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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 4494−4501
Simultaneous Detection of Antibiotic Resistance Genes on PaperBased Chip Using [Ru(phen)2dppz]2+ Turn-on Fluorescence Probe Bofan Li, Xiaoming Zhou,* Hongxing Liu, Huaping Deng, Ru Huang, and Da Xing* MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics South China Normal University, Guangzhou 510631, China
ACS Appl. Mater. Interfaces 2018.10:4494-4501. Downloaded from pubs.acs.org by UNIV OF TOLEDO on 09/28/18. For personal use only.
S Supporting Information *
ABSTRACT: Antibiotic resistance, the ability of some bacteria to resist antibiotic drugs, has been a major global health burden due to the extensive use of antibiotic agents. Antibiotic resistance is encoded via particular genes; hence the specific detection of these genes is necessary for diagnosis and treatment of antibiotic resistant cases. Conventional methods for monitoring antibiotic resistance genes require the sample to be transported to a central laboratory for tedious and sophisticated tests, which is grueling and time-consuming. We developed a paper-based chip, integrated with loop-mediated isothermal amplification (LAMP) and the “light switch” molecule [Ru(phen)2dppz]2+, to conduct turn-on fluorescent detection of antibiotic resistance genes. In this assay, the amplification reagents can be embedded into test spots of the chip in advance, thus simplifying the detection procedure. [Ru(phen)2dppz]2+ was applied to intercalate into amplicons for product analysis, enabling this assay to be operated in a wash-free format. The paper-based detection device exhibited a limit of detection (LOD) as few as 100 copies for antibiotic resistance genes. Meanwhile, it could detect antibiotic resistance genes from various bacteria. Noticeably, the approach can be applied to other genes besides antibiotic resistance genes by simply changing the LAMP primers. Therefore, this paper-based chip has the potential for point-of-care (POC) applications to detect various gene samples, especially in resource-limited conditions. KEYWORDS: antibiotic resistance genes, paper-based chip, loop-mediated isothermal amplification, [Ru(phen)2dppz]2+, multiple detection
1. INTRODUCTION Antibiotic resistant bacteria that are difficult to treat have been becoming increasingly common and have caused a severe global health crisis.1 Antibiotic resistance is encoded by particular genes, some of which can transfer among various bacteria via plasmid.2 With great clinical diagnostic value, the detection of antibiotic resistance genes is critical to provide a reference for doctors to treat patients with antibiotic drugs appropriately at the early stage of bacterial infections, thus improving the antibiotic curative effectiveness.3 Conventional methods for detecting antibiotic resistance genes include multiplex PCR,4 real-time PCR,5 electrochemical detection,6 and DNA microarray.7 While these assays provide precise results, they also require tedious operation, expensive instruments, and trained professional personnel to perform the procedure and analyze results. In recent years, great efforts have been made to develop point-of-care (POC) tests, since these assays provide equipment-free, robust diagnosis approaches for emergency situations.8−10 Accordingly, it is urgent to develop a method that could acquire antibiotic resistance genetic information from a POC test, especially in resource-limited regions. Paper-based devices, which can provide a POC platform for low-cost and disposable sensors to diagnose antibiotic © 2018 American Chemical Society
resistance genes simultaneously, have been widely applied for various analyte detection including metal ions,11 proteins,12 nucleic acids,13 and so forth. We have developed a series of paper-based biosensors for plant viruses,14 pathogenic bacteria,15 and miRNA16 detection, for instance, quantum dot-labeled strip biosensors exhibited a favorable sensitivity of 200 amol for miRNA-21 detection.16 However, a major challenge concerning the paper-based platform is how to integrate molecular amplification technologies into paper-based devices to allow more convenient and more efficient detection.17 Fortunately, some researches have revealed that paper as a substrate for nucleic acid amplification has some merits: (1) DNA can adsorb to cellulose, so paper provides an effective matrix for DNA capture; (2) paper has cellulose fiber networks that provide a high surface area to facilitate storage of dried reagents.18 Thus, the integration of nucleic acid amplification technologies with paper-based devices should be a crucial functionality of paper-based devices to conduct POC tests.19−21 Received: November 19, 2017 Accepted: January 11, 2018 Published: January 11, 2018 4494
