(CHAS) for Rapid, Colorimetric Detection of Enterohemorrhag

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Microfluidic Integration of a Cloth-Based Hybridization Array System (CHAS) for Rapid, Colorimetric Detection of Enterohemorrhagic Escherichia coli (EHEC) Using an Articulated, Centrifugal Platform Matthias Geissler,†,‡ Liviu Clime,†,‡ Xuyen D. Hoa,‡ Keith J. Morton,‡ Harold Hébert,‡ Lucas Poncelet,‡ Maxence Mounier,‡ Mylène Deschênes,§ Martine E. Gauthier,§ George Huszczynski,§ Nathalie Corneau,∥ Burton W. Blais,*,§ and Teodor Veres*,‡,⊥ ‡

Life Sciences Division, National Research Council of Canada, 75 de Mortagne Boulevard, Boucherville, QC J4B 6Y4, Canada Ontario Laboratory Network, Canadian Food Inspection Agency, Building 22, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada ∥ Bureau of Microbial Hazards, Health Canada, 251 Sir Frederick G. Banting Driveway, Ottawa, ON K1A 0K9, Canada ⊥ Institut National de la Recherche Scientifique, Centre Énergie, Matériaux, Télécommunications (INRS-EMT), 1650 Lionel-Boulet Boulevard, Varennes, QC J3X 1S2, Canada §

ABSTRACT: We describe the translation of a cloth-based hybridization array system (CHAS), a colorimetric DNA detection method that is used by food inspection laboratories for colony screening of pathogenic agents, onto a microfluidic chip format. We also introduce an articulated centrifugal platform with a novel fluid manipulation concept based on changes in the orientation of the chip with respect to the centrifugal force field to time the passage of multiple components required for the process. The platform features two movable and motorized carriers that can be reoriented on demand between 0 and 360° during stage rotation. Articulation of the chip can be used to trigger on-the-fly fluid dispensing through independently addressable siphon structures or to relocate solutions against the centrifugal force field, making them newly accessible for downstream transfer. With the microfluidic CHAS, we achieved significant reduction in the size of the cloth substrate as well as the volume of reagents and wash solutions. Both the chip design and the operational protocol were optimized to perform the entire process in a reliable, fully automated fashion. A demonstration with PCR-amplified genomic DNA confirms on-chip detection and identification of Escherichia coli O157:H7 from colony isolates in a colorimetric multiplex assay using rf bO157, f liCH7, vt1, and vt2 genes.

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based on multiplex PCR amplification of several markers in a single reaction tube, followed by rapid hybridization of the amplicons with an array of capture probes immobilized on polyester cloth strips and immunoenzymatic visualization of the hybridization events. This cloth-based hybridization array system (CHAS) has been adapted to the identification of E. coli O157:H72 and other enterohemorrhagic E. coli3 (EHEC) colony isolates and has been approved as a Compendium method by Health Canada in 2013.4 The advantages of using polyester cloth as a support for nucleic acid hybridizations include its low cost, short reaction times, and ease of handling. The detection of multiplex PCR products using an array of immobilized capture probes provides inherent confirmation of the amplicons by specific hybridization with complementary probes, the ability to discriminate amplicons regardless of size (i.e., yielding greater flexibility in primer design and more balanced amplifications for different targets in the reaction), the

he identification and characterization of pathogenic bacteria recovered from test samples during food safety investigations is a key operation in front-line food testing laboratories. An important example of a priority pathogen targeted by food safety testing programs is Escherichia coli O157:H7, which has been implicated in outbreaks of foodborne illnesses associated with the consumption of contaminated foods such as ground beef.1 In the event of an outbreak, it is imperative that production lots associated with the primary food vehicle are identified as quickly as possible in order to recall all contaminated products from the marketplace. Traditional techniques for the detection of E. coli O157:H7 in food rely on a time-consuming process involving preenrichment in a selective broth, followed by plating to reveal the presence of sorbitol-negative colonies, which are then purified and subjected to a battery of biochemical and serological tests to confirm their identity. Alternatively, the identification of food-borne colony isolates can be achieved through the detection of defining gene markers using polymerase chain reaction (PCR) techniques. For example, a timely colony identification procedure has been developed © XXXX American Chemical Society

Received: August 2, 2015 Accepted: September 28, 2015

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DOI: 10.1021/acs.analchem.5b03085 Anal. Chem. XXXX, XXX, XXX−XXX

