Simultaneous Detection of Antibiotic Resistance Genes on Paper

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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17653 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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ACS Applied Materials & Interfaces

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, Da Xing* MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China

* The contact information of the corresponding authors is Xiaoming Zhou, PhD, professor MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University E-mail: [email protected]

Da Xing, PhD, Professor MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University E-mail: [email protected]

Abstract Antibiotic resistance, the ability of some bacteria to resist antibiotic drug, has been a major global health burden due to the extensive use of antibiotic agents. Antibiotic resistance is encoded via respective genes, hence the specific detection of these genes is necessary for diagnostic and treat of antibiotic resistance cases. Conventional methods for monitoring antibiotic resistance genes require 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 ACS Paragon Plus Environment

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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 can simplify the detection procedure. [Ru(phen)2dppz]2+ was applied to intercalate into amplicons for product analysis, enabling this assay can 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 respectively from various bacteria. Noticeably, the approach can be applied to other genes besides antibiotic resistance genes detection 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 resistance bacteria that are difficult to treat have been becoming increasingly common and caused a severe global health crisis.1 Antibiotic resistance is encoded by respective genes, some of which can transfer among various bacteria via plasmid.2 With a great clinic 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 detection6 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, a mass of 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 ACS Paragon Plus Environment

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Accordingly, it is urgent to develop a method that could acquire antibiotic resistance genetic information at 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 resistance genes simultaneously, have been widely applied for various analytes detection containing metal ions,11 proteins,12 nucleic acids13 and so forth. We have developed a series of paper-based biosensors for plant virus,14 pathogenic bacteria15 and miRNA16 detection, for instance, quantum dots-labeled strip biosensor exhibited a favorable sensitivity of 200 amol for miRNA-21 detection.16 However, a major challenge concerning 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 acids 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 Isothermal nucleic acid amplification technologies have been widely raised concern 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 more efficient reaction than PCR.23 As one of these amplification assays, loop-mediated 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 hour.24 The usage of two pairs of primers makes LAMP possess transcendent specificity. Based on these properties, some researchers made attempt to introduce LAMP assay into the miniaturized microdevices such as glass,25 cyclic olefin copolymer26 and polystyrene.27 These favorable features make LAMP suitable ACS Paragon Plus Environment


to apply into paper-based devices for the improvement of detection efficiency. The conventional paper-based analytical methods for detecting the product of LAMP need to label signal probes on paper in advance, such as colloidal gold nanoparticles28 and calcein,29 which confront with some challenges, for instance, requiring expensive probe tagging moieties;30 nonspecific hybrid probes are difficult to clean up that result in a relatively high background signal.31 Although the intercalating dye SYBR Green Ι has been used to analyze the end products of LAMP for its character of label-free,32-33 there was some researches described that the ultraviolet fluorescence method generated a high ratio of false positives and was not recommended to be a LAMP read-out.34 Relatively, 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 result in intense fluorescence emission with a wavelength of 620 nm.35-36 Compared to above analytical methods, [Ru(phen)2dppz]2+ turn-on fluorescence detection possesses the properties of high sensitivity 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 spectrum region around 620–900 nm,37 thus facilitating to improve the 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 the simultaneous analysis of multiple antibiotic resistance genes for the first time. The nucleic acids amplification steps were simplified by fabricating the reaction discs with Whatman filter paper, which contained LAMP regents mix dried into the cellulose matrix. After sample was introduced into the reaction discs, put the chip in the ACS Paragon Plus Environment

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heating block immediately to proceed LAMP assay. Final detection of LAMP products used [Ru(phen)2dppz](PF6)2 and a handheld 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 macrolide-lincosamidestreptogramin resistance via rRNA methylases). Owing to the portable and low-cost of paper-based device, as well as LAMP technology and excellent fluorescence performance of [Ru(phen)2dppz]2+, the paper-based chip would have a great potential in POC detection of antibiotic resistance genes in resource-limited regions.