DOI: 10.1021/acsami.7b17653 ACS Appl. Mater. Interfaces 2018, 10, 4494−4501
Research Article
ACS Applied Materials & Interfaces
2. EXPERIMENTAL SECTION
Isothermal nucleic acid amplification technologies have been widely raised as a method to implement target amplification without the use of thermocycling.22 Moreover, as there is no need to change temperature, the amplification can be processed at the optimal condition of enzyme for a more efficient reaction than PCR.23 As one of these amplification assays, loopmediated isothermal amplification (LAMP), a highly sensitive assay, is operated at a constant temperature (60−65 °C) and can generate a billion copies of DNA products within 1 h.24 The usage of two pairs of primers makes LAMP possess transcendent specificity. Based on these properties, some researchers made attempts to introduce LAMP assays into miniaturized microdevices such as glass,25 cyclic olefin copolymer,26 and polystyrene.27 These favorable features make LAMP suitable to apply into paper-based devices for the improvement of detection efficiency. The conventional paper-based analytical methods for detecting the products of LAMP need to label signal probes on paper in advance, such as colloidal gold nanoparticles28 and calcein,29 which present some challenges, for instance, requiring expensive probe tagging moieties30 and involving nonspecific hybrid probes that are difficult to clean up resulting in a relatively high background signal.31 Although the intercalating dye SYBR Green I has been used to analyze the end products of LAMP for its label-free character,32,33 there was research showing that the ultraviolet fluorescence method generated a high ratio of false positives and was not recommended to be a LAMP read-out.34 The photoswitch probe [Ru(phen)2dppz]2+ (phen = 1,10-phenanthroline, dppz = dipyridophenazine), a transition metal complex, has an undetectable emission yield of photoluminescence in aqueous solution due to the protonation of the N atoms in phenazine, but when it intercalates into the major groove of DNA, the N atoms are protected by the intercalation of the phenazine ligand, thus resulting in intense fluorescence emission with a wavelength of 620 nm.35,36 Compared to the above analytical methods, [Ru(phen)2dppz]2+ turn-on fluorescence detection is highly sensitive and label-free. It is also worth noting that water and protein, the major absorbers of infrared and visible light, respectively, have low absorption coefficient in the spectral region around 620−900 nm,37 thus facilitating improved signal-to-noise of this assay. These features make the so-called “light-switch” probe [Ru(phen)2dppz]2+ suitable for LAMP product detection. In this study, we fabricated a paper-based chip integrated with LAMP assay and the “light switch” molecular probe [Ru(phen)2dppz]2+ for simultaneous analysis of multiple antibiotic resistance genes for the first time. The nucleic acid amplification steps were simplified by fabricating the reaction discs with Whatman filter paper, which contained LAMP reagent mix dried into the cellulose matrix. After a sample is introduced into the reaction discs, the chip is placed in the heating block immediately to initiate the LAMP assay. Final detection of LAMP products used [Ru(phen)2dppz](PF6)2 and a hand-held light source. For verifying the feasibility of the paper-based chip, we amplified the most common antibiotic resistance genes mecA (coding for methicillin resistance via penicillin binding protein 2a) and ermC (coding for macrolidelincosamide-streptogramin resistance via rRNA methylases). Owing to the portable and low-cost paper-based device, as well as LAMP technology and excellent fluorescence performance of [Ru(phen)2dppz]2+, the paper-based chip would have great potential in POC detection of antibiotic resistance genes in resource-limited regions.
