CA2853985A1 - Microfluidic detection of coliform bacteria - Google Patents

Abstract

A microfluidic system for detecting coliform bacteria in a water sample, the system including a silicon or PDMS substrate defining a microspot having a plurality of microwells, the microspot and microwells coated with at least one enzymatic substrate which results in a detectable change caused by the action of an enzyme produced by a coliform bacteria in the water sample.

Description

MICROFLUIDIC DETECTION OF COLIFORM BACTERIA
Field of the Invention [0001] The present invention relates to a microfluidic method and apparatus for detecting coliform bacteria, such as E. coli, in a sample.
Background of the Invention

[0002] Es.cherichia (E.coh) is a bacterium that is commonly found in the lower intestine of warm-blooded organisms [1,2]. E. coil can be found in, food products and contaminated drinkable water. Most E. coil are harmless, but some can cause serious food poisoning in humans, and are occasionally responsible for product recalls due to food contamination [3]. The CDC (Center for Disease Control and Prevention) estimates about 73,000 cases of E,coli infection occur each year in United States [3]. About 61 people die from the illness [2]. Greatest threat to drinkable water resources is contamination by microbial pathogens, so water quality is routinely assessed by testing for the presence of E. coli using pathogenic activity.

[0003] Three main groups of detections methods for E.coll: 1) Molecular whole-cell and surface recognition methods [4,5]; 2) Enzyme/substrate methods [6-8]; and 3) Nucleic acid detection methods [9]. Antibody-antigen binding [4,5] and receptor-ligand binding based techniques comes under molecular whole-cell and surface recognition methods.
Capture antibodies needs to be coated on the surface of the chips for capturing the E.
coli present in water samples. The production of specific antibodies is time consuming and expensive process.
Enzyme/substrate based methods are proven to be one of the best methods for improved specificity of bacteria detection. Fluorophore or color dye-tagged growth substrate in. specific broth is used in such techniques [6-8]. Upon growth specific enzymes of bacteria cleave the 'fluoroph.ore or color compound from the substrate, causing the fluorescence or color increase.
This type of detection takes 7 h to 24 h for detection. Buehler et al. [10]
identified specific enzymes for E.coli. .E.coh can produce either p-glu.euronidase [7, 10] or P-galactosidase, [11]
based on the type of E. coil strains. 'Hence, these enzymes have therefore been considered to be a suitable indicator for E. coif especially for the detection of fecal.
contamination of food and.
water. Nucleic acid methods, such as PCR (polymerase chain reaction) [9, 12-15], microarrays [16], and NASBA (Nucleic acid sequence based amplification) [17], are highly specific and sensitive techniques for detecting bacteria. Based upon known sequence complementarity, different specific strains of bacteria can be identified. There are .several other lab based conventional techniques (for example: membrane filtration, multiple tube fermentation, etc.) to detect E. coil in the contaminated water. All the above discussed methods are expensive, time consuming and not portable. In these cases, water samples need to be delivered to the labs for testing contaminants.

[0004] Therefore, there is a need to develop a hand-held, inexpensive and easy to use diagnostic system for fast and accurate results, Several groups have implemented microfluidic based biosensors [18-21] for rapid detection of E. coll. However, they are not able to detect low concentration of E. coil samples. Additionally some instruments such as impedance analyzer, microscopesor the like may be required for quantification.
Summary of the Invention

[0005] In the present work, we developed a new microfluidic method for detecting E. coil in contaminated water using "microspot with integrated wells" (MSIW). Improved and modified enzyme/substrate detection method is employed on the MSIW to enhance the sensitivity, specificity and selectivity of the detection method, In addition, amount of reagents and time required for the test has been reduced. MSIWs can detect low concentration of samples since there are no fluid movement within the MSIW. Methodology starts with fabrication of M,SfW
and then applying the specifically formulated enzymatic method on micro-wells.
Further measurements are conducted to quantify the color or fluorescence emitted by the wells(MSIW).
Initially the detection protocol was optimized in microcentrifuge tubes and it was found that approx. 70 min was required to detect E. co/i. The optimized protocol was then employed on MSIWs, which reduced the detection time to 10 to 15 min It was observed that MSIW reduces the reaction time compared to conventional centrifuge tube based method, likely due to the increase in reaction surface area and decrease in volume of the samples.

