2 show E. coli moving through the pores of the blotting paper strips (Panel C). Green colour indicates viable, motile bacteria getting attracted and moving through the bottom glucose strip (Panel B). The red colour denotes the non-viable cells due to contact with the middle Moringa strip (Panel A). The glucose concentration gradient is shown on the left.

[0082] In some characterization setups, the cardboard scaffold can be held with a clamp to maintain positioning of the water treatment apparatus. Only the lower edge of the BGS 102 (i.e. the first attraction zone of FIG. IB) was kept in contact with contaminated water. The elements of the one example setup is illustrated in Fig. 10 and described further below. The contact between the individual strips at the overlap regions was monitored to ensure the smooth transition of water and bacteria along the strips. Fluorescent stained bacteria, obtained through the procedure described previously, were used in these experiments by adding requisite amounts of bacteria to the sample water. For the purpose of characterization of the presently described water treatment apparatus, 60 mL water samples were used with an initial E. coli concentration of 1.5 X 10⁶ CFU/mL. [0083] The experimental time was varied to track the movement of the liquid/air interface to different positions on the paper strips, with a maximum experimental duration being 90 minutes. After the conclusion of the experiments, the paper strips was removed from sample water and kept inside a dark enclosure for imaging, as shown in Fig 11 and described further below. To confirm the presence of stained bacteria in the paper by fluorescence imaging, an excitation wavelength of 490 nm was used and images were captured with a camera equipped with a 575 nm bandpass filter.

[0084] Figure 3 is a graph showing bacterial removal efficiency of the water treatment apparatus of the present example as a function of chemoattractant, D-glucose (in the present example), concentration. A zero D-glucose concentration refers to the control experiment with an untreated paper strip (NS). For all the cases, initial bacteria concentration was 1.5 X 10⁶

CFU/mL. Error bars indicate the standard errors from three replicate experiments. The graph indicates that the paper strips dipped in 0.1 M D-glucose solution were the most efficient in removing E. coli from the water samples among the different D-glucose concentrations (0.0001 M, 0.001M, 0.01 M, 0.1 M and 1 M). Specifically, the GS laced with 0.1 M D-glucose removed 94.5% of the bacterial load, which was nearly three times more than the bacteria removal facilitated by the NS (indicated by the 0 glucose concentration in Fig. 3). The 33.3% bacterial removal obtainable with the NS can be attributed to the inherent capillary transport of E. coli suspension inside the porous paper. 1 M and 0.0001 M D-glucose concentrations were least effective among the GS with 64% and 67% bacteria removal, respectively, while both the 0.01 M and 0.001 M GS facilitated greater than 80% bacteria removal. Existing literature⁸⁸'⁹⁰ suggests that the optimum D-glucose concentration to promote E. coli chemotaxis in a chemotaxis assay is in the vicinity of 10^"3 M. Hence, it may be concluded that the paper substrate strips prepared using 0.1 M D-glucose solution are able to create a chemoattracting gradient similar to that caused by 10^"3 M D-glucose solution in capillary chemotaxis assays.

[0085] Figure 4 is a graph showing the bacteria removal efficiency of the 0.1 M GS as a function of bacterial concentrations (10², 10³, 10⁴, 10⁵ and 10⁶ CFU/mL E. coli). Error bars indicate the standard errors from three replicate experiments. The maximum bacteria removal efficiency of 99.5% was obtained for samples with an E. coli concentration of 104 CFU/mL. The results indicate the consistency of bacteria removal by GS, registering greater than 90%

2 5

efficiency for all the considered cases. Even for the worst cases of 10 and 10^J CFU/mL, only 7% of the initial contamination remained in the residual water. Hence, all subsequent experiments were conducted utilizing the most effective 0.1 M GS.

The 'trapping' process

[0086] It can be observed that the use of the porous substrate such as porous paper substrates can be useful for the trapping of bacteria. Besides facilitating the continuous migration of bacterial cells inside the porous substrate through capillary action and hence removing them from the bulk contaminated water, its permeable nature can also facilitate 'trapping' the same bacterial cells within its porous network. The substrate material chosen may be selected so that the pores are much larger than the size of individual cells, that the material does not hinder the movement of the cells away from the bulk water source. The characterization of the water treatment apparatus showed that the bacterial cells 'fished' from contaminated water may migrate from one end to the other end of the of the paper strips along with the liquid/air front (results not shown). Hence, the presence of a porous network may provide 'trapping' process, which could not have been achievable otherwise by using a substrate of impermeable nature.

'Killing ' o bacteria

[0087] Following the successful 'fishing' of bacteria from water, the water treatment process can proceed to 'killing' of the 'trapped' bacteria in the substrate using the bactericide. In the present example, the E. coli 'trapped' within the porous paper substrate may be 'killed' using MOCP. As noted earlier, the movement of the air/water interface along the strip allows bacterial to also migrate along the strip and into the MS containing MOCP.

[0088] Earlier studies with MOCP^38,107 has established that the flocculating and antimicrobial characteristics of the protein takes effect approximately 3 minutes after initiation of contact with a bacteria. This characteristic of MOCP may require a certain minimum length of the Moringa paper strip (MS) in the present example water treatment apparatus to allow the motile E. coli bacteria to be in contact with the MOCP for at least 3 minutes. By tracking the progress of the liquid/air interface, the length of the MS (i.e. treatment zone) may be determined. In the present example embodiment, a 55 mm long MS would provide a sufficient contact time for this purpose. Hence, the length of GS was also maintained at 55 mm to maintain uniformity. [0089] The inventors predicted that the E. coli cells after being 'fished' out of water by the BGS, would smoothly progress into the MS, where they would be neutralized owing to prolonged (greater than 3 minutes) contact with MOCP adsorbed onto the paper fibers. The principal function of the TGS is in facilitating the transfer of any viable E. coli cells, which didn't get killed in contact with MOCP located in the MS, to the topmost paper strip and essentially trapping them further. The position of the liquid/air interface and the corresponding bacterial distributions was tracked at different experimental times. The images obtained from this set of experiments are shown in Fig. 6. Specifically, Fig. 6 shows images indicating the positions of the liquid/air interface (shown by dotted line) and the corresponding bacterial distribution at different experimental times are shown in the top and the bottom panel, respectively. Yellowish- orange fluorescence on the paper strips, in the bottom panel, indicates the distribution of bacteria at the interface and away from it during different stages of the experiment. Fluorescence on the control strips (GS and NS) is shown on the right after 90 minutes from the start of the

experiment.

[0090] It can be observed that the E. coli cells may move with the air/water interface, rising steadily owing to capillary action through the porous paper strips, as indicated by the concentrated fluorescence intensity (emitted by the bacterial cells stained with fluorescing dyes Syto 9 and propidium iodide) tracing the liquid/air front in Fig. 6.

[0091] Control studies using two separate strips - NS and GS, each 140 mm long were also conducted. During the entire 90 minute span of the water front tracking experiments using control strips (images not shown), it may be observed that the bacterial cells generally followed the movement of the liquid/air interface. This is a classic example of motile bacterial cell behavior, which can also be observed in droplets of bacterial suspensions^{1 10} where the cells seek towards the droplet/air interface. The combined configuration of BGS-MS-TGS of the present example water treatment apparatus, on the other hand, indicated an interesting behaviour. During the first 5 minutes, a smooth front movement with the bacterial cells faithfully following the liquid/air interface may be observable, similar to that in the control experiments (e.g. see Fig. 6). However, as soon as the duration of the water front within MS extended beyond three minutes, a faint fluorescent trail behind the interface may be observed. After 10, 15 and 20 minutes of dipping the water treatment apparatus in the sample contaminated water, distinct regions with increasing fluorescence away from the interface and along the edges of the MS and TGS may be observed, indicating bacterial cell deposits in these areas. Close to the end of the experiment i.e., at the 60 and 90 minutes mark, a nearly complete separation of the bacterial cells from the air/water interface, as is evident from the widespread prevalence of fluorescing zones away from the interface in the TGS, can be observed. At the end of the experiment, the fluorescence intensity at the air/water interface in the TGS can be found to be low as compared to areas away from it, indicating a lower concentration of viable E. coli cells at the liquid/air interface area.

