key: cord-0769327-tgqlda6p
authors: Pham, Quang Nghia; Trinh, Kieu The Loan; Jung, Seung Won; Lee, Nae Yoon
title: Microdevice‐based solid‐phase polymerase chain reaction for rapid detection of pathogenic microorganisms
date: 2018-06-06
journal: Biotechnol Bioeng
DOI: 10.1002/bit.26734
sha: 3ca81f458426644a5a496829626d96798a910233
doc_id: 769327
cord_uid: tgqlda6p

We demonstrate the integration of DNA amplification and detection functionalities developed on a lab‐on‐a‐chip microdevice utilizing solid‐phase polymerase chain reaction (SP‐PCR) for point‐of‐need (PON) DNA analyses. First, the polycarbonate microdevice was fabricated by thermal bonding to contain microchambers as reservoirs for performing SP‐PCR. Next, the microchambers were subsequently modified with polyethyleneimine and glutaraldehyde for immobilizing amine‐modified forward primers. During SP‐PCR, the immobilized forward primers and freely diffusing fluorescence‐labeled reverse primers cooperated to generate target amplicons, which remained covalently attached to the microchambers for the fluorescence detection. The SP‐PCR microdevice was used for the direct identifications of two widely detected foodborne pathogens, namely Salmonella spp. and Staphylococcus aureus, and an alga causing harmful algal blooms annually in South Korea, Cochlodinium polykrikoides. The SP‐PCR microdevice would be versatilely applied in PON testing as a universal platform for the fast identification of foodborne pathogens and environmentally threatening biogenic targets.

The early detection of foodborne microbial agents is a critical concern in many interdisciplinary research fields. Among diverse pathogenic microorganisms, foodborne pathogens have accounted for the largest number of domestic and global outbreaks and are responsible for high rates of morbidity and mortality worldwide (Foudeh, Fatanat Didar, Veres, & Tabrizian, 2012; Turner et al., 2016) . Foodborne pathogens can also originate from the marine environment and include toxic algae, as well as harmful bacteria that could become a huge threat to human health (Visciano et al., 2016) .

The biotoxins that accumulate in contaminated seafood and the co-occurring pathogens that proliferate during harmful algal blooms may be passed on to higher trophic levels in the marine food network and eventually to humans (Oh et al., 2016) . Such pressures on human healthcare have stimulated the development of several diagnostic methods for the identification of microbial pathogens with high precision, short turnover time, and ease of operation (Foudeh et al., 2012; Law, Mutalib, Chan, & Lee, 2014) .

Nucleic-acid-based testing has played a central role in the detection of harmful microorganisms at the molecular level (Clerc & Greub, 2010; Craw & Balachandran, 2012; Niemz, Ferguson, & Boyle, 2011) . Attributed to the ability to quickly make several copies of genetic materials, the polymerase chain reaction (PCR) is widely used as a molecular diagnostic method in biomedical and biochemical research (Clerc and Greub, 2010; Craw and Balachandran, 2012; Law et al., 2014; Niemz et al., 2011; Oh et al., 2016; Turner et al., 2016; Visciano et al., 2016; Zhang & Ozdemir, 2009 & Muñoz, 2007) . Advances in lab-on-a-chip (LOC) techniques have enabled the miniaturization and integration of several functional components of PCR, allowing timely detection of biomolecular targets with a better analytical performance. Among several variations of PCR, solid-phase PCR (SP-PCR) is a promising method for developing integrated LOC platforms because it enables amplification followed by immediate detection of DNA.

This advantage allows the rapid on-site detection of amplicons in a time-saving manner by overcoming multiple manipulations required for post-analyses of PCR products (Khodakov & Ellis, 2014; Mercier, Slater, & Mayer, 2003; Shin, Perera, Kim, & Park, 2013) .