2.1. Materials and Reagents. The oligonucleotide primers with high performance liquid chromatography (HPLC) purification were purchased from Takara Biomedical Technology Co., Ltd., and suspended in ultrapure water. The sequences are listed in Table S1. Bst 2.0 DNA polymerase, 10× Isothermal Amplification Buffer, 10 mM dNTPs, and 100 mM MgSO4 were ordered from New England Biolabs and betaine from Sigma-Aldrich. [Ru(phen)2dppz](PF6)2 was synthesized as reported previously.38 Resistant strains methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli (engineered bacteria with plasmid gene mecA) were offered from Guangzhou Center for Disease Control and Prevention; susceptible strains Staphylococcus aureus (CMCC 26003), Listeria monocytogenes (CMCC 54007), and Salmonella (CMCC 50040) were purchased from Guangzhou Institute of Microbiology (Guangzhou, China). Filter paper (cat. no. 1002-185) was purchased from Whatman Inc. The magnetic plate used to fabricate the chip was a 0.75 mm thick ferritebonded synthetic rubber sheet obtained from McMaster-Carr. Plastic adhesive backing pad used as the laminate was ordered from Millipore (Billerica, MA). The heating device was homemade, containing a DC power supply (model EADP-36FB), a temperature controller (model XMTG-7401), and a copper heating block. All other reagents were analytical pure grade and provided by Aladdin Co., Ltd., and all buffers and chemical solutions were prepared in sterile water when required. 2.2. Fabrication of Paper-Based Chips. Each paper-based chip, schematic of which is shown in Figure 1a, contained five components from top to bottom: a top magnetic plate; a magnetic plate with 3 mm diameter alternating poles; filter paper cut into discs (diameter 3 mm) and adapted to magnetic plate with 3 mm poles, which form the discs for LAMP assay and fluorescence detection; laminate for disc support and fixation; a bottom magnetic plate. The attractive force produced by the two magnetic plates forms a seal to resist evaporation in combination with the mineral oil applied to the layer interfaces. 2.3. Preparation of Preamplification Chips. The gene-specific LAMP mix contained 0.2 μM outer primers (F3 and B3), 1.6 μM inner primers (FIP and BIP), 6 mM MgSO4, 1× Isothermal Amplification Buffer, 1.4 mM each dNTPs, 1 M betaine, 8 U of Bst 2.0 DNA polymerase, and sterile water. The paper-based chip was fabricated with 4 × 3 poles (Figure 1b), distributing 5 μL of LAMP mix equally in each pole. Then, the chip was left to air-dry for 2 h and stored at 4 °C. 2.4. Bacteria Culture, Genome Isolation, and LAMP Assay. Various bacteria were grown in broth medium at 37 °C overnight. The broth mixed 0.5 g of yeast extract, 1.0 g of tryptone, and 1.0 g of NaCl in 100 mL of water; the pH was adjusted to 7.0. The bacterial genome was isolated using the TIANamp Bacteria Genome DNA Kit and suspended in 100 μL of TE buffer. To perform LAMP assay in the paper-based chip, 5 μL of 1× Isothermal Amplification Buffer, which contained 1 μL of bacteria genome specimen, was added into each pole of the preamplification chip, and then 5 μL of mineral oil was applied to each pole to prevent liquid evaporation during the amplification. The top magnetic plate was placed on the chip to form a sealed structure, and the chip was placed into the copper heating block immediately. The optimal incubation was 64 °C for 55 min. The heating device is shown in Figure 1c. 2.5. Product Analysis by Fluorescence Detection. After incubation, the chip was first removed from the incubator and cooled down to room temperature. Second, the remaining mineral oil was removed, and then 4 μL of detection reagent containing 1 mM [Ru(phen)2dppz](PF6)2 was added into each spot, standing at room temperature for 5 min. Finally, the chip was put under a hand-held light source (450 nm wavelength) and the fluorescence emitted from [Ru(phen)2dppz]2+ was imaged. Images were then converted to grayscale and processed using ImageJ to quantify the intensity of gray value in each detection spot of the chip. Utilizing the classical calculation method, we determine LOD as the mean of the no template control (NTC) plus 3 times its standard deviation. The threshold value was used to determine positive and negative results. 4495
DOI: 10.1021/acsami.7b17653 ACS Appl. Mater. Interfaces 2018, 10, 4494−4501
Research Article
ACS Applied Materials & Interfaces