[0092] The above-described behavior can be explained from the detailed scanning electron microscopy (SEM) images of the paper strips used in the construction of the example water treatment apparatus, which is discussed subsequently. All the images shown in Fig. 6 are representative images, as the exact location and the shape of the interface would vary due to the inherent nature of randomness in the porous paper strips. The inventors refrained from quantifying the viable and non-viable bacterial cells based on the area-wise fluorescence intensity distribution of the paper strips because of certain challenges, which are discussed further below.

[0093] The inventors conducted other imaging analyses to further confirm that the fluorescence from paper strip used is indeed due to 'trapped' bacterial cells. For this purpose, direct evidence was gathered from scanning electron microscopy (SEM) images of the used paper strips. The fluorescing zones were identified and viewed under high vacuum settings of the electron microscope. The SEM images were first captured for the control strip (NS), as shown in Fig. 7 to establish a reference data set. Under such high vacuum setting of the SEM, the bacterial cells (E. coli in the present example) were not able to retain their characteristic rod like shape and appear fragmented in the images. Images taken all along the fluorescing interface of the control strip reveals a uniform distribution of bacterial cells on the individual paper fibers.

Except for the fluorescing liquid/air interface (see right panel in Fig. 6), no significant bacterial presence elsewhere on the NS was observable.

[0094] Subsequently, the three paper strips of the example water treatment apparatus were subjected to a similar inspection after the 'fishing, trapping and killing' process. Figure 8 displays the bacterial distributions observed in the TGS, MS and BGS, respectively. The left and right panels have different magnifications. The BGS did not show any fluorescence because all the bacterial cells had moved onto the MS and TGS as is evident from the SEM images, at the bottom panel of Fig. 8. In the ensuing investigation of the fluorescing regions for the MS, it was observable that deposits of aggregated bacteria, as shown in Fig. 8, middle panel were the source of fluorescence. These agglomerates are typical consequences of the flocculating action of

38 107

MOCP. As reported earlier ' , the mechanisms of bacterial neutralization by the MOCP is due to a combination of flocculation and membrane fusion processes leading to non-viable aggregates of bacterial cells. As the liquid/air interface crosses to the MS from the BGS, the MOCP, being water soluble, begins to interact with the bacterial cells at the interface. After the three minutes mark, both the flocculating and antimicrobial characteristics may start taking effect and the bacterial surface charges are neutralized causing them to form clusters. Shortly after, the neutralized and clustered bacteria are no longer able to follow the liquid/air interface and begin to trail behind. The large clusters which become comparable to the pore sizes of the substrate, are deposited towards the edges and away from the interface owing to hindrance caused by the paper fibers of the paper strips used in the example water treatment apparatus. The remaining viable bacterial cells may continue to follow the air/water interface up to the TGS where a reduced fluorescence intensity is observable. The smaller clusters are also pulled along up to the TGS where they lag behind the interface leaving a trail of nonviable bacteria cells spread across the TGS, as is evident from the images in the top panel of Fig. 8.

EXAMPLE 5 - DIP PEN WATER TREATMENT APPARATUS

[0095] Another example embodiment of the water treatment apparatus may take the form of a dip pen. Specifically, the three strips of the water treatment apparatus of Example 4, can be packaged into a cartridge with an outer enclosure in the shape of a pen, with one end closed and another end that defines an opening to allow exposure of the attraction zone to be dipped into water. In use, a user can then dip the pen to treat a glass of water, for example. In some dip pen embodiments, the pen could be immersed in the water for a specified time to remove the bacterial contaminant. Figure 9 showcases one such possible design and may be regarded as a "DipTreat" device. In some embodiments, the DipTreat device may be a kit comprising a pen- shaped enclosure and replaceable treatment strips. In other embodiments the "DipTreat" device may be a single-use, disposable device.

[0096] Embodiments of the DipTreat device may be a powerful hand-held tool for rapid treatment of water in remote communities and even for military applications, where in some embodiments one could dip this DipTreat device inside water collected in a glass (typically 250 mL size) for more than 3 min to get the water cleaned.

[0097]

Supplementary Information Regarding Empirical Characterization of the Water

Treatment Apparatus

[0098] Use of an experimental setup for testing the example embodiments of the water treatment apparatus described above will now be described. Specifically, aspects of the experimental and characterization setup are described in further detail in respect of the apparatus of Example 4 (treatment of E. coli using MOCP using paper as a substrate). However, it may be understood that other embodiments and/or configurations of the water treatment apparatus may be tested with the described experimental and characterization setup, with or without any modifications thereto. Accordingly, the description is provided for the purposes of explanation, and not limitation, of the present invention.

Experimental setup

[0099] Figure 10 shows the experimental setup used to test a water treatment apparatus such as the apparatus of Example 4 described above. A two-prong extension clamp (obtainable from VWR Canada), was attached to a clamp stand to hold the cardboard scaffold containing the three paper strips in position. The height of the clamp was adjusted to keep only the lower edge of the bottom glucose strip (BGS) dipped inside sample water. Similar clamp arrangements were used in tracking the liquid/air interface in control experiments using single strips of NS and GS and in determining the efficiency of different GS. Glass beakers containing 60 mL of sample water were used in all the experiments.

Imaging system

[00100] The images of the bacterial dispersions in the chemotaxis assay were captured with an Omax digital camera mounted to an optical microscope using a Tou View software (ToupTek Photonics). A 0.5X reduction lens was connected to the camera using an appropriate adapter for reducing the image size to match the sensor of the digital camera. [00101] To capture images of the fluorescence distribution on paper strips, a dark enclosure was created to prevent interference from ambient light. The light source and DSLR camera were affixed to the top of the enclosure while the paper strips were placed on the bottom wooden support (see Fig. 11). In order to capture the fluorescent emissions from tagged E. coli cells, a 575 nm bandpass filter was attached to the camera. The LED light source was attached with a collimator lens to narrow the light rays to our region of interest.

Analysis of fluorescence distribution on porous substrates

[00102] One of the challenges in performing fluorescence experiments with paper substrates containing both the bacteria and bactericide MOCP is that the latter is a protein that is also able to fluoresce along with the tagged bacteria within an overlapping wavelength. The inherent fluorescence of most proteins arises due to the presence of the amino acids tryptophan and tyrosine^{1 11}. When excited by a light source at 295 nm, the emission spectra of purified

1 12

MOCP are found to be dominated by tryptophan emissions with an emission maximum at the wavelength of 343 +/- 2 nm. Moreover, existing literature suggests that the emission spectrum of MOCP and Syto 9 (used for staining E. coli cells) overlap around the 440 nm-450 nm

region^112,1 '³. Therefore, if the paper strips are excited with a light of wavelength 490 nm (outside the emission spectrum of MOCP), the inventors would only observe fluorescent emissions from Syto 9 and propidium iodide used to dye the bacterial cells, devoid of any contribution from tryptophan and tyrosine, which would then confirm the presence and distribution of bacteria at the fluorescing regions. Based on the studies of Stocks^{1 13} and Kwaambwa and Maikokera^{1 12}, the inventors also hypothesized that a 302 nm excitation wavelength would reveal a fluorescent distribution on the paper strips with contributions from both MOCP and the stained bacteria. This was confirmed in in the images shown in in Fig. 12. The strips of the example water treatment apparatus was dismantled and laid out as three separate strips: bottom glucose strip (BGS), middle Moringa strip (MS), and top glucose strip (TGS) for visualization. In the first 5 minutes, the liquid/air interface has moved into MS, as indicated in the images with appreciable fluorescence for both the 302 nm and 490 nm excitation sources. After 15 minutes, the water front has moved in the TGS, as indicated by the position of the liquid/air interface. At the end of the experiment (90 minutes), a broader spread of fluorescence is observable to indicate the presence of bacterial clusters along with water soluble MOCP that has been transported from MS to TGS. Comparison between the left and the middle panels of Fig. 12 corresponding to the strips of paper substrate obtained from the example water treatment apparatus shows greater intensity of fluorescence under UV light, indicating the contribution of MOCP. The control strip (GS) at the right-most panel did not show any extra fluorescing regions under UV light confirming that no additional fluorescence arises in the absence of MOCP.