Since the invention of SP-PCR, the technique has been developed in the form of microarray-based approaches and further incorporated into LOC systems (Chin et al., 2017; J. Hoffmann, M. Trotter, Stetten, Zengerle, & Roth, 2012; J. Hoffmann, S. Hin, Stetten, Zengerle, & Roth, 2012; Kersting, Rausch, Bier, & von Nickisch-Rosenegk, 2014) . Most of the reported SP-PCR LOC platforms have focused on the surface modification of solid substrates for the immobilization of solid-support primers. Despite the noticeable improvements highlighted in these systems, disadvantages remain, which need to be overcome. Specifically, a microarray platform for the genotyping of human mutation genes was developed (Damin, Galbiati, Ferrari, & Chiari, 2016) . However, that platform requires the synthesis of a copolymer coated onto the substrate surface for the immobilization of primers, which demands extensive and time-consuming preparation steps. An organosilanization method was also introduced to immobilize primers (J. Hoffmann, S. Hin et al., 2012) . Although this approach is compatible with SP-PCR, the reproducibility of the coating chemistry depends on highly controlled operative conditions. Besides, the use of bulky apparatuses for the plasma activation of the substrate surface before the coating procedures used in both the abovementioned studies may not meet the conditions of resource-poor laboratories. These shortcomings hamper the realization of an SP-PCR microdevice.

To address these challenges, this study aims to fabricate a thermoplastic SP-PCR microdevice using a chemically robust and thermostable surface modification for primer grafting. For the fabrication, the polycarbonate (PC) microdevice was hot embossed to contain microchambers. The amine groups of polyethyleneimine (PEI) were first coated onto PC via aminolysis to form the urethane linkages without requiring prior surface oxidation of PC. The PEIcoated PC was then activated with glutaraldehyde (GA) to produce a reactive surface for the covalent tethering of amine-modified primers.

The microdevice was used for the simultaneous amplification and detection of two major foodborne pathogens, namely Salmonella spp. and Staphylococcus aureus (S. aureus). Furthermore, to examine the universal applicability of the microdevice, we applied the platform for the detection of a representative harmful marine microalga,

Cochlodinium polykrikoides (C. polykrikoides). After on-chip PCR, the amplicons were directly detected inside the microchambers by fluorescence imaging without having to take out the amplicons. The surface modifications of the PC substrates were characterized by water contact angle measurements, and the density of the coated amine functional groups was also assessed by fluorescence measure- Surface modification was carried out inside the closed microchambers. First, the microchambers were incubated with an aqueous solution of 5 wt% PEI for 1 hr at room temperature. Afterward, the microchambers were washed with deionized water and completely dried with compressed air. The aminated surfaces of the microchambers were activated with freshly prepared 2.5% (v/v) GA solution diluted in phosphate buffer (pH 7.0) containing 10 mM sodium cyanoborohydride, which functions as a reducing agent, for 2 hr at room temperature. The activated microchamber surfaces were thoroughly rinsed with phosphate buffer (pH 7.0) to remove unreacted GA and then dried completely with compressed air.

Finally, a solution of 0.5-µM amine-modified primers prepared in phosphate buffer (pH 7.0) was directly introduced into the PHAM ET AL. 

To analyze the stability of the surface coatings on PC under thermal cycling condition, PEI-GA-coated microchambers were immobilized with PCR amplicons amplified off-chip from 10 ng (3.25 × 10 6 copies) DNA template. The amplicons were produced to have both amine groups and Alexa 488 dyes at the 5′ and 3′ ends, respectively. The microchambers were then filled with the solutions of PCR buffer and water and were subjected to the thermal cycling conditions used for F I G U R E 1 (a) Overall scheme of the fabrication and surface modification of the SP-PCR microdevice for primer grafting. A PC substrate containing three microchambers was embossed with another flat one. Microchambers were successively modified with PEI and GA, and amine-modified primers of corresponding microbe targets were directly immobilized onto the microchamber surfaces. (b) SP-PCR procedures for the detection of foodborne pathogens and alga. PCR mixtures were introduced into the microchambers afterward to perform SP-PCR. Synthesized amplicons remained covalently attached to the solid surface, and the amplified target signals were collected using fluorescence imaging. Green and red fluorescence indicate the signals of Alexa 488 and Alexa 647 dyes, respectively. GA, glutaraldehyde; PC, polycarbonate; PEI, polyethyleneimine; RT, room temperature; SP-PCR, solid-phase polymerase chain reaction [Color figure can be viewed at wileyonlinelibrary.com] SP-PCR. Fluorescence signals of the immobilized amplicons were measured before and after 45 cycles of thermal treatment using an Olympus IX71 inverted fluorescence microscope and were analyzed with ProgRes ® Capture Pro 2.8 software (Jenoptik, Jena, Germany).