The detection strategy was based on the fact that in aqueous solution, the fluorescence of [Ru(phen)2dppz]2+ is quenched via the protonation of N atoms. When it binds with double stranded DNA, due to the planar phenazine ligand interaction with the base pairs in the major groove of DNA, the N atoms in phenazine are protected, which generates a change in microenvironment of [Ru(phen)2dppz]2+ that improves the population of luminescent state, leading to intense fluorescence emission at 620 nm.39 In addition, the intensity of fluorescence is proportional to the amount of [Ru(phen)2dppz]2+−DNA complex, which makes it possible for quantitative detection of antibiotic resistance genes in bacteria.40 Using a hand-held light source with a wavelength of 450 nm to excite the fluorescence of [Ru(phen)2dppz]2+−DNA complex, one can image the result with a camera. An overview of the paper-based chip for multiple antibiotic resistance gene detection is illustrated in Figure 1B. Antibiotic resistance genes mecA and ermC were diagnosed, in addition to a control nucleic acid fragment of 16S rDNA common to most bacteria. 3.2. Time Course Studies of the Paper-Based Amplification. LAMP assay is a highly efficient amplification technology, and we found when it was integrated into the paper-based chip, the amplification could be completed within 60 min. To quantitatively reveal the speed of paper-based amplification and estimate the optimal incubation time, the genome from MRSA (106 copies) was used to perform parallel tests for mecA and ermC detection. We conducted amplification in 48 chips; they were isolated sequentially from the heating block every 5 min and then [Ru(phen)2dppz](PF6)2 was added immediately to detect fluorescence signals. Negative controls without genome were set to judge whether the result of amplification was reliable. In the fluorescence photographs of the test spots (Figure 2A), the fluorescence signals could be observed clearly in the spots with genome after 30 min incubation, and the fluorescence intensity increased gradually with the extension of time. The fluorescence signal data (in grayscale) was plotted along with time in minutes as shown in Figure 2B; t0 was defined as the moment when the chips were put into the heating block. We found that the curves of negative controls kept at a low level of fluorescence, meaning there were no amplification products in these spots during incubation. The curve of ermC positive assay rose at first, probably because the loop primers of ermC had a higher hybridization efficiency than those of mecA, while the curve of mecA positive assay maintained a higher level of fluorescence, based on the fact that the amplicon length of mecA (512 bp) was longer than that of ermC (297 bp), resulting in more [Ru(phen)2dppz](PF6)2 combined with the former. The fluorescence of mecA, as well as ermC, reached a maximum at nearly 55 min. The signal-to-noise ratio of mecA at 55 min was 18.723, while that of ermC was 15.474. The results of real-time PCR for mecA and ermC amplification are shown in Figure S4, and proved that the loop primers could amplify mecA and ermC well. The final fluorescence intensity of mecA assay was stronger than that of ermC assay in RT-PCR, which was similar to paper-based amplification. However, the difference of the final fluorescence intensity between mecA and ermC was not remarkable in the paper-based amplification, and the curves of mecA positive assay in RT-PCR increased faster than those of ermC. We considered it was caused by the difference amplification efficiency between LAMP and PCR, and the distinction between LAMP primers (inner primers and outer primers) and RT-PCR primers (only outer primers). These results showed that the paper-based chip
Figure 1. Schematic illustration of the paper-based chip analysis system. (A) The device contains the paper-based chip and a homemade heating device, exploded view (a) shows the five layers of the chip architecture; the topless assembled device (b) can accommodate multiple samples and controls; the heating device (c) contains a heating controller and heating block. Schematic B shows the simultaneous detection of antibiotic resistance genes on the paperbased chip: (I) bacteria cell lysis; (II) addition of LAMP reagents; (III) addition of bacteria genome; (IV) isothermal amplification; (V) addition of [Ru(phen)2dppz](PF6)2; (VI) fluorescent detection using a hand-held light source.