[0006] In one aspect, the invention comprises a microfluidic system for detecting coliform bacteria in a water sample, comprising a silicon or PDMS substrate defining a microspot having a plurality of microwells, the microspot and each microwell coated with at least one enzymatic substrate which results in a detectable change caused by the action of an enzyme, which enzyme is indicative of the presence of a coliform bacteria.

[0007] In another aspect, the invention comprises a method of detecting a coliform bacteria in a water sample, comprising the steps of depositing a volume of the sample in each of a plurality of microwells arranged in a microspot, wherein the microspot and each microwell is coated with at least one enzymatic substrate which results in a detectable change caused by the action of an enzyme, which enzyme is indicative of the presence of a coliform bacteria, and detecting the change or absence of the change.

[0008] In one embodiment, the coliform bacteria is E. coil.

[0009] Additional aspects and advantages of the present invention will be apparent in view of the description, which follows. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Brief Description of the Drawings

[00010] The invention will now be described by way of an exemplary embodiment with reference to the accompanying simplified, diagrammatic, not-to-scale drawings:

[00011] Figure 1A shows a schematic of one embodiment of a MSIW; Figure 1B
shows a scanning electron microscope (SEM) image of the microwells inside a silicon based MSIW;
Figure 1C shows a SEM image of the microwells inside a PDMS based MSIW.

[00012] Figure 2 shows microcentrifuge tube tests with E. coil with Red-gal after incubating the samples for 12 hours (a) at room temperature and (b) at 37 C; Label 1 and l' on the tubes indicate the Ecoli concentration of 10 cfu/ml, Labels 2 and 2' belongs to E.
coil concentration of 100 cfu/m1 and label3 and 3' related to Eco/i concentration of 1000 cfu/ml.

[00013] Figure 3 shows an optical image of the coated PDMS based MSIWs after reacting with E. coll.

[00014] Figure 4 shows graphs of intensity of color/fluorescent development over incubation time for reaction. (a) Color intensity produced by Red-gal substrate after enzymatically reacting with E. coil (b) Fluorescence intensity produced by MUG substrate after enzymatically reacting with E. coll.

[00015] Figure 5 shows (a) SEM image of dried PDMS MSIWs after complete reaction of E.
coil with Red-gal; (b) Exploded view of dried well of MSIW
Detailed Description of Preferred Embodiments

[00016] When describing the present invention, all terms not defined herein have their common art-recognized meanings. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention. The following description is intended to cover all alternatives, modifications and equivalents that are included in the spirit and scope of the invention, as defined in the appended claims.

[00017] The present invention relates to a microfiuidic system and method for detecting E.
coil in a sample. The microfiuidic system comprises a microspot with integrated wells. As used herein, a "microspot" comprises an array of microwells formed in a substrate, such as a silicon or PDMS substrate. The microspot may be a well or depression, or may be defined by a raised border, In one embodiment, the microspot is a circular well, approximately 2 mm in diameter, and the microwells are about 40 to 120 um in diameter and spaced approximately 80 to 150 pm apart (measured from the center of each microwell), in a regular or irregular pattern. Thus, a microspot may feature approximately 200 to 450 microwells, as shown schematically in Figure 1.

[00018] The microwells may be formed in a silicon or PDMS substrate using conventional photolithography or soft lithography methods known in the art. In general terms, an oxide layer is added to the substrate and then the microwell pattern added and etched.