The effect of temperature on the stability of the surface coatings was evaluated by analyzing the fluorescence signals before and after the thermal treatment using the Minitab version 16 (Minitab, State College, PA) with 95% confidence intervals, as mentioned above.

For foodborne pathogens, liquid cultures of Salmonella spp. and S. aureus were grown in NB and MHB media, respectively, at 37°C for 16 hr. One milliliter of the overnight culture was then collected and centrifuged at 15,000g for DNA extraction. Isolation of genomic DNA (gDNA) was carried out using the Wizard Genomic DNA purification kit. Samples of the alga C. polykrikoides were directly collected from the ocean during its blooming season in 2013, and algal gDNA was extracted using the RNeasy Mini Kit. Extracted gDNA was stored at 4°C.

In this study, the flat heat block of a commercialized thermal cycler (Gene-Touch TC-E-96GA, Bioer) was used to conduct SP-PCR. A PC substrate was placed on the heat block, and the surface temperatures were measured using an infrared camera (FLIR Thermovision A320, Wilsonville, OR). To evaluate the temperature distribution, 10 spots were randomly selected within the microchamber area for the measurement at each temperature zone. The average temperature was analyzed using ThermaCAM Researcher 2.8 software.

After immobilizing amine-modified primers, the surfaces of the microchambers were blocked with 2 mg ml -1 BSA for 1 hr at room temperature. The microchambers were then washed with deionized water and completely dried with compressed air. SP-PCR was applied on the PC microdevice to amplify invA, nuc, and large subunit ribosomal RNA (LSU rRNA) genes of Salmonella spp., S. aureus, and C. polykrikoides, respectively. The amplification of each target was performed in a 20-µl reaction mixture containing 10 ng of DNA template (3.25 × 10 6 copies of gDNA), 10× Taq reaction buffer, 10 mM of each deoxynucleotide, 1.5 mg ml -1 BSA, 100 nM of forward primer, 400 nM of fluorescencelabeled reverse primer, and 1.25 U of Taq DNA polymerase. After introducing the PCR mixtures into the microchambers, the inlet and outlets were clamped, and the microdevice was placed on the heat block of the thermal cycler. SP-PCR was performed with an initial denaturation at 100°C for 5 min followed by 30 thermal cycles at 100°C for 35 s, 63°C for 35 s, and 76°C for 35 s. In this study, amplification of three targets was performed at the same annealing temperature of 63°C. After SP-PCR, the microchambers were washed, dried, and observed under the fluorescence filters.

As control experiments, DNA amplifications were also performed on pristine microchambers under the same temperature conditions of SP-PCR but without the primer preimmobilization process. The obtained PCR amplicons were also cross checked by agarose gel electrophoresis. The amplicons were stained with ethidium bromide and detected using the Gel Doc EZ system (Bio-Rad). 

We evaluated the effects of PEI concentration (1%, 5%, and 10%) and the PEI modification time (30, 45, and 60 min) on the density of amine groups coated on PC. As shown in Supporting Information Figure S1 , the surface amine density increased with the increase in the PEI concentration and the modification time but decreased after 45 and 60 min of coating with 10% PEI. This reduction probably occurred because a higher PEI concentration and longer modification time might have resulted in the stronger polymer entanglement of PEI chains, reducing the interactions between the amine groups of coated PEI and FITC, as well as the final amine density of PEI coated on the PC surface (Pan et al., 2014) . The amine density reached 37.13 ± 0.91 nmol cm -2 after 60 min of modification with 5% PEI, which was the highest measured density and was significantly different from the other treatments (P < 0.05). Therefore, we finally selected this treatment condition for the modification of PC with PEI.