3. RESULTS AND DISCUSSION 3.1. Principle of the Method. The core architecture of the paper-based chip, illustrated in Figure 1a, consisted of three major layers: a top magnetic layer, which combined with mineral oil in the layer interfaces created a seal to prevent evaporation of the reagents during amplification; the middle reaction layer consisting of a magnetic plate with hollow arrays (each cavity with a diameter of 3 mm) and filter-paper discs, which were cut into appropriate size to adapt to the arrays, providing a substrate where LAMP reagents were dried and amplification was performed; the bottom layer containing a laminate to form the foundation of arrays and a magnetic plate to form attractive force with the top layer. The LAMP mix was introduced into each spot of the chip, dried, and stored at 4 °C for further use. The results of the experiments for optimizing the reaction conditions are shown in Figure S1, Figure S2, and Figure S3. We found that LAMP assay could achieve a good result under conditions of 8 mM Mg2+ and 64 °C. The optimal amplification time for the paper-based chip is presented in the next section. After the genome was isolated from antibioticresistant bacteria and added into the chip, LAMP assay was conducted at 64 °C for 55 min. In the final detection, we applied [Ru(phen)2dppz](PF6)2 to detect the LAMP products. 4496
DOI: 10.1021/acsami.7b17653 ACS Appl. Mater. Interfaces 2018, 10, 4494−4501
Research Article
ACS Applied Materials & Interfaces
3.3. Simultaneously Analysis of Antibiotic Resistance Genes. To assess the feasibility of simultaneous amplification and detection of multiple antibiotic resistance genes in the paper-based chip, MRSA containing antibiotic resistance genes mecA and ermC and a control gene, 16S rDNA, was selected as target bacteria. For facilitating the presentation of the detection results, we constructed a rhombic test spot pattern as shown in Figure 3A. Each chip involved four spots, namely, spot 1 and spot 2 containing 16S rDNA primers, spot 3 containing ermC primers, and spot 4 containing mecA primers. The genome of MRSA (106 copies) was added into test spots to perform LAMP assay. After incubation, the results of gene-specific LAMP were first read-out by naked eye using a hand-held light source. As quality control of the paper-based chip, the amplified result of 16S rDNA reflects whether the chip is credible. In Figure 3a, the genome was added into every test spot except for spot 1, it presented the ideal situation that negative control (spot 1) had undetectable fluorescent signal while test groups (spots 2, 3, and 4) possessed intense fluorescence. In Figure 3b, the genome was added only into spot 2, and the fluorescence only appeared in spot 2, as was expected, indicating that there was amplification product only when the test spot contained the genome. In Figure 3c, the genome was introduced into spots 2 and 3, while in Figure 3d, spots 2, 3, and 4 all contained the genome; the fluorescent phenomena confirmed the above conclusion, and we also found that the amplification of ermC and mecA in the paper-based chip had favorable results, as well as 16S rDNA. Images were converted to grayscale and quantified using ImageJ. The gray value of every spot in Figure 3b,c,d was measured and integrated into a histogram, as shown in Figure 3B. The fluorescence intensity of spots with target genome were obviously higher than the control group in the absence of target, indicating that amplification in the paperbased chip possessed high signal-to-noise ratio. Noticeably, this analytical method can be applied to other genetic targets besides antibiotic resistance gene detection by simply changing LAMP primers. It can be predicted that the paper-based chip would be widely used to simultaneously detect various nucleic acids samples in the future. 3.4. Analytical Sensitivity of the Paper-Based Chip. Various amounts of genome from MRSA were used to estimate
Figure 2. Real-time amplification of multiple LAMP for mecA and ermC. (A) Fluorescence intensity of amplification changes with time. The “+” symbols and “−” symbols indicate positive assay adding 106 copies of MRSA genome and the negative control (no MRSA genome), respectively. Round dashed frames in the graph reflect the location of detection spots in the chip. (B) Real-time amplification curve of multiple LAMP on the paper-based chip.
could be used to detect genes with outstanding results in 1 h, and the optimal incubation time of this assay was 55 min. Compared to conventional LAMP assay, which can be accomplished with 30−60 min in aqueous solution, the paper-based LAMP has no requirement to take more time to be completed and, significantly, it can simplify the preparation of reagents in testing ground with a substitute of preamplification chips, as well as reduce the subsequent analytical operation just with fluorescence probe introduced into spots of the paper-based chip and imaged.