2. Experimental section 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 were listed in Table S1. Bst 2.0 DNA polymerase, 10 × Isothermal Amplification Buffer, 10 mM dNTPs, 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 Resistance strains methicillin resistant staphylococcus aureus (MRSA) and Escherichia coli (engineering bacteria with plasmid gene mecA) were offered from Guangzhou Center for Disease Control and Prevention, susceptive 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 0.75 mm thick ferrite-bonded 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 home-made, containing a DC power supply ACS Paragon Plus Environment


(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 contained five components from top to bottom, namely: a top magnetic plate; a magnetic plate with a 3 mm diameter of alternating poles; the filter paper was 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; a laminate for discs support and fixation; a bottom magnetic plate, which is shown in Fig. 1a. The attractive force produced by two magnetic plates form a seal to resist from evaporation in combination with the mineral oil applied to the layer interfaces.

2.3. Preparation of pre-amplification chips The gene-specific LAMP mix contained 0.2 µM of outer primers (F3 and B3), 1.6 µM of inner primers (FIP and BIP), 6 mM of MgSO4, 1× Isothermal Amplification Buffer, 1.4 mM of each dNTPs, 1 M of Betaine, 8 U of Bst 2.0 DNA polymerase and sterile water. When the paper-based chip was fabricated with 4 × 3 poles (Fig. 1b), distributing 5 µL LAMP mix equally in each pole. Then, the chip was left to air dry for 2 h and stored in 4 °C.

2.4. Bacteria culture, genome isolated 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 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 paper-based chip, 5 µL of 1 × Isothermal Amplification ACS Paragon Plus Environment

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Buffer which contained 1µL bacteria genome specimen was added into each pole of pre-amplification chip and then 5 µL mineral oil was appended into each pole to prevent liquid evaporation during the amplification. Pack the top magnetic plate on the chip to form a sealed structure, and put the chip into the copper heating block immediately. The optimal incubation was 64 °C for 55 min. The heating device is shown in Fig. 1c.

2.5. Product analysis by fluorescence detection After incubation, the chip was firstly removed from the incubator and cooled down to room temperature. Secondly, getting rid of remained mineral oil and then 4 µL detection reagent containing 1 mM [Ru(phen)2dppz](PF6)2 was added into each spot, standing at room temperature for five minutes. Finally, the chip was put under a handheld light source (450 nm wavelength) and imaged the fluorescence emitted from [Ru(phen)2dppz]2+. Images were then converted to grayscale and processed using Image J to quantify the intensity of gray value in each detection spot of chip. Utilizing the classical calculation method, we determine LOD as the mean of the no template control (NTC) plus 3 times of their standard deviation. The threshold value was used to determine positive and negative results.

3. Results and Discussion 3.1. Principle of the method The core architecture of the paper-based chip, illustrated in Fig. 1a, consisted of three major layers: a top magnetic layer, which combined with mineral oil in the layer interfaces, creating a seal to prevent evaporation of the reagents during amplification; the middle reaction layer consisted 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 ACS Paragon Plus Environment


reagents were dried and amplification was performed; the bottom layer contained 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 in 4 °C for further use. The results of the experiments for optimizing the reaction conditions were shown in Fig. S1, Fig. S2 and Fig. S3. We found that LAMP assay could get a good result at the condition of 8 mM Mg2+ and 64 °C. The optimal

Fig. 1. Schematic illustration of the paper-based chip analysis system. (A) The device contains the paper-based chip and a home-made 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 paper-based chip: I. Bacteria cell lysis; ACS Paragon Plus Environment

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II. Add LAMP reagents; III. Add bacteria genome; IV. Isothermal amplification; V. Add [Ru(phen)2dppz](PF6)2; VI. Fluorescent detection using a handheld light source.

amplification time of the paper-based chip was presented in the next section. After the genome was isolated from antibiotic-resistant bacteria and added into the chip, conduct LAMP assay under 64 °C for 55 min. In the final detection, we applied [Ru(phen)2dppz](PF6)2 to detect the LAMP products. 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 up with double stranded DNA, due to the planar phenazine ligand can interact with the base pairs in 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 handheld light source with a wavelength of 450 nm to excite the fluorescence of [Ru(phen)2dppz]2+-DNA complex, and imaging the pictures by a camera. An overview of the paper-based chip for multiple antibiotic resistance genes detection was illustrated in Fig. 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 amplified 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 the parallel tests for mecA and ermC detection. We ACS Paragon Plus Environment


conducted amplification in 48 chips respectively, they were isolated sequentially from heating block every five minutes and then added [Ru(phen)2dppz](PF6)2 immediately to detect fluorescence signals. Negative controls without genome were set to judge whether the result of amplification was reliable. In