[00019] The microwells are coated with the desired enzyme substrate using an enzyme substrate solution. During a positive assay, the enzyme substrate is hydrolyzed or cleaved by a coliform or an E.coli specific enzyme, which results in a detectable change in colour or fluorescence. In one embodiment, the microwells are coated with a solution comprising 4-Methylumbellifery1-13-D-glucuronide (4-MUG) substrate, 6-Chloro-3-indoly1-0-D-ga1actoside (Red-Gal) substrate in N,N-dimethylformamide (DME), ferric chloride in deionized water and bacteria protein extraction reagent.

[00020] E. coil produces P-D-glucuronidase, which hydrolyzes 4-MUG to 4-MU, which fluoresces blue color under exposure to long wavelength ultraviolet light (350-360 nm). E. coil (along with other coliforms) may produce I3-galactosidase, which hydrolyzes Red-gal to produce the development of red color, visible to the naked eye.

[00021] In use, a small volume, for example 5 pl, of the aqueous sample in question may be added to each microwell. Optionally, the microwell may be incubated briefly at a slightly elevated temperature, for example, about 37 C. Color and/or fluorescence development may be monitored visually or using commercially available readers. The maximum intensity of the color or fluorescence may be indicative of the concentration of coliform or E. coil in the sample.

[00022] Without restriction to a theory, it is believed that increased density of the substrates (MUG or Red-gal) in the microwells reduces the time and concentration of E.
coil required for detectable reactions. Due to high density of wells in MSIW, the reaction surface area is increased compared to other microfluidic approaches using the same sample volume. As a result, the microspot with integrated microwells may enhance the signal intensity even with low concentration samples.

[00023] Exemplary embodiments of the 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.

[00024] Example 1 ¨ Methods and Materials

[00025] E.coli samples of different strains (0157, Castellani and Chalmers (ATCC 11229), Dh5a) were obtained from University of Alberta, Edmonton, Canada. 4-MUG
substrate (4-Methylumbellifery1-13-D-glucuronide, trihydrate) was purchased from Bioworld, Dublin, OH, USA. Red-gal (6-Chloro-3-indoly1-13-D-galactoside) was purchased from Research Organics, Cleveland, OH, USA. Lauryl Tryptose Broth (LTB) and Bacteria protein extraction reagent (B-PER) were obtained from Fisher Scientific, Canada. N,N-Dimethylformamide (DMF), Anhydrous Fenic Chloride (FeC13)were bought from Sigma-Aldrich, USA. 100-mm-diameter silicon (Si) substrate was purchased from Silicon Valley Microelectronics Inc., Santa Clara, CA, USA. Polydimethylsiloxane (PDMS) was obtained from Dow Corning Corporation, Midland, MI, USA.

[00026] Fabrication of MSIP: We used silicon and polydimethylsiloxane (PDMS) based MSIWs in this work. The silicon MSIW were fabricated using the standard photolithography process. MSIW fabrication is similar to the fabrication of micropillars reported elsewhere [22,23]
but briefly described here. A 100-mm-diameter Si substrate was taken and cleaned with Piranha solution. Further, a ¨ 0.52-micron thick oxide layer was thermally grown on top of it followed by patterning of MSIWs on silicon/silicon-dioxide substrate with standard photolithography using HPR504 (Fuji-film Electronic Materials Inc., Mesa, Arizona) positive photoresist (PPR).
Subsequently oxide and silicon layers are anisotropically etched in plasma-reactive ion etchers (dry etching technique, DRIE). After etching the silicon for about 70 um, the PPR on the substrate was stripped off using acetone and the substrate was thoroughly cleaned in Branson PPR stripper. Then oxide layer was removed using plasma-reactive ion etchers.
PDMS based MSIWs were fabricated using standard soft lithography process as described elsewhere [24]. A
micro-spot of 2 mm diameter with square/staggered configuration of wells is considered in this work. A schematic of MSIW and SEM image of the wells inside the silicon MSIW
and PDMS
MSIW is shown in Figs. lA and 1B/1C respectively.