We further confirmed the surface modification of the microchamber with PEI and GA by determining the ability of the PEI-GAcoated microchamber to capture PCR amplicons. The amplicons were produced to have both amine groups and Alexa 488 fluorescence dyes, as mentioned previously. Figure 3a shows the schematic of immobilizing the amplicons on the PEI-GA-coated PC. The use of PEI to form stable urethane linkages on the PC surface before GA crosslinking was necessary to conjugate aminemodified PCR amplicons because neither the pristine PC surface nor PC surface modified only with GA could capture PCR amplicons after washing, as shown in Supporting Information Figure S2a ,b, respectively. Also, PEI was more effective than 3-aminopropyltriethoxysilane (APTES) in functionalizing the PC surface with amine groups and forming more stable bonds against hydrolytic cleavage (Supporting Information Figure S2c ). This was probably because PEI has much higher amine content with the ratio of primary, secondary, and tertiary amines being 1:2:1, as compared with primary amine-bearing APTES. Since primary and secondary amines can potentially react with GA (Bai et al., 2006) , 

An important criterion of SP-PCR is that chemical linkages between immobilized primers and the surface coatings can withstand hydrolytic cleavage and thermal degradation under the thermal cycling conditions. Primary amines functionalized on PC were reported to suffer from hydrolytic cleavage during the amplification reaction (M. Jang, C. K. Park et al., 2014) , which could return PC to its innate hydrophobic characteristic. However, this tendency was mediated in case of PEI, as demonstrated through the ignorable hydrophobic recovery of PEI-coated PC after 30 PCR cycles (Supporting Information Figure S3 ). This was probably because interactions between PEI and PC take place at both primary and secondary amines (Lee & Ram, 2009 ), resulting in more stable urethane linkages between PEI and PC.

The surface coatings of PEI and GA and the grafted amine-modified PCR amplicons were further evaluated under the thermal cycling conditions. Figure 4a ,b displays the schematic of the thermal stability test of the surface coating and the immobilized amplicons, which was amplified off-chip from 10 ng (3.25 × 10 6 copies) DNA template. As shown in the fluorescence images in Figure 4c ,d, the fluorescence intensities were relatively identical before and after thermal treatment. Figures S2-S4) , the continuous changes in temperature during the thermal cycling program resulted in a nonsignificant reduction in the fluorescence intensity after thermal treatment (P > 0.05). Since 85.3% of the fluorescence signal remained after 45 thermal cycles, the linkages between the surface coatings and the immobilized amplicons were highly stable under the thermal cycling conditions. The thermal-induced fluorescence decrease in this study was comparable or even lower to that reported from a study using a copolymer coated on silicon substrates (Damin et al., 2016) . A greater loss of fluorescence (31.1%-55.6%) under thermal cycling conditions was also reported for the immobilization of DNA on cyclic olefin copolymer or polymer, polypropylene, polydimethylsiloxane, and glass using 1,4-diphenylene diisothiocyanate, a homobifunctional cross-linker (J. Hoffmann, S. Hin et al., 2012) . Fluorescence decreases of 40%-56% were reported for DNA immobilization on glass using self-synthesized linkers of benzene-1,3,5-triacetic acid and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide after 40 thermal cycles (Fedurco, Romieu, Williams, Lawrence, & Turcatti, 2006) and various sulfonated analogs, which are water-soluble heterobifunctional linking molecules, after 50 thermal cycles (Adessi et al., 2000) . The preliminary data obtained in this study showed that thermally stable bonds were established between the immobilized primers and the solid substrate, which confirms the capability of the surface modification strategy to realize SP-PCR using our developed PC microfluidic platform. Table 1 . In addition, the number of desired targets can be increased by fabricating more reaction chambers on the same substrate. As mentioned above, two typical reactions that take place during SP-PCR are liquid-phase and solid-phase amplifications.

Liquid-phase amplification usually proceeds efficiently because of the free diffusion of the aqueous primers and outcompetes the solid priming reactions hampered by the steric hindrance of dense solidsupport primers (Adessi et al., 2000) . To achieve a high efficiency of 2200 | PHAM ET AL.