Fig. 2. Real-time amplification of multiple LAMP for mecA and ermC. (A) Fluorescence intensity of amplification changes with time. The ‘+’ symbols and ‘-’ symbols indicates 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.

fluorescence photograph of the test spots (Fig. 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 Fig. 2B, t0 was defined as the moment when the chips put into the heating block. We found that the curves of negative controls kept at a low level of fluorescence, ACS Paragon Plus Environment

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meaning there were not amplification products in these spots during incubation. The curve of ermC positive assay rose at first—probably caused by that the loop primers of ermC had a higher hybridization efficiency than mecA—while the curve of mecA positive assay maintained at a higher level of fluorescence, based on the fact that the amplicon length of mecA (512 bp) was longer than ermC (297 bp), result in more [Ru(phen)2dppz](PF6)2 combined with the former. Meantime, the fluorescence of mecA reached maximum at nearly 55 min, as well as ermC. The signal-to-noise ratio of mecA at 55 min was 18.723 while ermC was 15.474. The result of real-time PCR for mecA and ermC amplification was shown in Fig. S4, which proved the same consequence that the loop primers could amplify mecA and ermC well. The final fluorescence intensity of mecA assay was stronger than 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 as paper-based amplification, and the curves of mecA positive assay in RT-PCR upraised faster than 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 above results showed the paper-based chip could be used to detect genes with outstanding results in one hour, and the optimal incubation time of this assay was 55 min. Compared to conventional LAMP assay that can be accomplished with 30-60 min in aqueous, the paper-based LAMP has no necessary to take more time to be completed and, significantly, it can simplify the preparation of reagents in testing ground with a substitute of pre-amplification chips, as well as reduce the subsequent analytical operation just with fluorescence probe introduced into spots of the paper-based chip and imaged.

3.3. Simultaneously analysis of antibiotic resistance genes ACS Paragon Plus Environment


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, 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 spots pattern as shown in Fig. 3A. Each chip involved four spots, namely: spot 1 and spot 2 contained 16S rDNA primers; spot 3 contained ermC primers; spot 4 contained 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 firstly read-out by naked eye using a handheld light source. As the quality control of the paper-based chip, the amplified result of 16S rDNA reflects whether the chip is credible. In Fig. 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 group (spot 2, 3 and 4) possessed intense fluorescence. In Fig. 3b, the genome was added only into spot 2, and the fluorescence was merely appeared in spot 2, as was expected, indicating that there was amplification product only when the test spot contained the genome. In Fig. 3c, the genome was introduced into spot 2 and 3, while in Fig. 3d, spot 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 quantifying the gray value of test spots using Image J. The

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Fig. 3. Simultaneous detection of multiple antibiotic resistance genes. In photographs (A), diagram (a) represents the all-positive result of the multiple amplification in ideal situation presented via cartoon. Numbers denote different genes-specific amplification: 1. no template control of 16S rDNA; 2. 16S rDNA; 3. ermC; 4. mecA. Photograph (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 (A), quantify the intensity of gray value in each spot. gray value of every spot in above Fig. 3b, 3c and 3d was measured and integrated into a histogram, as shown in Fig. 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 paper-based chip possessed high signal-to-noise ratio. Noticeably, this analytical method can be applied to other genetic targets besides antibiotic resistance genes detection by simply changing LAMP primers. It can be predictable 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 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 respectively, and every spots per 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 for 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 Fig. 4A and Fig. 4B, fluorescence photographs of the test spots could be clearly observed ACS Paragon Plus Environment


and the fluorescence intensity increased with the raise of genome from MRSA. Due to the fiber structure in the filter paper revealed irregular distribution, some parts of 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 Fig. 4C and Fig. 4D), we found that the fluorescence intensity reflected the amount of amplification products in appropriate scale of genome, which was from 100 copies to12500 copies for mecA gene, while from 200 copies to 25000 copies for ermC gene. We also

Fig. 4. Analytical sensitivity of antibiotic resistance genes 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 was repeated for five times. Round dashed frames in the graph reflect the ACS Paragon Plus Environment

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location of detection spots in the chip. Histogram (C) and (D) indicate the fluorescence intensity of the paper chip (A) and (B), quantify the intensity of gray value in each spot. Red dashed line denotes the LOD.