Figure 3. Simultaneous detection of multiple antibiotic resistance genes. In photographs A, diagram a represents the all-positive result of the multiple amplification in an ideal situation presented via cartoon. Numbers denote different gene-specific amplification: (1) no template control of 16S rDNA; (2) 16S rDNA; (3) ermC; (4) mecA. Photographs b, c, and d denote the different positive results with the target added in turn: (b) 2 positive; (c) 2 and 3 positive; (d) 2, 3, and 4 positive. Round dashed frames in the graph reflect the location of detection spots in the chip. Histogram B shows the fluorescence intensity of the multiple amplification in part A, quantifying the intensity of gray value in each spot. 4497
DOI: 10.1021/acsami.7b17653 ACS Appl. Mater. Interfaces 2018, 10, 4494−4501
Research Article
ACS Applied Materials & Interfaces
Figure 4. Analytical sensitivity of antibiotic resistance gene detection using the paper-based chip. Quantitative amplification based on the paper chip for the detection of mecA (A) and ermC (B), each column represents the test repeated five times. Round dashed frames in the graph reflect the location of detection spots in the chip. Histograms C and D indicate the fluorescence intensity of the paper chips A and B, quantifying the intensity of gray value in each spot. Red dashed line denotes the LOD.
the sensitivity of the paper-based chip. We designed the chip with 6 columns of test spots, different concentration of genome was added into each column, and every spot in a column contained the identical amount of genome. To verify the reliability of the assay results, each column had 5 test spots to repeat the analysis five times. After incubation, [Ru(phen)2dppz](PF6)2 was introduced into every spot and imaged. Antibiotic resistance genes mecA and ermC were selected as targets. As shown in Figure 4A,B, fluorescence photographs of the test spots could be clearly observed, and the fluorescence intensity increased with the increase of genome from MRSA. Because the fiber structure in the filter paper revealed irregular distribution, some parts of the paper would absorb more of the DNA products, giving rise to the heterogeneous distribution of fluorescence. However, we measured the fluorescence intensity of the whole detection spot, and generally the intensity of fluorescence was proportional to the amount of DNA products, which made it credible for quantitative detection. Through analyzing the interrelated grayscale of the test spots (see Figure 4C,D), we found that the fluorescence intensity reflected the amount of amplification products in appropriate scale of genome, which was from 100 copies to 12500 copies for mecA gene and from 200 copies to 25000 copies for ermC gene. We also measured the LOD of the paper-based chip, using the mean of the negative control plus 3
times of its standard deviation. The LOD was 100 copies for mecA and 285 copies for ermC, meaning the paper-based chip for antibiotic resistance gene detection reached sensitivity as few as 100 copies; hence it could be applied to analyze practical samples based on the fact that the amount of genome in antibiotic resistant bacteria collected from patients is much more than the hundreds.41 Other methods such as electrochemical microchip integrated with PCR assay could detect genomic DNA from Salmonella enterica with a poorer LOD of 300 copies42 than our paper-based chip. Compared with the thermoplastic microfluidic sensor for multidrug resistant Tuberculosis detection with the LOD of 50 copies of genome,43 the sensitivity of our paper-based chip was slightly lower than it, but the paper-based chip was more portable and convenient with fewer operating steps, and noticeably, this chip was suitable for multiple gene detection simultaneously. Improving the fabrication technique of chip or employing preferable fluorescent signal probe may make it possible for more sensitive detection in the future. 3.5. The Specificity of Assay. To verify that the paperbased chip could detect antibiotic resistance genes from different bacteria, 106 genome copies of MRSA, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Salmonella were used as targets to conduct LAMP assay and fluorescence detection. We designed the pattern of test spots similar to a 4498