[00103] The paper strips illuminated by a 302 nm ultraviolet light source (UV Lamp, Cole Parmer, Canada) showed increased fluorescence when compared with those imaged using a 490 nm light source. The increased fluorescence obtainable using former excitation source is inclusive of the additional fluorescent emissions from tryptophan residues within the MOCP protein. The protein, being water soluble, is transported from the MS to the TGS along with the bacterial aggregates leading to larger fluorescing zones within the TGS (see images on the left panel of FIG. 12 after 15 minutes dipping time). The control strip (GS) did not exhibit any additional fluorescing zones under illumination 302 nm with the excitation source, thereby confirming the absence of any protein. The fluorescence in the control strips is solely due to the fluorescent stained bacteria.

[00104] A few of key points in analyzing the fluorescent distributions caused by the MOCP and fluorescent tagged bacterial cells captured by the example water treatment apparatus can be noted. For the configuration of the example water treatment apparatus, quantification of the live and dead cells from these images based on the fluorescence intensity distribution were not carried out because of the porous nature and the thickness of the blotting paper strips used as the substrate. Also the fluorescence dye Syto 9 has been known to show strong bleaching effect¹¹⁴ which considerably decreases the fluorescing intensity with time, giving unrealistic results. Secondly, in crude Moringa oleifera seed extract were used for the presented example. The Moringa seeds contain nearly 27% proteins by mass¹⁰⁴ of which only 1.2% is constituted by MOCP¹⁰⁷. This introduces a other water soluble proteins which are unaccounted for within the seed extract, which do not possess any flocculating or antimicrobial properties but contribute towards the fluorescence owing to the ubiquitous presence of tryptophan and tyrosine. Although using a 302 nm excitation wavelength minimizes the fluorescence emissions of tyrosine, the overlapping tryptophan emissions from the other water soluble proteins, make it almost impossible to distinguish the exact locations of MOCP in the used paper strips. However, selection of a suitable substrate, bactericide, bacterial dye and corresponding excitation sources in alternative embodiments of the water treatment apparatus, may permit quantification of live/dead cell counting by fluorescence. The characterization of these components may similarly be carried out as described in the present disclosure to determine appropriate preparation and imaging parameters to enable fluorescence quantification of live/dead cells.

Comparison with current products on the market

[00105] A table summarizing current products on the market in comparison to dip and treat strips according to some embodiments of the present disclosure is provided in Table 1 below:

Table 1 : Comparison of dip and treat device with current products on the market

Technology Targeted Power

Products Price Efficiency

Used Contaminants Source

Filtration

bottles, filtration . .. ~, . Bacteria and

Microfiitration $20 - $150 99% ^N0t _j straws, gravity protozoa required filters

Bacteria, virus,

Ultrafiltration protozoa, Required

Filtration bottles and chemicals, $60 - $400 >99% in some adsorption particulates, cases

heavy metals

Variable,

depend-

Chemical pills Chemical Bacteria and Not

$10 - $ 20 ing on

and solutions treatment protozoa required

water

sources

Bacteria,

bottles and Ultraviolet

protozoa and $70 - $150 > 99% Required pens light

virus

Bacterial

chemotaxis

Dip and treat

and Bacteria $12 (per _{¾ g9%} Not strips (present

contact-killing coli) 100 strips) ^{" "} ' required research)

antimicrobial

agent

Conclusions

[00106] Described herein is an apparatus and method of 'fishing, trapping and killing' bacterial cells which has a significant potential in point-of-use water treatment. The results described herein suggest that the 'fishing, trapping and killing' mechanisms can be integrated into a water treatment device comprising of a suitable chemoattractant and bactericide laced on a porous substrate material. In the example water treatment apparatus described herein, blotting paper strips laced with natural chemoattractant and bactericide materials D-glucose and Moringa oleifera seed extract, respectively, were used implemented the 'fishing, trapping and killing' mechanisms to treat water contaminated with E. Coli. Specifically, example apparatus is capable of exploiting the chemotactic response of E. coli towards D-glucose to lure approximately 99.5% of the bacterial load from sample water onto the example water treatment apparatus. The configuration of top and bottom D-glucose strips help in trapping the bacteria within the paper substrate where the E. coli cells are killed by the centrally placed Moringa strips due to the bactericidal properties of the MOCP found within the Moringa oleifera seed extract.

[00107] The 'fishing, trapping and killing' mechanisms of the example water treatment apparatus can be characterized using fluorescence imaging of the fluorescent tagged bacterial cells and MOCP. The distribution of the fluorescent tagged E. coli cells on paper strip substrates can further be characterized through detailed SEM imaging. Overall, the findings demonstrate a sustainable water treatment system can be implemented using compounds that can be used to facilitate the 'fishing, trapping and killing' mechanisms. The described example water treatment apparatus, may be configured to use the antimicrobial activity of MOCP and the chemoattracting properties of D-glucose to treat E. coli bacteria. The described example water treatment apparatus incorporating the identified compounds was effective in 'fishing, trapping and killing' E. coli in a relatively simple and efficient manner. The use of a chemoattractant to lure bacteria from water towards an antimicrobial protein in conjunction with scaffold to provide support has significant potential to be a 'clean and green' technology as well as an economical solution towards removing pathogens to turn contaminated water into potable water.

[00108] For the treatment of bacteria, the efficiency of the water treatment apparatus can be enhanced by increasing the concentration of the bactericide. For example, in the case of treatment of E. coli with MOCP, preparing the water treatment apparatus with higher Moringa oleifera extract concentrations may thereby incorporate more MOCP molecules to augment bacteria neutralization . [00109] As noted previously, the described 'fishing, trapping and killing' technique can be generalized for a large number of deleterious waterborne pathogens by using a combination of effective chemoattractants and contact killing antimicrobials which can be integrated with the substrate material. Moreover, the described techniques can be expanded to remove pathogens from other forms contaminated liquids such as milk and fruit juices. This strategy can also find use towards targeted pathogen removal from water or any liquid media through the use of species specific chemoattractants. The use of non-toxic compounds such as D-glucose and Moringa oleifera in the described example embodiments help ensure that no harmful chemicals are further leached into the sample water. The described water treatment apparatus and methods of treating water can be implemented as a compact point-of-use product for water treatment in limited resource communities across the globe.

Detection of Water For Bacterial Contamination

[00110] According to another aspect of the present disclosure, there is provided a simple, low-cost device for detecting pathogens such as the E. coli in water samples by performing enzymatic reactions on the porous substrate. Specifically, in some illustrative embodiments, the inventors implemented the E. coli detection capabilities of Mobile Water Kit (MWK) ³⁷ on a strip-based detection device. In some particular embodiments, the detection device may use blotting paper strips as the porous substrate. A hydrophobic barrier can be created at one edge of the detection device using a suitable material such as paraffin wax and the like. Creation of the barrier can be followed by depositing the MWK chemical solution to create a reaction zone, adj cent to the hydrophobic barrier. The hydrophobic barrier may inhibit the spread of the chemicals and water. The substrate were completely dried inside the fume hood. The other end of the detection device may be coated with a chemoattractant, such as 0.1 M D-glucose

(dextrose) solution. The detection device with a glucose coated end can be dipped in E. coli contaminated water for detecting E. coli bacteria. E. coli in the water sample is attracted towards the detection device due to a chemo taxis mechanism and the E. coli trapped in the paper strip will percolate towards the other end of the detection device due to capillary action. The E. coli bacteria may concentrate at the top edge of the detection device and react with the MWK chemicals to produce an observable indicator, to indicate their presence. In some embodiments, the appearance of a colour in at least one portion of the detection device may indicate the presence of bacteria of interest in water samples.

[00111] In the broadest embodiment, the detection device comprises a strip of a long, narrow piece of cellulose blotting paper, having a chemoattractant (at one edge), a hydrophobic barrier (at the other edge), and custom formulated chemical reagents at a reaction zone

(immediately below the hydrophobic barrier). In some embodiments, the hydrophobic barrier comprises wax.

[00112] When the detection device is dipped in water, the bacteria of interest in the water sample is attracted toward the detection device due to a chemotactic response mechanism, followed by movement along the detection device toward the reaction zone due to a capillary wicking mechanism of the substrate, and finally the capillary motion is arrested at the top edge of the detection device by the hydrophobic barrier. The bacteria of interest concentrated at the reaction zone of the detection device can react with chemical reagents in the reaction zone to produce an observable change. In some embodiments the observable change may be a change of colour on a portion of the detection device corresponding to the reaction zone, when dipped into water samples indicates, can indicate the presence of contamination in the water.