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lem.8,10,11 A computer-controlled, centrifugal platform was developed that executes a predetermined sequence of spinning and articulation steps to feed and rinse the cloth substrate in a fully autonomous fashion. We demonstrate detection of E. coli O157:H7 colonies using the microfluidic CHAS (μCHAS) procedure by targeting key marker genes for this pathogen.

simultaneous detection of a large number of different amplicons, and the ease of visualization of the probe reactions (i.e., development of colored spots). A disadvantage of the CHAS protocol in its original embodiment (Table 1) is the Table 1. Standard CHAS Protocol step #

operation

1

incubation with digoxigenin (DIG)-labeled amplicon wash with phosphate-buffered saline containing 0.05% (v/v) Tween 20 (PBST) incubation with anti-DIG Fab fragments− peroxidase (POD) conjugate in PBST-Bb wash with PBST incubation with tetramethylbenzidine (TMB) membrane peroxidase substratec

2 3 4 5 total:

volumea (mL)

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duration (min)

1.5

15

5 × 1.5

n.a.

1.5

10

5 × 1.5 1.5

n.a. 0°) indicates rotation in a clockwise E

DOI: 10.1021/acs.analchem.5b03085 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 4. Stroboscopic images of the chip at different stages of the process. Colored solutions were used to provide visual contrast: PCR product, red; PBST, green; anti-DIG POD conjugate, yellow; TMB, blue. (a) Home position. The chip is still at rest and the cloth substrate remains fully immersed in solution containing the PCR product. (b) The platform begins to rotate. (c) The cloth substrate is being rinsed. (d) Anti-DIG POD conjugate is transferred into the hybridization unit. (e) The chip resumes rotation to dry the cloth substrate. (f) Wash buffer and TMB are relocated and begin to transfer into the destination reservoirs (3) and (7), respectively. (g) Increasing the rotational speed completes the transfer process. (h) The chip resumes its home position. (i) TMB is passed onto the hybridization unit. (j) After spots have been developed, the cloth substrate is dried through a final spin step. The array contained VT1 and VT2 probes and was exposed to PCR-amplified vt1 and vt2 target genes. Void segments on the cloth substrate were left intentionally blank.

Figure 5. E. coli identification assays performed using μCHAS. (a) Scheme detailing the arrangement of the probes on the cloth substrate. Probe/ target combinations are as follows: O157, rf bE gene; H7, f liC gene; VT1, vt1 gene; VT2, vt2 gene; IAC, internal amplification control. (b−g) Photographs of cloth substrates showing the results of the μCHAS process using PCR products from colony lysates of various bacteria with different verotoxin genotypes. (b) E. coli O157:H7 [O157, H7, VT1, VT2, IAC]. (c) E. coli O157:H7 [O157, H7, VT2, IAC]. (d) E. coli O26:H11 [VT1, VT2, IAC]. (e) E. coli O5:NM [VT1, IAC]. (f) E. coli O6:H34 [VT2, IAC]. (g) S. urbana [IAC]. Scale bar: 5 mm.

equilibrate pressure and facilitate circulation of fluid within the system. Operation. The implementation of the μCHAS procedure is detailed in Table 2, where spinning frequencies and chip orientation are provided for each step. Figure 4, in addition, shows the chip at different stages of the process. The chip is mounted on the platform after hybridization and contains all the necessary components in the respective reservoirs (Figure 4a). Transfer compartment (7) remains empty initially. The overall volume accommodated on the chip accounts for 1.0 mL, which represents a significant reduction with respect to the 19.5 mL that are used in the standard method (Table 1). During the

initialization phase (steps 1−6), liquid is conditioned within each reservoir while the PCR product is removed from the hybridization unit once the platform starts rotating (Figure 4b). The cloth substrate is then rinsed (steps 7−9) through articulation of the chip in a clockwise direction (Figure 4c). Anti-DIG POD conjugate solution is engaged (steps 10−13) by orienting the chip in the opposite direction (Figure 4d). The narrow exit channel at the lower end of the hybridization unit (100 μm in width and depth) reduces the flow rate with respect to the incoming supply, making it possible to keep the cloth substrate fully immersed in conjugate solution when the platform is brought to a standstill. We maintained the same F