[00027] Fabricated MSIW were coated with specifically formulated enzyme substrate solution (mixture of 100 [1,1 of 1 % (w/v) 4-MUG and/or Red-Gal substrate in LTB, 10 ,1 of 1 % (w/v) ferric chloride in DI water, 100 u.1 B-PER) Mixture of above prepared solution is dispensed into WSW, which is kept at room temperature or 1 h for coating of the mixture to be effective. After coating, the MSIW were cleaned with LTB.

[00028] Enzymatic Test in MicroCentrifuge Tubes. Before the protocol for detection of E.
coli is conducted for MSIW, the chemistry is first tested on micro-centrifuge tubes of 1.5 ml size (purchased from Fisher scientific, Canada). The color or fluorescence producing conditions with different concentrations of chemical reagents, substrates and E. coli samples are optimized. We tried with different E.coli samples in LTB medium (0157, Castellani and Chalmers (ATCC
11229), Dh5a) with Red-gal and 4-MUG substrates. In all experiments, E,coii (Castellani and Chalmers) has been used for convenience and testing. Enzyme substrate solution was prepared as above, with a mixture of 4-MUG substrate, Red-Gal substrate, ferric chloride solution and B-PER, Then E. coil sample of known concentration is added and incubated at room temperature (or at 37 C) for development of color or fluorescence. The presence of E. coil as red color with Red-gal substrate is observed within 70 min of incubation. This shows that some strains of E.coli has P-galactosidase enzyme which cleaves the Red-gal compound to produce red color. With time, the intensity of the red color increased up to 12 h and then remained constant.
The intensity of the red color produced by different concentrations of E. coil with Red-gal substrate solution is shown in Fig. 2.
[000291 Detection of E.coli: MSIWs coated in accordance with the example described above were used for detecting E. coll. Different concentrations of E. coil samples of 1-5 tl volume were dispensed on the coated MSIW and kept at room temperature, Glucuronidase A
(gusA) gene in E. coil encodes P-D-Glucuronidase (GUS) to hydrolyze the substrate 4-MUG (in the coating) enzymatically which leads to the generation of the fluorogenic compound 4-MU.
The presence or absence of an active P-galactosidase in E. coil was detected by Red-Gal (substrate), which produces a characteristic red color when cleaved by P-galactosidase enzyme, thereby providing an easy means of distinguishing the presence or absence of E. coil in water.
Using portable optical color/fluorescent readers (Lateral flow reader and ESElog, Qiagen, Germany) [25], the average color/fluorescent intensity emitted by the MSIWs was measured. Based on intensity, the level of contamination can be predicted for early warnings.
[00030] The developed protocols were applied on to the MSIWs and measured the intensity of the developed color/fluorescence after enzymatic reaction. Color (red) identification in silicon based MSIWs is difficult compared to PDMS based MSIWs, since silicon material itself emits a color which interferes with red color produced in the reaction. Hence we used PDMS based MSIWs for the most of the present work. Figure 3 shows the optical image of the PDMS based MSIWs after treating E. coil with RedGal substrate. A light red-color was obtained at the time of enzymatic reaction within 5 to 10 min. It was observed that the intensity of the color increased over time. It was observed that MSIW reduces the reaction time compared to conventional centrifuge tube based method, probably due to an increase in the reaction surface area to sample volume ratio, The average color or fluorescent intensity emitted by MSIW after enzymatically reacting with respective substrates was measured using portable optical readers. Figure 4 shows the increase in color or fluorescent intensity emitted by MSIWs with increase in incubation time.
It is observed that intensity values increased linearly within first few minutes and then slowly increased (non linearly) up to a maximum value, and then remained relatively constant . This means that in first few min, E. coli was reacting with enzymatic substrates and the reaction was completed after 10 to 15 min.
[00031] A scanning electron microscope was employed to observe the coating and reaction zones of MSIWs. Figure 5 shows a dried PDMS MSIWs (after complete reaction with E. coli). It was found that coating of microwells was not uniform. Although non-uniformly coated microwells may provide qualitative results and rough quantitative results, it would be preferable to have uniformly coated MSIWs to allow for accurate measurement of intensity values, which may be correlated to concentration levels of the bacteria.
References The following references are incorporated herein by reference (where permitted) as if reproduced in their entirety. All references are indicative of the level of skill of those skilled in the art to which this invention pertains.
REFERENCES
[11] Makrides, S., 1996. "Strategies for achieving high-level expression of genes in escherichia coil". Microbiological Reviews, 60(3), pp. 512-538.
[2] Griffin, P., Ostroff, S., Tauxe, R. , Greene, K., Wells, j., Lewis, J., and Blake, P., 1988.
"Illness associated with Eschericlaia coli 0157:h7 infections, a broad clinical spectrum". Annals of Internal Medicine, 109(9), pp. 705-712.
[3] http://www.cd.c.goviecoli/., (Accessed January 10, 2013).
[4] Shelton, D., and Karns, j., 2001. "Quantitative detection of Escherichia coli o157 in surface waters by using inmninomagnetic electrochemilumineseenee". Applied and environmental microbiology, 67(7), pp. 2908-2915.