F I G U R E 5 (a) On-chip PCR results of three microbial targets performed in both pristine and coated microchambers. Lane M is the 100-bp DNA ladder. Lanes 1-3, 4-6, and 7-9 show the results of amplified target genes of S. aureus, Salmonella spp., and C. polykrikoides, respectively. Lanes 1, 4, and 7 are negative controls. Lanes 2, 5, and 8 are target genes amplified with the pristine microchambers, whereas lanes 3, 6, and 9 are those amplified with the coated microchambers. (b) Fluorescence images of SP-PCR results of S. aureus, Salmonella spp., and C. polykrikoides and their corresponding negative controls (without DNA templates). SP-PCR, solid-phase polymerase chain reaction [Color figure can be viewed at wileyonlinelibrary.com] solid-phase amplification performance, an asymmetric balance of the aqueous forward and fluorescence-labeled reverse primers was adopted. The addition of floating forward primers at a low concentration is advantageous in reducing the steric constraint commonly associated with SP-PCR (Adessi et al., 2000; Khan, Poetter, & Park, 2008) as well as minimizing the competition between the aqueous and surface-grafted primers. In this study, different ratios of aqueous forward and fluorescence-labeled reverse primers were examined, and the ratio of 1:4 was found to suit our experimental setup (Supporting Information Figure S5 ). Since PHAM ET AL. Figure S6 ). Also, a procedure of step-by-step surface coating followed by the immobilization of amine-modified primers is crucial for realizing on-chip SP-PCR (Supporting Information Figure S7 ).

Besides, the limit of detection of SP-PCR microdevice was determined using 10-fold dilution series of S. aureus gDNA varying from 3.25 to 3.25 × 10 6 copies. For comparison, homogeneous PCR, which was normal on-chip PCR, was also performed similarly to evaluate the efficiency of SP-PCR. The collected on-chip PCR products were captured by the PEI-GA-coated microchambers, which were then washed to measure the fluorescence signals of the immobilized amplicons. To confirm the accuracy of dilutions, off-chip PCR reactions were first carried out using a commercial thermal cycler, and the amplicons were analyzed using gel electrophoresis. As shown in Supporting Information Figure S8 , the intensities of target bands decreased with decreasing concentration of DNA template, confirming the precision of the dilution series. Figure 6 shows the sensitivity comparison between SP-PCR and homogeneous PCR and their corresponding fluorescence images. As shown in Figure 6a , the efficiency of SP-PCR was generally lower than that of the homogeneous PCR probably due to the steric interactions between the freely diffusing primers and solid-support primers in SP-PCR, as mentioned above. Despite this lower efficiency, the fluorescence signals of the immobilized amplicons in SP-PCR were clearly observed compared with homogeneous PCR. The lowest amount of DNA template at which fluorescence signal can still be observed was 32.5 copies of gDNA. The signals increased with increasing amount of DNA template and almost remained stable when the amount of DNA template was 3.25 × 10 5 copies, indicating the saturation of the surface coating with the amplicons.

In summary, we developed a LOC platform for the performance of SP-PCR to detect two foodborne agents and a microalga. The simple and reliable immobilization of amine-modified primers for SP-PCR was successfully realized by a robust two-step surface modification of PC with PEI and GA. The chemical coatings on the surface of the microchambers exhibited a high stability under thermal cycling condition with 85.3% of the immobilized PCR amplicons remaining after the thermal treatment. The obtained results showed the possibility of using a monolithic plastic platform for the simultaneous amplification and detection of not only prominent foodborne pathogens but also microalgae that annually cause harmful algal blooms. This versatility ensures the wide and universal applicability of the introduced platform as a portable PON testing device, while extending its ranges to other molecular analyses, including gene expression, mutation analysis, and genotyping. 

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SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article

The authors declare that there is no conflict of interests.

http://orcid.org/0000-0001-5303-9740Kieu The Loan Trinh http://orcid.org/0000-0002-5318-4742Nae Yoon Lee http://orcid.org/0000-0001-5029-4009