DOI: 10.1021/acs.analchem.5b03085 Anal. Chem. XXXX, XXX, XXX−XXX

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CONCLUSION Translation of the CHAS into a compact microfluidic chip format has been mediated by the concept of articulation and its implementation on a centrifugal platform. The ability to independently change the orientation of the microfluidic circuitry around a secondary rotation axis makes it possible to displace fluids on demand from virtually any location on the chip, thus allowing for efficient management of the footprint. Another important difference of our platform compared to many other centrifugal systems relates to the fact that complexity has been shifted from the chip toward the instrument: instead of driving a sophisticated microfluidic system using a simple spinning shaft, we developed a more elaborate platform with integrated stepper motors and electronic circuitry to operate a simple and straightforward device. This scenario greatly enhances the possibilities of process integration and automation since microfluidic functions are primarily controlled through velocity and chip orientation rather than feature dimensions or wettability. Likewise, the composition of the solutions that participate in a particular process can be changed without affecting their transfer behavior, making articulation more versatile than capillarybased systems. A simple microfluidic design also translates into low-cost, high-throughput manufacturing schemes for the chip to be disposable in an economic fashion. Increasing the number of chips that can be processed at a time (e.g., by stacking multiple chips on top of each other) would further enhance the utility of the system for rapid testing in food safety laboratories.

incubation time as for the standard CHAS, though it is plausible that steady flow would enhance the interaction between the antibody moiety of the conjugate and the immobilized DIG label on the hybridized PCR product. The cloth substrate is dried once the platform resumes rotation (Figure 4e). The next stage involves relocation of both PBST and TMB (steps 14−19) to render these components available in reservoirs (3) and (7), respectively (Figure 4f−h). Although the siphon structures connected to these reservoirs are both primed through clockwise articulation, the difference in αc is large enough so that the cloth substrate can be washed (steps 20−22) before TMB is released in a second step (Figure 4i). During the development stage (steps 23−27), the cloth substrate is incubated (using a typical duration of 1 min). The appearance of a blue-colored spot (Figure 4j) indicates successful hybridization between the probe and its complementary DIG-labeled amplicon, thus, revealing the presence of the target gene. E. coli Identification Assay. We identify the target pathogen (E. coli O157:H7) through the detection of gene markers rf bO157, f liCH7, vt1, and vt2 (encoding the O and flagellar antigenic determinants and verotoxin genes 1 and 2, respectively), which together are definitive for this organism (though the VT profile can vary from carriage of either or both types). Bacterial isolates are subjected to a procedure targeting all of the gene markers in a single multiplex PCR incorporating a detectable label (DIG), which is subsequently revealed after hybridization of the PCR products with an array of ampliconspecific capture probes individually spotted on the polyester cloth substrate (Figure 5a). The assay also features an internal amplification control (IAC) to test for PCR inhibition. The functioning and reliability of the PCR primers and probes were validated in previous work using the standard CHAS format.2 We subjected five verotoxigenic E. coli (VTEC) strains with different genotypic profiles for the O157, H7, VT1, and VT2 traits to the μCHAS procedure. A non-VTEC bacterial strain, Salmonella enterica serovar Urbana, was also included as a negative control. All of the VTEC strains gave the expected pattern of CHAS reactivity for the different markers. Positive results were obtained for the O157 and H7 features with both E. coli O157:H7 strains, with variable results for either VT1 or VT2 according to their genotypes (Figure 5b,c). None of the three other VTEC strains produced positive results for O157 and H7, which is expected since these strains belong to different serotype designations, whereas variable results for VT1 and VT2 were observed according to their respective genotypes (Figure 5d−f). None of the gene markers were evident for the S. urbana strain (Figure 5g), which is expected since this organism is unrelated to VTEC. The IAC produced a signal with all of the bacterial strains, validating the results obtained with the sample devoid of the target gene markers (i.e., S. urbana). Our findings suggest that μCHAS provides a high degree of specificity in the detection of gene targets associated with E. coli O157:H7 and other VTEC pathogens. The colorimetric spots, which can be easily discerned by eye, are well-defined in terms of contrast for positive signals, validating the μCHAS process from an analytical point of view. The absence of notable background signal on the cloth suggests that single-step rinsing under fluidic conditions is sufficient to replace for the multiple wash steps that are used in the standard CHAS process.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work (M.G. and L.C.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the GRDI-funded program “Strengthening Food and Water Safety in Canada through an Integrated Federal Genomics Initiative”. We thank our colleagues Emmanuel Roy (NRC), Hélène Roberge (NRC), and Caroline Miville-Godin (NRC) for technical assistance and useful discussions.

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REFERENCES

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DOI: 10.1021/acs.analchem.5b03085 Anal. Chem. XXXX, XXX, XXX−XXX