[5] Varshney, M., and Li, Y., 2007. "Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle- antibody conjugates for detection of Escherichia con. o157: H7 in food samples". Biosensors and Bioelectronics, 22(11), pp.
2408- 2414.
[6] Sarhan, IL, and Foster, H., 2008. "A rapid ftuorogenic method for the detection of escherichia coli by the production of 0-glucuronidase". õJournal of Applied Microbiology! , 70(5), pp. 394-400.
[7] Pettibone, G., 1992, The use of Iauryl .tryptose broth containing 4-methylumbelliferyl-{3-d-glucuronide (mug) to enumerate escherichia coli from freshwater sediment", Letters in applied microbiology, /S(5,), pp, 190-192.
[8] Gaudet, 1., Florence, L., and Coleman, R., 1996. "Evaluation of test media for routine monitoring of escherichia coli in nonpotable waters.". Applied and environmental microbiology, 62(11), pp. 4032-4035.
[9] Bellin, T., Pulz, M., Matussek., A., Hempen, H., and Gunzer, F., 2001.
"Rapid detection of enterohemorrh.agic Escherichia coli by real-time per with fluorescent hybridization probes", journal of clinical microbiology, 39(1)õ pp. 370-374.
[10] Buehler, H., Katzman, P., and Doisy, E., 1951. "Studies onf3-glucuronidase from e. coli.".
In Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, NY), Royal Society of Medicine, 76, pp. 672-676.
[11] Edberg, S., and Edberg, M., 1988. "A defined substrate technology for the enumeration of microbial indicators of environmental pollution,". The Yale journal of biology and medicine, 61(5), pp. 389-396.
[12] thek.we, A., Watt, P., Grieve, C., Sharma, V., and Lyons, S., 2002.
"Multiplex fiuorogenic real-time per for detection and quantification of escherichia coli 0157: H7 in dairy wastewater wetlands". Applied and Environmental Microbiology, 68(10), pp. 4853-4862.
[13) Jothikumar, N., and Griffiths, M., 2002. "Rapid detection of escherichia coli o157:117 with multiplex real-time per assays". Applied and environmental microbiology.
68(6), pp. 3169-3171, [14] Bhagwat, A., 2003. "Simultaneous detection of Escherichia coli 0157: H7, listeria monocytogenes and salmonella strains by real-time per". International journal offood microbiology, 84(7), pp. 217-224.
[15] lbekwe, A., and Grieve, C., 2003. "Detection and quantification of escherichia coli o 157:
117 in environmental samples by real-time per"õlournal of applied microbiology, 94(3), pp. 421-431.
[16] Chandler, 1D., Brown, j., Call, D., Wunschel, S., Grate, J., Holman, D., Olson, L,, Stottlemyre, M., and Bruckner-Lea, C., 2001. "Automated im.munomagnetic separation and mieroarray detection of e. coli o157:147 from poultry carcass rinse".
International journal (Wood microbiology, 70(1 ), pp. 143-154.