[00113] As described in greater detail subsequently, the performance of some

embodiments of the detection device is checked with different known concentrations of E. coli contaminated water samples using different dip and wait times. These embodiments of the detection device have also been tested with different interfering bacteria {i.e. strains of bacteria that are not considered to be of interest) and chemical contaminants. The inventors observe that the different interfering contaminants do not have any impact on the ability of the detection device to detect the bacteria of interest.

[00114] A schematic illustration of an embodiment of the E. coli detection device is provided in Figs. 13A and 13B. Referring first to Fig. 13 A, shown therein is an embodiment of detection device 1300A for detecting bacterial contamination in a water sample. In some embodiments, the bacteria of interest is E. coli bacteria. In other embodiments, the detection device may be functionalized with suitable agents to detect other types of bacteria and/or pathogens. [00115] In the present embodiment, the substrate of the detection device 1300A is blotting paper, for example, Whatman gel blotting paper, Grade GB003. In other embodiments, other suitable porous substrate materials, such as those identified previously for the water treatment device may similarly be used. The substrate may be shaped to a desired size. In the example detection device of Fig. 13 A, a piece of paper substrate material can be diced into 75 mm ^χ 5 mm size strips. One edge of the detection device can be coated with a hydrophobic material, such as wax and the like, to form a hydrophobic barrier 1302. The hydrophobic barrier 1302 may be used to prevent the spread of the chemicals (e.g. reactants used for detection of the bacteria) and bacteria trapped in the reaction zone 1304 from further migration along the detection device through capillary action. In some embodiments, the fibers within the hydrophobic barrier 1302 are also coated with a hydrophobic material.

[00116] The reaction zone 1304 may be positioned adjacent to the hydrophobic barrier 1302, and may be said to form "below" the hydrophobic barrier. The region of the detection device designated as the reaction zone 1304 may be formed by depositing a compound suitable for detecting the bacteria of interest. For example, in some embodiments, the reaction zone 1304 can be formed by depositing thereon a MWK chemical mixture for detecting E. coli , which has been described previously by Gunda et al.²⁷' ²⁸ In some embodiments, 100 μ , of MWK chemical mixture may be used. In other embodiments a different amount of MWK chemical mixture may be used, depending on the dimensions of the reaction zone 1304.

[00117] The other (i.e. , second edge) edge of the detection device can be coated with a chemoattractant to form an attraction zone 1306 to attract any bacteria or pathogens present in the water sample to the detection device 1300A. In some embodiments, where the bacteria of interest is E. coli, D-glucose (dextrose) at 0.1 M concentration may be used as a suitable chemoattractant, as described previously. In some embodiments, the attraction zone 1306 may be formed by dispensing 100 μΐ, of 0.1 M D-glucose. Other chemoattractant compounds of suitable concentrations may be used for detection of other pathogens. This second edge can be referred to as the "attraction zone" since, as noted previously, the chemoattractant acts as a chemotaxis agent to attract the bacteria of interest towards the detection device. The substrate can be dried, for example, under a fume hood or other suitable drying device before use, in which the detection device is dipped into water. [00118] It may be further noted that in some embodiments, such as the embodiment of

Fig. 13 A, there may be a non-functionalized region 1308 or an inactive zone of substrate material. Under use, water along with any bacteria may travel along this non-functionalized region 1308 to the detection zone 1304. In other embodiments however, such a non- functionalized region 1308 does not exist such that the reaction zone 1304 and the attraction zone 1306 are immediately adjacent to each other similar to the barrier zone 1302 being immediately adjacent to the reaction zone 1304.

[00119] In yet other embodiments, the detection device may be fabricated using portions of substrates attached together. For example, in some embodiments, as shown in FIG. 13B, the detection device 1300B may be assembled using two smaller portions of substrate material, a first portion 1320 and a second portion, in overlapping contact to form a contact zone 1324, similar to the water treatment device of Fig. IB.

[00120] To test a water sample, the attraction zone 1306 containing the chemoattractant can be introduced the water sample. The chemoattractant in the attraction zone 1306 may then disperse into the water and to establish a chemoattractant concentration gradient in the water sample. This chemoattractant gradient may induce a chemotactic response of one or more strains of bacteria present in the water, including the bacteria of interest. The response of the bacteria to the presence of the chemoattractant gradient includes movement towards the attraction zone 1306. This chemotactic movement can result in an increase in the movement or migration of bacteria towards the detection device.

[00121] The use of the attraction is thus similar to the use of the attraction zone 102 of water treatment device 100B of FIG. 2, to 'fish' the bacteria. The sample water along with any bacteria may percolate into the porous matrix of the detection device by capillary action to direct water and bacteria from the attraction zone 1306 to the reaction zone 1304 provided on the device, the latter zone being operable to for the presence of bacteria in the sample of water. Once the water front reaches the hydrophobic barrier of the detection device, the barrier may inhibit further progression of water and migration of bacteria beyond the reaction zone. The detection device can then be removed from the water and kept aside on a flat surface. Any bacteria trapped in the reaction zone can thus react with the detection chemicals in the reaction zone and to generate a visually observable change of a region of the detection device corresponding to the reaction zone. In some embodiments, MWK chemicals may be used for detection of E. coli. A positive test result for the presence of E. coli may take the form of a visual change in colour from white to a pinkish red colour. In other words, the appearance of pinkish red colour in the reaction zone 1304 indicates the presence of E. coli bacteria in the water sample being tested.

[00122] Figures 14A and 14B illustrate examples embodiments of detection devices that may be used to detect E. coli. The presence of visually observable colour change in an area 1320 of the substrate corresponding to the reaction zone 1304 of the exemplary embodiments of the detection device is a result of the presence of a known concentration of E. coli in the

contaminated water samples. Figure 14B illustrates that it may be observed that there is an appearance of pinkish red colour (by way of example) at the area 1320 corresponding to the reaction zone 1304 of the E. coli detection device to represent the presence of E. coli. However, it may be noted that the colours described in the present disclosure are by way of example only. Depending on the type of reactants used, the observable colour change may be colours other than pinkish red. Figure 14A illustrates that there is no colour change in the area 1320 of the E. coli detection device when the water sample is only deionized water in the absence of bacteria.

[00123] Figure 15 shows the appearance of pinkish red colour (by way of example) within an area 1320 of the substrate designated as the reaction zone 1304 of some embodiments of the E. coli detection device for various water samples contaminated with known concentrations (CFU/100 mL) of E. coli (ATCC 11229) after 2 hours of incubation at room temperature. It may be noted that, in the examples shown, the colour within the area 1320a-1320h can vary in intensity based on the concentration of bacteria in the water samples and how much time the detection device is dipped into the water. In the present example, the intensity of the colour at area 1320a corresponding to the highest bacteria concentration is greatest relative to the remaining detection devices, and other areas 1320b-g have decreasing intensity. In area 1320h, corresponding to no bacteria, it may be said that the there is no colour change. In the present embodiment, it may be observed that, generally, the colour intensity decreases with a decrease in the concentration of E. coli. However, in other embodiments, the reactant used in the reaction zone may respond to the concentration of bacteria by changing from one colour to another instead of varying in intensity, in a manner similar to how litmus paper may change colour depending on the pH which is reflective of the concentration of hydrogen ions present in a sample solution. [00124] Figure 16 shows a graph of response times (the amount of time one has to wait for the results such as a colour change) of some embodiments of the E. coli detection device as a function of E. coli concentration. Specifically the graph portrays the comparison of E. coli testing response times, the time to the appearance of an observable pinkish red colour at the reaction zone 1304, with various samples of water spiked with known concentrations of E. coli. It can be observed that the appearance of pinkish red colour at the reaction zone of E. coli detection device for samples with 4* 10⁶ CFU/lOOmL to 4xl0⁵ CFU/lOOmL may happen in 10 to 15 min. In one embodiment, the E. coli detection device is expected to detect E. coli

concentration at 4 CFU/l OOmL in 2 hours.

[00125] The methods and systems of the various embodiments of present invention are described in the following Examples, which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.

EXAMPLE 1 - CAPABILITIES OF MWK ON THE MODIFIED DIPTREAT DEVICE

[00126] An example embodiment of the detection device is described herein which implements the E. coli detection capabilities of MWK³⁷ on a modified DipTreat³⁸ device, described above, to create a simple platform for E. co/z^'/coliform detection in water.