DOI: 10.1021/acsami.7b17653 ACS Appl. Mater. Interfaces 2018, 10, 4494−4501
Research Article
ACS Applied Materials & Interfaces
mentioned. In Figure 5c, the genome of Escherichia coli, which is the engineered bacteria with plasmid gene mecA, was selected, and we found that spots 2 and 6 produced fluorescent signal, indicating Escherichia coli possesses 16S rDNA and mecA gene. The fluorescence of spot 6 represented that the paperbased chip could detect genes on plasmid as well as in the genome. In Figure 5d,e,f, the genomes of Staphylococcus aureus, Listeria monocytogenes, and Salmonella were introduced into the chip, respectively, showing the same result that only spot 2 displayed fluorescence, indicating that the antibiotic resistance genes mecA and ermC were not present in these three bacteria. The interrelated histograms of the test spots are shown in Figure 5B; the gray value of every spot in Figure 5b−f was measured. Based on above phenomena, in addition to the advantage of simultaneous detection, the paper-based chip was capable of detecting antibiotic resistance genes from various bacteria. 3.6. Real Sample Detection. We did the experiment to verify the validity of the paper-based chip in real sample detection. For the reason that the blood or pus from patients with MRSA was difficult to acquire and MRSA in diluted broth resuspended in human serum could simulate the real sample, we did the tests in simulating the actual sample environment. The cultured MRSA was centrifuged from 100 μL of lysogeny broth and resuspended into 1 mL of human serum. The genome DNA was isolated using the TIANamp Bacteria Genome DNA Kit and quantified by measuring the OD260. Here, the number of genome copies was 1 × 105. Then it was applied onto the paper-based chip for gene amplification and fluorescence detection. As shown in Figure 6A, the fluorescence intensity of the positive assays was notably stronger than negative controls. While the results of the positive assays were consistent, two of the negative controls, ermC third and mecA second, produced a little more fluorescence than other controls. It might be that the highly efficient LAMP assay caused the amplification of the primer dimer. Although a few negative controls produced weak fluorescence, the positive assay still had much higher fluorescence intensity compared to it, and in general, this method had good signal-to-noise ratio for real sample analysis. For quantitative analysis, the fluorescence intensity of each spot was measured (shown in Figure 6B). Consistent with the results of Figure 2 and Figure 3, the intensity of ermC and 16S rDNA appeared to be little different while the intensity of mecA was higher. The experimental results verified that the paper-based chip is capable of multiplex antibiotic resistance gene detection from a complex biological sample.
Mercedes logo, as shown in Figure 5A, each chip consisted of 6 spots: spots 1 and 2 contained 16S rDNA primers; spots 3 and
Figure 5. Specificity assay of the paper-based chip. Numbers in fluorescence photographs A denote different gene-specific amplification: (1) 16S rDNA negative control; (2) 16S rDNA experimental group; (3) ermC negative control; (4) ermC experimental group; (5) mecA negative control; (6) mecA experimental group. The results of the specificity assay for five bacteria: (a) all-positive result of the multiple amplification presented ideally via cartoon; (b) 2, 4, and 6 positive for Methicillin-resistant Staphylococcus aureus; (c) 2 and 6 positive for Escherichia coli; (d) 2 positive for Staphylococcus aureus; (e) 2 positive for Listeria monocytogenes; (f) 2 positive for Salmonella. Round dashed frames in the graph reflect the location of detection spots in the chip. (B) Fluorescence intensity of bacteria-specific amplification from part A, quantifying the intensity of gray value in each spot.