[17] Heijnen, L., and Medema, G., 2009. "Method for rapid detection of viable escherichia coli in water using real-time nasba". water research, 43(12), pp. 3124-3132.
[18] Varshncy, M., Li, Y., Srinivasan, B., and Tung, S., 2007. "A label-free, microfmidics and interdigitateci array microelectrode-based impedance biosensor in combination with.
nanoparticles hnmunoseparation for detection of Escheriehia coli o157: H7 in food samples".
Sensors and Actuators B: Chemical, 128(1), pp, 997107.
[19] Yoon, J., Han, J., Choi, C., Bui, M., Sinclair, R., etal., 2009. "Real-time detection of escberichia coli in water pipe using a microlluidie device with one-step latex immunoaggiutination assay.". Transactions of the A.SABE, 52(3), pp. 1031-1039.
[20] Hossain, S., Ozimok, C., Sicard, C., Aguirre,=S., Ali, M., Li, Y., and Brennan, .1., 2012, "Multiplexed paper test strip for quantitative bacterial detection".
Analytical and bioanalytical chemistry, pp, 1-10, [21] Fan, X,, White, 1., Shopova, S., Zhu, H., Suter, J., and Sun, Y., 2008.
"Sensitive optical biosensors for unlabeled targets: A review". Analytica Chi mica Ada, 620(1-2), pp. 8-26.
[22] Gunda, N. S. K., Joseph, J., Tamayol, A., Akbari, M., and Mitra, S. K., 2012. "Measurement of pressure drop and flow resistance in mierochannels with integrated micropillars".
Microlluidics and Nanopuldics, 14(3-4), pp. 711-721, [23] Gunda, N. S. K., Bern, 13., Karadimitriou, N., Mitra, 5, K., and Hassanizadeh, S., 2011.
"Reservoir-on-a-chip (roc): A new paradigm in reservoir engineering". Lab on a Chip -Miniaturisation for Chemistry and Biology, 11(22), pp. 3785-3792.
[24] Kumar, G.N,S., Mitra, S.K., and Rao, V., 2009. "Fabrication of dielectrophoretic mierofiuidic device". ASME 2009 7th International Conference on .Alanochannes, Microchannels, and Minichannels 2009, IC1VMM2009, PART
A(ICNMM2009-82170), pp. 113-11.9.
[25] Wildeboer, D., Arnirat, L,, Price, R., and Abuknesha, R., 2010, "Rapid detection of escherichia coli in water using a hand-held fluorescence detector". Water research, 44(8), pp. 2621-2628.
[26] Rasmussen, S. R., Larsen, M. R,, and. Rasmussen, S. E., 1991. "Covalent immobilization of dna onto polystyrene microwells: the molecules are only bound at the 5' end".
Analytical biochemistry, 198(1), pp. 138-142.
[27] Ostuni, E., Chen, C. S,, Ingber, D. .E., arid Whitesides, G. M., 2001.
"Selective deposition of proteins and cells in arrays of mierowells". Langmuir, 17(9), pp, 2828-2834.
[28] Steinitz, M., 2000. "Quantitation of the blocking effect of tween 20 and bovine serum albumin in elisa microwells". Analytical Biochemistry, 282(2), pp. 232-238.

Claims (2)

WHAT IS CLAIMED IS:

1. A microfluidic system for detecting coliform bacteria in a water sample, comprising a silicon or PDMS substrate defining a microspot having a plurality of microwells, the microspot and microwells coated with at least one enzymatic substrate which results in a detectable change caused by the action of an enzyme, which enzyme is indicative of the presence of a coliform bacteria.

2. A method of detecting a coliform bacteria in a water sample, comprising the steps of depositing a volume of the sample in each of a plurality of microwells arranged in a microspot, wherein the microspot and each microwell is coated with at least one enzymatic substrate which results in a detectable change caused by the action of an enzyme, which enzyme is indicative of the presence of a coliform bacteria, and detecting the change or absence of the change.