[00127] The materials and methods for preparing the example strip-based detection device were as follows:

1. Preparation of glucose strips: 200 mL of 0.1 M D-glucose (dextrose) solution was prepared under normal laboratory conditions (22°C). Blotting paper strips (Size: 55 mm x 14 mm, Grade GB003, Whatman®) were completely immersed in the glucose solution and kept on a shaker at 100 rpm for 2 hours. Thereafter, the strips were removed and completely dried (4-5 hours) inside a fume hood. These strips were used in the experiment.

• * 37

2. Preparation of the MWK chemicals: Refer to the journal article of Gunda et al. .

3. Preparation of MWK detection strip: Blotting paper strips (Size: 55 mm x 14 mm, Grade GB003, Whatman®) were used for this purpose. A hydrophobic barrier was created approximately 15 mm from the bottom edge of the strip using paraffin wax coating. This was followed by depositing 200 μΐ, of the MWK chemical solution in the 15 mm by 14 mm space at the bottom of the strip. The wax barrier prevented the spread of the chemicals through capillary action. The strips were completely dried inside the fume hood (3 hours) or other suitable drying device.

4. Device configuration: A glucose strip and a MWK strip were attached end to end on a cardboard scaffold with double-sided tape as shown in Figs. 17A and 17B. This configuration ensured that the portion with the MWK chemicals were in direct contact with the glucose strip.

5. Experimental Procedure: E. coli cells were grown in Lauryl Tryptose Broth (LTB) using previously described techniques . Different volumes of deionized water were seeded with E. coli to prepare model contaminated water. The bottom portion of the glucose strip of E. coli testing was immersed into sample water and the colour change detected over the period of experiment.

[00128] Using 40 mL volumes of sample water with a high bacterial concentration of 10⁶ and 10⁵ CFU/mL, distinct colouration of the MWK strip was observed after 90 minutes of experimental time, as shown in FIG. 17A and 17B, respectively. For higher bacterial

concentration, the colour of area 1330 located in the bottom glucose strip outside of the area 1320a corresponding to the reaction zone may change so as to be be observable as well, which may be indicative of the leaching of MWK chemicals to the top portion of the glucose strip that is in contact with the MWK strip. The first visuallay observable appearance of colour in area 1320a and 1320b corresponding to the reaction zone for both of these cases (i.e. 10⁶ and 10⁵ CFU/mL) was recorded approximately 50 minutes after the start of the experiment. In the present example, it may be observed that the colour intensity of area 1320a is greater than area 1320b because of the higher concentration of bacteria in the former. Hence, E. coli testing the device of the present embodiment can act as a 'litmus test' to assess the presence of bacterial

contamination, capable of providing a new detection method for E. coli on paper strips. Also, it is not restricted to any specific volume of water - as long as there is E. coli in any volume of water, the bottom strip (laced with D-glucose or another suitable chemoattractant) will always attract the bacteria. The attracted baceria may then travel along the length of the paper strip through capillary action and then react with the MWK chemicals to produce colour change.

[00129] As shown in Fig. 18, MWK detection strips were prepared and configured in the manner previously described. Specifically, in the example, hydrophobic Paraffin wax melted on paper strips in area 1340 was used to create a hydrophobic barrier 1302 at one end (i.e. the top part). In the present case, 200μί of MWK chemicals were added to area 1320 the bottom part of the strip. On drying, a yellowish colour (by way of example) may be observed. In other embodiments, other colours inherent to the detection compound may be observed. In yet other embodiments, the detection compound may be colourless until it reacts with a target bacteria. Figure 19 shows the uneven nature of the wax coating on a batch of prepared paper strips.

[00130] Figures 20 and 21 show the changes in colours observable in area 1320 corresponding to the reaction zone immediately at the end of an experiment where the strips were dipped in water samples with known concentrations of E. coli. For example, Fig. 20 shows the pinkish red colour (by way of example) observable in area 1320 for a concentration of approximately 10⁵ CFU/mL, and Fig. 21 shows the resultant changes in colour resulting a relatively more intense pinkish red colour (by way of example) observable in area 1320 for a higher concentration of approximately 10⁶ CFU/mL.

[00131] Figure 22 shows the changes in colours and differences in colour intensity observable in areas 1320a and 1320b of the substrate corresponding to the reaction zone after 1 hour. In the present example, The strip on the left shows the intensity of colour observed for the lower concentration of E. coli, (pinkish red, by way of example) and the strip on the right shows the intensity of colour observed for the higher concentration of E. coli (a pinkish red of relatively greater intensity, by way of example)

EXAMPLE 2 - E. COLI DETECTION DEVICE

[00132] In another embodiment of the E. coli detection device, the following alternative materials can be used: Whatman gel blotting paper (0.8 mm thickness, Grade GB003), enzymatic substrate Red-Gal (6-Chloro-3-indolyl- ?-D-galactoside) and N,N-Dimethylformamide (DMF) were procured from Sigma Aldrich, Canada. Lauryl Tryptose Broth (LTB) (BD 224150), Bacteria protein extraction reagent (B-PER), Veal Infusion Broth (BD 234420), Bacto 70 Yeast Extract (BD 212750), Brain Heart Infusion Broth (BD 237500), and Nutrient Broth (BD 234000) were purchased from Fisher Scientific, Canada.

[00133] Bacteria strains such as E.coli Castellani and Chalmers (American Type Culture Collection (ATCC) 11229), Enterococcus faecalis (E. faecalis) (ATCC 19433), Salmonella enterica subsp. enter tea (S. enterica) (ATCC 14028) and Bacillus subtilis (B. substilis) (ATCC 33712, Mil 12 strain) were obtained from Cedarlane, Burlington, ON, Canada. E. coli K-12 strains were purchased from New England Biolabs, Ipswich, Massachusetts, USA. E.coli ATCC 1 1229 and E. coli K-12 were grown in LTB medium as well as in nutrient broth medium at 37°C in incubator (Lab Companion SI-300 Benchtop Incubator and Shaker, GMI, Ramsey, Minnesota, USA) for 24 hours. B. subtilis bacteria strains were cultured in a growth medium consisting of Veal Infusion Broth and Yeast Extract (5: 1 ratio) at 30°C in an incubator for 24 hours whereas E. faecalis and S. enterica were grown in brain heart infusion broth medium and nutrient broth medium, respectively. Deionized water was used to prepare the respective broth medium.

[00134] Broths were sterilized in an autoclave at 121°C prior to using them for culturing the respective bacteria. Serial dilutions were prepared in deionized water to make bacteria concentrations in the range of 2 - 2 x 10⁶ CFU/mL. Water samples with known concentrations of bacteria were utilized to check the performance of E. coli detection device of the present embodiment.

[00135] Sodium fluoride, EMD ferric chloride (hexahydrate), and EMD sodium chloride were procured from Fisher Scientific, Canada. Sodium nitrate, iron Chloride hexahydrate, ammonia persulfate, sodium iodide, sodium sulfate, potassium hydroxide, sodium bromide, sodium phosphate, and calcium propionate were purchased from Sigma Aldrich, Canada.

Standard fluoride solution (lppm), fluoride solution (lOppm), cadmium and lead were obtained from Hanna instruments, Woonsocket, RI, USA.

EXAMPLE 3 - PREPARATION OF CUSTOM FORMULATED CHEMICAL COMPOSITION

[00136] In another embodiment of the detection device, a new chemical composition for the reaction zone capable of detecting E. coli is presented. Specifically, the formulation may be prepared by dissolving 100 mg of solid media (1 :1 mixture of LTB and Red-Gal) in 4 mL of liquid media (1 :2:5 mixture of DMF, B-PER, and deionized water). The enzymatic substrate Red-Gal can be used to detect E. coli that secretes /?-galactosidase enzymes. A chromogenic compound Red-Gal (6-chloro-3-indolyl- ?-D-galactoside) contains two components: 6-chloro-3- indolyl and β-D-galactoside. The ?-galactosidase enzyme produced by E, coli hydrolyses this complex Red-Gal molecule resulting in the release of pinkish red colour producing dimerized 6- Chloro-3-indolyl compound. The release of pinkish red colour may thus be visually observable to indicate the presence of E. coli in a sample of water. The inclusion of B-PER in custom formulated chemical reagents may be made to accelerate the extraction of ?-galactosidase enzymes by lysing the E. coli bacteria cells without denaturing the bacterial enzymes.