4. CONCLUSIONS In conclusion, we designed an inexpensive device for antibiotic resistance gene detection that enables LAMP assay integrated with the “light switch” molecule [Ru(phen)2dppz]2+ into the paper-based chip for the first time and tested its performance of speed, sensitivity, and specificity for gene analysis. The basis of fabrication used patterned paper sheets that contained the reaction mixture in multiple paper spots with the ability of paper to facilitate the transport of liquid by capillary effect, in conjunction with convenient isothermal amplification, replacing most of the instruments needed to conduct nucleic acid amplification tests (NAATs), and could be operated by nonprofessionals with minimal user steps. The compressive force of the magnetic plates and mineral oil between the layers
4 contained ermC primers; spots 5 and 6 contained mecA primers. The genome of bacteria was added into spots 2, 4 and 6, while spots 1, 3, and 5 were set as NTC. In such a spot pattern, the experimental group and negative control of each respective gene were close to each other, facilitating to form an obvious contrast. Fluorescence detection followed isothermal amplification. Figure 5a showed the ideal situation that every test spot with genome produced intense fluorescence; however, the NTC groups showed no fluorescent signal. In Figure 5b, the genome of MRSA was selected as specimen, and we found the fluorescence result was the same as Figure 5a, indicating that MRSA possesses 16S rDNA, mecA, and ermC as previously 4499
DOI: 10.1021/acsami.7b17653 ACS Appl. Mater. Interfaces 2018, 10, 4494−4501
Research Article
ACS Applied Materials & Interfaces
Figure 6. Real sample analysis using paper-based chip. (A) Fluorescence image of the paper-based chip for ermC, 16S rDNA, and mecA detection; ermc, 16S rDNA, and mecA indicates the spots in each row had the corresponding LAMP primers added, respectively. The “+” symbols and “−” symbols indicate positive assays adding 105 copies of MRSA genome and the negative control (no MRSA genome), respectively. Round dashed frames in the graph reflect the location of detection spots in the chip. Identical assay was conducted in three spots of each row. (B) Histogram reflecting the fluorescence intensity of detection spots in part A.
Technology Plan [201508020003], and the Program of the Pearl River Young Talents of Science and Technology in Guangzhou [2013J2200021], the Special Support Program of Guangdong Province (2014TQ01R599), and the Outstanding Young Teacher Training Program of Guangdong Province (HS2015004).
created a dynamic seal that resisted evaporation, which allowed incubation of the amplification reaction to be accomplished at 64 °C within 1 h. The paper-based chip took advantage of the intrinsic fantastic merit of the “light switch” molecule [Ru(phen)2dppz]2+, which was used to intercalate into the major groove of double stranded DNA for label-free fluorescence detection. It had a LOD of 100 copies for mecA gene and 285 copies for ermC gene from MRSA. Meanwhile, the paper-based chip could be used to detect antibiotic resistance genes from MRSA, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Salmonella respectively. The feasibility for multiple antibiotic resistance gene detection of MRSA real sample was also confirmed. This research demonstrated an available application of the paper-based chip in detecting antibiotic resistance genes from MRSA, and we believe it has great potential for higher throughput and more diverse detection of genes, particularly for POC diagnostics in resource-limited regions in the future.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b17653. Oligonucleotide sequences of LAMP primers and optimization experiments (PDF)
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REFERENCES
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AUTHOR INFORMATION
Corresponding Authors
*Xiaoming Zhou. E-mail:
[email protected]. *Da Xing. E-mail:
[email protected]. ORCID
Xiaoming Zhou: 0000-0001-5597-4804 Da Xing: 0000-0002-5098-0487 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China [21475048], the National Science Fund for Distinguished Young Scholars of Guangdong Province [2014A030306008], the Project of Guangzhou Science and 4500
DOI: 10.1021/acsami.7b17653 ACS Appl. Mater. Interfaces 2018, 10, 4494−4501
Research Article
ACS Applied Materials & Interfaces
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DOI: 10.1021/acsami.7b17653 ACS Appl. Mater. Interfaces 2018, 10, 4494−4501