measured the LOD of the paper-based chip, using the mean of the negative control plus 3 times of their 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 the 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 resistance bacteria collected from patients is much more than the hundreds.41 Other methods such as electrochemical microchip integrated with PCR assay, which 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 genome,43 although the sensitivity of our paper-based chip was slightly lower than it, the paper-based chip was more portable and convenient with fewer operating steps, and noticeably, this chip was suitable for multiple genes 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 the paper-based chip could detect antibiotic resistance genes from different bacteria, 106 copies genome of MRSA, staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Salmonella were respectively used as target to conduct LAMP assay and fluorescence detection. We designed the pattern of test spots similar to a Mercedes logo, as shown in Fig. 5A, each chip consisted of 6 spots: spot 1 and 2 contained 16S rDNA primers; spot 3 and 4 contained ermC ACS Paragon Plus Environment


primers; spot 5 and 6 contained mecA primers. The genome of bacteria was added into spot 2, 4 and 6, while spot 1, 3 and 5 were set as NTC. In such a spots pattern, experimental group and negative control of respective gene were close to each other, facilitating to form an obvious contrast. Fluorescence detection was followed with isothermal amplification. Fig. 5a showed the ideal situation that every test spot with genome could be observed intense fluorescence, however the NTC groups without fluorescent signal. In Fig. 5b, the genome of MRSA was selected as specimen, and we found the fluorescence result was the same as Fig. 5a, indicating that MRSA possess 16S rDNA, mecA and ermC as previously mentioned. In Fig. 5c, the genome of Escherichia coli that is the engineering bacteria with plasmid gene mecA was selected and we found that spot 2 and 6 emerged fluorescent signal, indicating


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Fig. 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 in (A), quantify the intensity of gray value in each spot.

Escherichia coli possess 16S rDNA and mecA gene. The fluorescence of spot 6 represented that the paper-based chip could detect genes of plasmid as well as genome. In Fig. 5d, 5e and 5f, the genome of Staphylococcus aureus, Listeria monocytogenes and Salmonella were introduced into the chip respectively, showing the same result that only spot 2 displayed fluorescence, indicating the antibiotic resistance gene mecA and ermC were not existed in these three bacteria. The interrelated histogram of the test spots was shown in Fig. 5B, the gray value of every spot in above Fig. 5b, 5c, 5d, 5e and 5f 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 resuspend in human serum could simulate the real sample, we did the tests in ACS Paragon Plus Environment


simulating the actual sample environment. The cultured MRSA was centrifuged from 100 µL lysogeny broth and resuspended into 1 ml 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 was 1 × 105 copies. Then it was applied into the paper-based chip for gene amplification and fluorescence detection. As shown in Fig. 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 3rd and mecA 2nd, emerged 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 appeared week 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 were measured (shown in Fig. 6B).

Fig. 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 were added corresponding LAMP primers respectively. The ‘+’ symbols and ‘-’ symbols indicates 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 ACS Paragon Plus Environment

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fluorescence intensity of detection spots in (A).

Consistent with the result of Fig. 2 and Fig. 3, the intensity of ermC and 16S rDNA appeared to be little difference while the intensity of mecA was higher. The experimental results verified that the paper-based chip is capable of multiplex antibiotic resistance genes detection from complex biological sample.

4. Conclusions In conclusion, we designed an inexpensive device for antibiotic resistance genes 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 genes 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 the convenient isothermal amplification, replaced most of the instruments needed to conduct nucleic acid amplification tests (NAATs), and could be operated by non-professionals with minimal user steps. The compressive force of the magnetic plates and mineral oil between the layers created a dynamic seal that resisted evaporation, which allowed incubation of the amplification reaction to be accomplished at 64 °C within 1 hour. 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 genes detection of MRSA real sample ACS Paragon Plus Environment


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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 a great potential for higher throughput and more diverse detection of genes, particularly for POC diagnostic in resource-limited regions in the future.

ASSOCIATED CONTENT Supporting Information The oligonucleotide sequences of LAMP primers and optimal experiments supplied as Supporting Information is included. The Supporting Information is available free of charge on the ACS Publications website.

Notes The authors declare no competing financial interest.

Acknowledgment 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 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).

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