EXAMPLE 4 - PREPARATION OF E. COLI DETECTION DEVICE

[00137] In another embodiment of the detection device, the substrate is blotting paper that is cut into 70 mm x 5 mm size strips. While the length of detection device chosen, i.e., 70 mm, is sufficient for the capillary inhibition to occur, a person skilled in the art will appreciate that other lengths that allow for capillary inhibition to occur can be used in other embodiments. Blotting paper is made of pure cellulose produced entirely from the high quality cotton linters with no additives. In other embodiments, other types of materials known to a skilled person in the art may be used. In this embodiment, the blotting paper has a weight of 320 g/m², wet strength of 300 mm water column, and water absorbency of 740 g/m². The blotting paper ensures the proper wicking and uniform capillary action.

[00138] One edge of the detection device can be coated with a suitable hydrophobic material such as wax to form a hydrophobic barrier. As noted previously, the wax barrier prevents the further spreading of the chemicals and bacteria in the reaction zone through capillary action. The reaction zone is formed below the hydrophobic barrier by depositing the 100 L of above mentioned custom formulated chemical composition (Red-Gal, B-PER and LTB) using pipette and followed by drying under normal laboratory conditions (temperature around 23°C) for one hour. After coating the detection device with the custom formulated chemical composition in the reaction zone, the opposite edge of the detection device is coated with D-glucose (dextrose) by dispensing 100 μΐ, of 0.1 M D-glucose and then allowed to be dried at room temperature (23°C) for one hour. This edge may also be known as the attraction zone since D-glucose acts as a chemotaxis agent to attract the bacteria towards the detection device. The resulting detection devices were completely dried for one hour under a fume hood before dipping them into a water sample. EXAMPLE 5 - TESTING WATER SAMPLES WITH E. COLI DETECTION DEVICE

[00139] In another embodiment of the detection device, the device was used to test for E. coli in a water sample. To perform the test, the edge with the attraction zone of the E. coli detection device can be dipped into the E. coli contaminated water. The D-glucose in the attraction zone disperses into the sample contaminated water and forms a concentration gradient in the water. This gradient creates the chemotactic movement of E. coli bacteria from the surrounding water and it eventually increases the migration of bacteria towards the E. coli detection device . The water along with bacteria (attracted to the edge of the attraction zone of the detection device) percolates into the porous matrix of the substrate due to capillary action. Once the water front reaches the hydrophobic barrier (e.g. barrier 2302 of Fig. 23B) of the E. coli detection device, the detection device can be removed from the water and kept aside on a flat surface. The bacteria trapped in the reaction zone can react at room temperature with chemicals in reaction zone and produce the visually observable changes.

[00140] Figures 23B and 23C illustrate the colour change that may be observed visually at areas 2320b and 2320c of the substrate corresponding to the reaction zone 2304 of E. coli detection device because of the presence of E. coli (ATCC11229) in contaminated water.

Specifically it may be observed that there is a pinkish red colour (by way of example) in area 2320c of the reaction zone 2304 of the device of Fig. 23C, which represents the presence of E. coli. In the present case, a known concentration of E. coli was used in the testing, such that, if desired, it may be possible to correlate colour intensity to bacteria concentration. A controlled study was further conducted in which the E. coli detection device was tested in deionized water at room temperature with no E. coli which resulted in no visually observable colour change in the reaction zone as shown in area 2320b of Fig. 23B.

[00141] Figure 23A shows the scanning electron microscope image of the porous paper matrix substrate of used in some embodiments of the E. coli detection device. In the example •shown, the paper is a randomly distributed network of paper fibres with an estimated porosity of 65% to 73%.

[00142] Figure 24 shows the appearance of visually observable pinkish red colour at area 2300 of the substrate corresponding to the reaction zone 2304 of the E. coli detection device for various indicated concentrations of E. coli (ATCC 11229) contaminated water samples after 2 hours at room temperature. It is to be noted that the colour intensity can vary based on the concentration of bacteria in water samples and how much time the E. coli detection device is dipped into the water. It may be observed that the colour intensity decreases with the decrease in the concentration of E. coli.

[00143] The performance of the E. coli detection device can be evaluated based on the dip time and wait time. Dip time may refer to the amount of time the E. coli detection device is in contact with the water samples (e.g. the attraction zone being dipped into the water sample) whereas wait time (response time) is the amount of time one has to wait for the results (i.e.

appearance of pinkish red colour) after removing the E. coli detection device from the water samples.

[00144] Figure 25 is a graph that can be used to portray the comparison of wait (response) times for the visually observable appearance of pinkish red colour at the reaction zone of some embodiments of the E. coli detection device as a function of various dip times and for different water samples spiked with known concentrations of E. coli. The average wait times with error bars are provided in Fig. 25. It can be observed that the visually observable appearance of pinkish red colour at reaction zone of E. coli detection device for samples with 2xl0⁵ CFU/mL to 4xl0⁴ CFU/mL happens in 60 to 65 min (wait time) corresponding to a dip time of 2 min.

[00145] It may be observed from the graph that the wait time may decreases with the increase in dip times. The increase in dip time allows a higher number of E. coli bacteria to accumulate at the reaction zone, which in turn decreases the wait time needed to produce the colour due to presence of E. coli bacteria. It can also be observed that the lower concentrations of E. coli in contaminated water samples take longer wait (response) times compared to higher concentrations of E. coli.

[00146] In this embodiment, the space between the attraction zone and the reaction zone (e.g. the non-functionalized zone 1308 of Fig. 13) will generally not influence the performance of the E. coli detection device if the detection device is kept within the contaminated water samples for a sufficient period of time. In some embodiments, the detection device can be dimensioned at an optimal length that is required to maintain the stability of the detection device to sustain the water absorbency for a longer time. In some embodiments, the length of the detection device is 70mm. [00147] The disclosure below describes the mathematical modelling of the operational dynamics of embodiments of the detection device operable to detect E. coli in water. However, the principles described may be applicable to detection devices operable to detect other types of bacteria in water. The wicking of E. coli contaminated water into porous paper matrix follows the Washburn-Lucas equation and may be given as^43"48,

L² = ϋίΙ η^*

(1) where, L is the distance moved by the fluid front, γ is the effective surface tension (which includes the effect of any contact angle dependency), D is the average pore diameter of paper, t is the time and η* is the effective viscosity of the E. coli contaminated water. Effective viscosity depends on the concentration of E. coli bacteria. The effective viscosity of E. coli contaminated water can be provided as⁴⁹: c 1 + 2λ(2 + λ)

< = n 2~ψ„ (1 + λ)³

(2) where, η is the viscosity of water without E. coli bacteria, φ is volume fraction occupied by E. coli bacteria in water, ε₀ is the amplitude of the strain rate, c is the point force representing the flagellum, λ is the length of the run between tumbles, representing bacteria motility. By neglecting the motility effects, one can obtain the effective viscosity of the E. coli contaminated water as:

[00148] E. coli bacteria are usually rod-shaped and range between 0.25 - 1.0 μιη in diameter and approximately 2.0 μνα long, with a bacterial volume of 0.6 - 0.7 ιη^{3 50}. Based on the concentrations of bacteria (2 xlO⁵ CFU/mL to 200 CFU/mL) used in the examples described herein, the volume fraction occupied by E. coli bacteria in water can varies from 1.4x10^" to 1.4xl0^"10, which in turn dictates that there is a negligible effect of bacterial suspensions on the viscosity of the contaminated water. Therefore, for further analysis, one may need to decouple the hydrodynamic effects from the reaction kinetics responsible for the appearance of the pinkish red colour on the detection device. The initial rate of interaction of Red-Gal substrate with β- galactosidase enzyme can be described by the Michaelis-Menten equation⁵¹,

where, _cat is turnover number and E₀ is concentration of /?-galactosidase enzyme (released from E. coli bacteria), K_m is Michaelis constant and S is the concentration of Red-Gal substrate. It is clear that the wait time for colour appearance is dependent on the interaction of the Red-Gal substrate with ?-galactosidase enzyme. The presence of B-PER at the reaction zone helped to accelerate the production of ?-galactosidase enzyme from E. coli. While the Red-Gal substrate is used in this embodiment, other substrates known to a person skilled in the art may also be used.

EXAMPLE 6 - EFFECT OF E. COLI GROWTH MEDIUM ON PERFORMANCE OF THE E.

COLI TESTING DEVICE

[00149] In order to study the effect of E. coli growth medium on performance of the E. coli detection device, the two E. coli bacteria strains ATCC1 1229 and K-12 were grown in an LTB medium as well as in a nutrient broth medium. Water samples contaminated with these strains of E. coli bacteria were tested with an embodiment of the E. coli detection device. It was observed that the reaction zone E. coli detection device produced pinkish red colour with both kinds of samples. However, the E. coli bacteria cultured in the LTB medium generated a higher intensity of colour compared to the bacteria grown in the nutrient broth medium.

EXAMPLE 7 - EFFECT OF INTERFERING BACTERIA AND CHEMICAL

CONTAMINANTS

[00150] The effects of interfering bacteria and chemical contaminants on an embodiment of the E. coli detection device were also tested. Figure 27 is a chart that illustrates the results obtained by using an embodiment of the E. coli detection device to test 40 different water samples. The E. coli detection device was tested with water samples containing several interfering bacteria, including B.subtilis, E. faecalis and S. enterica.

[00151] For Category A (Samples # 1-3) water samples, i.e., water samples containing only interfering bacteria (B.subtilis, E.faecalis or S.enterica) and without E. coli bacteria do not produce any colour. On the other hand, the E. coli detection device produced colour for water samples that contained both interfering bacteria and E. coli (i.e., Category B Samples # 4-7). It may be observed that the interference bacteria had no effect on the detection of E.coli with the E. coli detection device.

[00152] Similarly, testing the E. coli detection device with water samples containing several kinds of chemical contaminants with and without E. coli bacteria were also carried out. The E. coli detection device did not produce any colour changes when it was tested with water samples (Category C, water samples # 8-23 ) containing different chemical contaminants.

However, the E. coli detection device was able to produce the colour (pinkish red colour) to indicate the presence of E. coli when the device was tested with water samples containing E. coli along with different chemical contaminants (Category D, water samples # 24-39). On the basis of these results, the embodiment of the E. coli detection device tested provides a degree of specificity as it did not react with the chemicals (Red-Gal, B-PER and LTB) coated on the E. coli detection device and the contaminants do not interfere with the E. coli bacteria when they were interacting with chemicals (Red-Gal, B-PER and LTB) on the E. coli detection device.

[00153] Similarly, testing of the E. coli detection device with negative control, i.e., deionized water, without having any bacteria and chemical contaminants (Category E Sample # 40) were carried out. It was found that the pinkish red colour was not produced by the E. coli detection device for this negative control. Accordingly, the E. coli detection device may function properly under different kinds of water samples for both positive and negative controls as well as with interfering bacteria and chemical contaminants.

[00154] Figure 26 illustrates the use of another embodiment of the E. coli detection device for the detection of E. coli bacteria in water samples. In the illustrated embodiment, the attraction zone, reaction zone and barrier zone may be enclosed in a case to provide structural support in a "pen-like" DipTest tool. Specifically an enclosure in the shape of a pen may be provided to enclose the detection device, with one end closed and another end that defines an opening to allow exposure of the attraction zone to be dipped into water. When in use, a user can dip the E. coli detection device in water for testing purposes. The device can be immersed in water for a certain time and then be removed from the water and placed on a flat surface for the result. [00155] The example embodiments of the E. coli detection device making use of paper strips as the substrate as described herein may operate as a litmus paper for determining whether or not a water sample is free from bacterial contamination.

[00156] The E. coli detection device may be useful in remote locations where one can dip this device and find whether the water is safe to use or not. In particular, the E. coli detection device can be used for checking the quality of water a variety of locations including, but not limited to, swimming pools, lakes, rivers, and beaches.

[00157] In summary, described herein is a novel E. coli detection device, similar to a litmus test, for detection of E. coli bacteria in water samples. The E. coli detection device can be fabricated relatively easily and may be relatively simple to use for testing the water samples.

[00158] As described herein, in some embodiments, for a dip time of 2 min, the E. coli detection device may be able to detect the concentrations of E. coli as low as 200 CFU/mL in 180±20 min of wait time, and higher concentrations such as 2xl0⁵ CFU/mL within 75±12 min of wait time. However, for a dip time of 90 min, the E. coli detection device may be able to detect concentrations of E. coli as low as 200 CFU/mL in 54±8 min of wait time and higher

concentrations such as 2xl0⁵ CFU/mL within 28±5 min of wait time.

[00159] Further optimizations in terms of the concentration of individual chemical ingredients used here may be implemented so that one can have a field deployable device to provide a "yes/no" litmus test for E. coli concentration as low as 1-10 CFU/100 mL, thereby meeting the U.S. EPA standards .

[00160] In some embodiments, the E. coli detection device can be carried in a pocket and used to test the water samples whenever required. The E. coli detection device can also easily be disposed of, after completion of a test, with minimal effort. In other embodiments, the pocket- device may be in the form of a kit, in which strips may be inserted into a main body, such as the main body depicted in Fig. 26, of the device for testing of each sample. The testing strips may be replaced to allow testing of further samples so that the main body is reusable.

[00161] In some embodiments, the E. coli detection device can be adapted and integrated with further developments in the detection of other bacteria and pathogens and used not just for water samples but for many other liquids such as milk, wine, juices, and the like, and food items such as frozen meat and cheese.

[00162] The examples and corresponding diagrams used herein are for illustrative purposes only. The principles discussed herein with reference to determination of equilibrium dissociation constants can be implemented in other systems and apparatuses. Different configurations and terminology can be used without departing from the principles expressed herein. For instance, steps, equipment, components, and modules can be added, deleted, modified, or re-arranged without departing from these principles.

[00163] Unless the context clearly requires otherwise, throughout the description and the claims: "comprise," "comprising," and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to" . "Connected," "coupled," or any variant thereof means any connection or coupling, either direct or indirect, between two or more elements; the coupling or connection between the elements can be physical, logical, or a combination thereof. "Herein," "above," "below," and words of similar import, when used to describe this specification shall refer to this specification as a whole and not to any particular portions of this specification. "Or" in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list. The singular forms "a," "an," and "the" also include the meaning of any appropriate plural forms.

[00164] Where a component is referred to above, unless otherwise indicated, reference to that component should be interpreted as including as equivalents of that component, any component which performs the function of the described component (i.e., that is functionally equivalent), including components which are not structurally equivalent to the disclosed structure which performs the function in the illustrated exemplary implementations of the invention.

[00165] Specific examples of systems, methods and apparatuses have been described herein for purposes of illustration. These are only examples. The methods and apparatuses provided herein can be applied to systems and apparatuses other than the examples described above. Many alterations, modifications, additions, omissions and permutations are possible within the practice of this invention. This invention includes variations on described

implementations that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and/or acts with equivalent features, elements and/or acts;

mixing and matching of features, elements and/or acts from different implementations;

combining features, elements and/or acts from implementations as described herein with features, elements and/or acts of other technology; omitting and/or combining features, elements and/or acts from described implementations.

[00166] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention. Furthermore, it should be understood that the empirical results described previously were provided for the purposes of explanation, and not limitation, of the present invention.

[00167] It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions, omissions and sub-combinations as may reasonably be inferred. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

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[00168] All publications mentioned herein are hereby incorporated by reference in their entireties. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

Claims

WHAT IS CLAIMED IS

1. A water treatment apparatus for treating water contaminated with bacteria, the apparatus comprising: a first attraction zone containing a chemoattractant for receiving bacteria from the contaminated water;

a treatment zone containing a bactericide for neutralization of the bacteria, the treatment zone being connected to the first chemoattractant zone to receive bacteria migrating from the first attraction zone to the treatment zone; and

a second attraction zone containing the chemoattractant connected to the treatment zone to receive viable bacteria migrating from the treatment zone to the second attraction zone, the first and second attraction zones being separated by the treatment zone.

2. The water treatment apparatus of claim 1, wherein each of the first attraction zone, second attraction zone and treatment zone is disposed on a porous substrate material.

3. The water treatment apparatus of claim 1, wherein the first attraction zone is insertable into contaminated water to induce a chemotactic response of the bacteria in the contaminated water to migrate to the apparatus.

4. The water treatment apparatus of claim 1, wherein the second attraction zone induces a chemotactic response of the viable bacteria in the treatment zone to migrate the second attraction zone.

5. The water treatment apparatus of claim 2, wherein: the first attraction zone is provided on a first substrate portion, the treatment zone is provided on a second substrate portion, and the second attraction zone is provided on a third substrate portion, each substrate portion being a separate piece of substrate material; and the first substrate portion is connected to the second substrate portions by overlapping contact and the second substrate portion and the third substrate portion are connected by overlapping contact.

6. The water treatment apparatus of claim 5, wherein for each overlapping contact, a contact zone is defined by corresponding edges of each of the two overlapping substrate portions such that a length of the contact zone defined by a separation the corresponding edges is at least 5mm.

7. The water treatment apparatus of claim 2, wherein the first attraction zone, second attraction zone and the treatment zone are established on a common substrate portion.

8. The water treatment apparatus of claim 2, wherein the porous substrate material comprises at least one pore having a volume greater than the volume of a bacterium to entrap at least one bacterium therein.

9. The water treatment apparatus of claim 2, wherein each of the first and second attraction zones and the treatment zone are rectangular in shape so that the longitudinal arrangement of the first and second attraction zones and the treatment zone resembles an elongated water treatment strip.

10. The water treatment apparatus of claim 9, wherein the porous substrate material is blotting paper.

1 1. The water treatment apparatus of claim 9, wherein the porous substrate material is a polymer.

12. The water treatment apparatus of claim 10, wherein the treatment zone of the elongated water treatment strip is at least 55 mm in length.

13. The water treatment apparatus of claim 10, wherein the contaminated water moves along the blotting paper from the first attraction zone to the treatment zone and to the second attraction zone by capillary action to facilitate migration of bacteria from the attraction zone to the treatment zone and migration of viable bacteria from the treatment zone to the second attraction zone.

14. The water treatment apparatus of claim 1 , wherein the chemoattractant is D-glucose.

15. The water treatment apparatus of claim 14, wherein a concentration of D-glucose in the first attraction zone is 0.1 M.

16. The water treatment apparatus of claim 14, wherein concentrations of D-glucose in the first and second attraction zones are 0.1 M.

17. The water treatment apparatus of claim 1 , wherein the bactericide is moringa oleifera cationic protein.

18. The water treatment apparatus of claim 17, wherein the moringa oleifera cationic protein is adsorbed onto the surface of a porous substrate material.

19. The water treatment apparatus of claim 1 further comprising a scaffolding structure to support the first and second attraction zones and the treatment zone.

20. The water treatment apparatus of claim 1 further comprising a case that encloses the entire treatment zone, the entire second attraction zone and a substantial portion of the first attraction zone.

21. The water treatment apparatus of claim 20, wherein the case has an enclosed first end and an open second end, and the first attraction zone extends outside of the case through the open second end.

22. A method of treating water contaminated with bacteria with a water treatment apparatus, the method comprising: establishing a chemoattractant concentration gradient in the water to induce a chemotactic response of the bacteria in the water to migrate towards a first attraction zone of the water treatment apparatus; directing migration of the bacteria to a treatment zone of the treatment apparatus, the treatment zone being connected to the first attraction zone; entrapping the bacteria within the treatment zone; and exposing the bacteria to a bactericide in the treatment zone to neutralize the bacteria.

23. The method of claim 22, wherein the establishing the chemoattractant concentration gradient in the contaminated water comprises applying a chemoattractant to the attraction zone of the water treatment apparatus and dipping the attraction zone into the contaminated water and maintaining the treatment zone above the contaminated water.

24. The method of claim 23 further comprises providing a second attraction zone in the water treatment apparatus containing the chemoattractant, wherein the second attraction zone is connected to the treatment zone to draw viable bacteria away from the treatment zone and the first attraction zone, and wherein the first and second attraction zones are separated by the treatment zone.

25. The method of claim 23, wherein the chemoattractant is D-glucose in the first attraction zone is D-glucose with a concentration of 0.1 M.

26. The method of claim 24, wherein the chemoattractant in each of the first and second attraction zones is D-glucose with a concentration of 0.1 M.

27. The method of claim 24, wherein the chemoattractant in each of the first and second attraction zones is D-glucose and a concentration of D-glucose in the first attraction zones is different from a corresponding concentration of D-glucose in the second attraction zone.

28. The method of claim 22, wherein the bactericide is moringa oleifera cationic protein.

29. The method of claim 28, wherein method further comprises exposing the bacteria to the moringa oleifera cationic protein for at least 3 minutes by providing the treatment zone in a porous substrate having pores greater than the size of at least one bacterium to entrap the at least one bacterium therein.

30. The method of claim 24, the method further comprises providing a porous substrate for establishing the first and second attraction zones and the treatment zone.

31. A test strip for detecting bacteria in a water sample, the test strip comprising: an attraction zone at a first end of the test strip containing a chemoattractant for receiving water and bacteria contained in the water; a reaction zone connected to the attraction zone and located between the first end and a second of the test strip to receive bacteria migrating from the attraction zone to the reaction zone for detection; and a barrier zone at the second end of the test strip to inhibit progression of water and migration of bacteria along the test strip, the barrier zone being separated from the attraction zone by the reaction zone.

32. The test strip of claim 31 , wherein the attraction zone is insertable into the water to induce a chemotactic response of the bacteria in the water to the test strip.

33. The test strip of claim 31 , wherein the reaction zone contains a reagent that produces a visually observable change in the presence bacteria.

34. The test strip of claim 33, wherein the reagent of the reaction zone is MWK chemicals.

35. The test strip of claim 33, wherein the visually observable change is a change in a colour of the reagent.

36. The test strip of claim 31 , wherein the substrate material is porous.

37. The test strip of claim 36, wherein the porous substrate material is blotting paper.

38. The test strip of claim 36, wherein the porous substrate material is a polymer.

39. The test strip of claim 31, wherein the chemoattractant is D-glucose.

40. The test strip of claim 39, wherein the D-glucose concentration in the attraction zone is 0.1 M.

41. The test strip of claim 31 , wherein the barrier zone comprises a hydrophobic material applied to the second end of the test strip.

42. The test strip of claim 41 , wherein the hydrophobic material is paraffin wax.

43. The test strip of claim 31, wherein the test strip comprises two substrate portions such that

the attraction zone is provided on a first substrate portion; the reaction zone and barrier zone are provided on a second substrate portion; and the attraction zone and reaction zone are in overlapping contact.

44. The test strip of claim 43, wherein each substrate portion is rectangular in shape having a common width.

45. The test strip of claim 31 , wherein the attraction zone, reaction zone and barrier zone is disposed on a common strip of substrate material.

46. The test strip of claim 45, further comprises a default zone between the reaction zone and attraction zone.

47. The test strip of claim 31 further comprising a scaffolding structure to supporting the attraction zone, reaction zone and barrier zone.

48. The test strip of claim 31 further comprising a case that encloses the entire barrier zone, reaction zone and a substantial portion of the first attraction zone.

49. The test strip of claim 48, wherein the case has an enclosed first end and an open second end, and the attraction zone extends outside of the case through the open second end.

50. A method of testing a water sample for bacterial contamination using a test strip, the method comprising: inducing a chemotactic response of the bacteria present in the water towards an attraction zone at a first end of the test strip containing a chemoattractant by establishing a chemoattractant concentration gradient in the water; providing a reaction zone for testing the water for the presence of bacteria;

directing progression of the water and migration of the bacteria from the attraction zone at the first end of the test strip to a reaction zone; and inhibiting progression of the water and migration of the bacteria to a second end of the test strip and away from the reaction zone by establishing a barrier zone at the second end of the test strip.

51. The method of claim 50, wherein the attraction zone is insertable into the water being tested to induce the chemotactic response of the bacteria in the water to the test strip.

52. The method of claim 50 further comprising generating a visually observable change in the reaction zone in the presence of bacteria in the reaction zone.

53. Systems comprising any new, inventive feature, combination of features, or

subcombination of features disclosed herein.

54. Methods comprising any new, inventive step, act, combination of steps and/or acts, or sub-combination of steps and/or acts described herein.

55. Kits comprising any new, inventive feature, combination of features, or subcombination of features disclosed herein.